WO2006025801A2 - Methods for cancer diagnosis - Google Patents

Abstract

We disclose a method of diagnosis of a cancer in an individual, the method comprising detecting modulation of expression of a Sprouty1 sequence. A method of treatment or prophylaxis of a cancer in an individual is also disclosed, the method comprising modulating the amount of a Sprouty1 polypeptide or nucleic acid in a cell of an individual. The methods are suitable for detection and treatment of breast cancer, based on our demonstration that Sprouty1 expression is down-regulated in this disease.

Description

METHODS

FIELD

This invention relates to the field of medicine. In particular, it relates to treatment and diagnosis of diseases, in particular breast cancer, as well as compositions for such use.

BACKGROUND

In the Western world and the developed countries of Asia, breast carcinoma is the second leading cause of cancer-related death in women (18). Breast cancer tops the cancer list for women in Singapore, with 700-800 new cases being diagnosed each year (19). In the USA, 180,000 women are diagnosed annually with new cases of breast cancer (18). Despite better diagnosis and routine screening around a quarter of the cases will die from their disease.

The identification of genes and biochemical pathways involved in breast oncogenesis are of utmost importance in the rational molecularly based preventative and therapeutic approaches. Until recently, no specific molecular alteration has been observed in even the majority of breast cancers.

The BRCAl and BRCA2 genes involved in hereditary breast cancers do not appear to play a role in sporadic cases, which represent by far the majority of the cases. Amplification or over-expression of oncogenes [c-myc, erbB2, cyclin DΪ and epidermal growth factor receptor (EGF-R)] (20, 21) and loss of tumour suppressor genes [p53, PTEN, PTCH (patched), MKK4] (22-25) occur in only a fraction of the cases. Recently a gene encoding a novel cytokine, HIN-I, was identified via SAGE (Serial Analysis of Gene Expression) as a candidate breast tumour suppressor gene that is not expressed and is hypermethylated in the majority of breast cancers (26). HIN-I is inactivated in pre¬ invasive tumours, such as DCIS and LCIS, and its methylation is high (over 70%) in early- stage tumours, which makes it a very good marker for early detection of breast cancer. Several other genes have been demonstrated to be hypermethylated in breast carcinomas, including; pi 6, E-cadherin, BRCAl, oestrogen receptor, GSTPl (glutathione S-transferase Pl), MDGI (mammary-derived growth factor inhibitor), HoxA5 and 14-3-3- σ (27-34) . However relatively low numbers of tumours were ranked positive for hypermethylation of these genes with the exception ofl4-3-3-σ that has abnormal methylation levels in around 50% of invasive carcinomas. The silencing of HIN-I is however not specific to breast tumours as it is also found in lung and prostate carcinomas (26).

Accordingly, there is a need for markers for breast cancer detection, and in particular, those which are tissue specific for breast tissue.

It is known that down-regulation of expression of Sprouty2 in breast cancer tissue compared to normal breast tissue. WO 2004/029295 discloses the use of Sprouty2 nucleic acids and polypeptides in the treatment of breast cancer, as well as the detection of breast cancer in an individual.

SUMMARY

According to a 1^st aspect of the present invention, we provide a method of diagnosis of a cancer in an individual, the method comprising detecting modulation of expression of a Sproutyl sequence in the individual, or any part of the individual.

Preferably, the cancer comprises breast cancer. Preferably, the method comprises detecting down-regulation of Sproutyl expression in a breast cell or tissue of or from the individual.

Preferably, a Sproutyl nucleic acid is detected by means of a probe comprising at least a portion of a nucleic acid having the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. Preferably, the method comprises detecting a Sproutyl polypeptide, preferably by means of an antibody to Sproutyl, in a sample comprising a breast cell or tissue from the individual. Preferably, the expression of Sproutyl in the sample is compared to the expression of Sproutyl in a control breast cell known to be non-cancerous. Preferably, a down- regulation of Sproutyl expression in the sample compared to the control breast cell is diagnostic of breast cancer, or susceptibility to breast cancer.

There is provided, according to a 2^nd aspect of the present invention, a method for identifying a pre-cancerous breast cell, comprising detecting a reduced level of a Sproutyl polypeptide and/or a Sproutyl nucleic acid in the cell, or an extract thereof.

In preferred embodiments, the method further comprises detection of modulation, preferably down-regulation, of expression of a Sprouty2 sequence, preferably by means of a probe comprising at least a portion of a nucleic acid having the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 7, or an antibody to Sprouty2.

We provide, according to a 3^rd aspect of the present invention, a specific binding agent for Sproutyl for use in a method of diagnosis of a cancer, preferably breast cancer.

Preferably, the specific binding agent is selected from the group consisting of: (a) a nucleic acid probe comprising a Sproutyl nucleic acid, or a fragment thereof capable of hybridisation to a Sproutyl sequence; (b) a nucleic acid probe having the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a fragment thereof; (c) a primer comprising between 10 to 15 residues from a sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, preferably having a sequence selected from the sequences set out in Tables IA or IB; (d) a pair of primers comprising a forward primer selected from sequences depicted in Table IA together with a reverse primer selected from sequences depicted in Table IB; and (e) an anti- Sproutyl antibody.

As a 4^th aspect of the present invention, there is provided a combination of a specific binding agent according to the 3^rd aspect of the invention and a specific binding agent for Sprouty2, for use in a method of diagnosis of a cancer, preferably breast cancer. We provide, according to a 5^th aspect of the present invention, a method of treatment or prophylaxis of a cancer in an individual, the method comprising modulating the amount of a Sproutyl polypeptide or nucleic acid in a cell of an individual.

Preferably, the cancer comprises a breast cancer. Preferably, the amount of a Sproutyl polypeptide or nucleic acid is increased, preferably specifically, in a breast cell of the individual.

Preferably, the amount of a Sproutyl polypeptide or nucleic acid is not substantially increased in any other cell or tissue type.

The present invention, in a 6* aspect, provides a method of manipulating a cell, the method comprising the steps of: (a) detecting a reduced level of a Sproutyl polypeptide or nucleic acid in a cell, or an extract thereof; (b) increasing the level of a Sproutyl polypeptide or nucleic acid in the cell.

Preferably, the cell is derived from or present in an individual at risk of developing breast cancer. Preferably, the expression or activity of an endogenous Sproutyl sequence is up-regulated. Preferably, a control sequence, preferably a promoter and/or an enhancer sequence of Sproutyl is replaced with an endogenous control sequence.

Preferably, the expression of a Sproutyl sequence is up-regulated in a breast cell of the individual, but not substantially in any other cell or tissue type. Preferably, an expression construct capable of delivering breast cell specific expression of Sproutyl is introduced into a cell of the individual.

In a preferred embodiment, the method further comprises modulating, preferably increasing, the amount of a Sprouty2 polypeptide or nucleic acid in a cell, preferably a breast cell, of an individual, preferably such that the amount of a Sprouty2 polypeptide or nucleic acid is not substantially increased in any other cell or tissue type, preferably by means of an expression construct capable of delivering breast cell specific expression of Sprouty2. Preferably, the method comprises introducing a nucleic acid according to the 7^th aspect of the invention below to a cell of the individual.

In a 7^th aspect of the present invention, there is provided a nucleic acid construct comprising a Sproutyl gene or a coding portion thereof, together with one or more control elements selected from the group consisting of: (a) a tumour specific promoter selected from the group consisting of: vascular endothelial growth factor (VEGF) promoter, vascular endothelial growth factor receptor- 1 (VEGFR-I) promoter, VEGFR-2 promoter, c-erbB2 promoter, L-plastin promoter, Bcl-2 promoter and MUCl promoter; (b) a breast tissue specific promoter selected from the group consisting of: human α-lactalbumin (ALA) promoter, ovine β-lactoglobulin (BLG) promoter and a long terminal repeat (LTR) of a mouse mammary tumour virus (MMTV); (c) an inducible promoter selected from the group consisting of: a stress gene promoter, a heat shock protein (HSP) promoter and a multidrug resistance gene-1 (MDR-I) promoter.

In preferred embodiments, the nucleic acid construct further comprises a Sprouty2 gene or a coding portion thereof.

Preferably, the method further comprises administering a Sproutyl polypeptide or nucleic acid to the individual. In a preferred embodiment, the method further comprises administering a Sprouty2 polypeptide or nucleic acid to the individual.

According to an 8^th aspect of the present invention, we provide a host cell comprising a nucleic acid construct according to the 7^th aspect of the present invention.

We provide, according to a 9^th aspect of the invention, a breast cell transformed with a nucleic acid construct according to the 7^th aspect of the present invention.

There is provided, in accordance with a 10^th aspect of the present invention, a transgenic non-human animal comprising a transgene which does not express, or expresses a reduced level, of Sproutyl . In preferred embodiments, the transgenic non-human animal further comprises a transgene which does not express, or expresses a reduced level, of Sprouty2.

As an 11^th aspect of the invention, we provide use of a transgenic non-human animal according to the 10^th aspect of the present invention as a model for breast cancer.

We provide, according to a 12^th aspect of the invention, a method of identifying a molecule capable of binding to a Sproutyl polypeptide, the method comprising contacting a Sproutyl polypeptide with a candidate molecule and determining whether the candidate molecule binds to the Sproutyl polypeptide.

According to a 13^th aspect of the present invention, we provide a method of identifying a modulator of Sproutyl expression, the method comprising contacting a cell with a candidate molecule, and detecting elevated expression of Sproutyl in or of the cell.

There is provided, according to a 14^th aspect of the present invention, a method of identifying a drug, the method comprising exposing a transgenic animal according to the 10^th aspect of the invention to a candidate molecule, and detecting or monitoring the development or lack thereof of a breast cancer in the transgenic animal.

Preferably, the method further comprises isolating and/or synthesising the molecule.

We provide, according to a 15^th aspect of the present invention, a molecule identified, isolated or synthesised by a method according to the 12^th, 13^th or 14^th aspect of the invention..

According to a 16^th aspect of the present invention, we provide use of a molecule capable of modulating, preferably down-regulating, the expression of a Sproutyl sequence, preferably a molecule according to the 15^th aspect of the invention, in a method of treatment of a breast cancer. According to a 17^th aspect of the present invention, we provide a molecule capable of modulating, preferably down-regulating, the expression of a Sproutyl sequence, preferably a molecule according to the 15^th aspect of the invention, for use in a method of treatment of a breast cancer.

We provide, according to an 18^th aspect of the present invention, a nucleic acid having the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a fragment thereof capable of specifically hybridising to a Sproutyl sequence, preferably in combination with a nucleic acid having the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 7 or a fragment thereof capable of specifically hybridising to a Sprouty2 sequence.

According to a 19^th aspect of the present invention, we provide a primer comprising between 10 to 15 residues from a sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, preferably having a sequence selected from the sequences set out in Tables IA or IB, preferably in combination with a primer comprising between 10 to 15 residues from a sequence shown in SEQ ID NO: 4 or SEQ ID NO: 7.

As an 20^th aspect of the invention, we provide a Sproutyl polypeptide or nucleic acid, or a combination of such with a Sprouty2 polypeptide or nucleic acid, for use in a method of treatment of a cancer, preferably breast cancer, in an individual.

According to a 21^st aspect of the present invention, we provide an antibody capable of specific binding to Sproutyl, or a combination of such with an antibody capable of specific binding to Sprouty2, for use in a method of treatment of a cancer, preferably breast cancer, in an individual.

In highly preferred embodiments, the Sproutyl comprises human Sproutyl, preferably comprising a human Sproutyl nucleic acid having a GenBank accession number NP_005832 or AAH63856, or a human Sproutyl polypeptide having a GenBank accession number 043609. There is provided, according to a 22^nd aspect of the present invention, a diagnostic kit for detecting breast cancer in an individual, or susceptibility of the individual to breast cancer, comprising means for detection of Sproutyl expression in the individual or a sample taken from him or her.

Preferably, the means for detection is selected from the group consisting of: a

Sproutyl polynucleotide or a fragment thereof; a complementary nucleotide sequence to Sproutyl nucleic acid or a fragment thereof; a Sproutyl polypeptide or a fragment thereof, or an antibody to a Sproutyl, preferably comprising an anti-human Sproutyl antibody, more preferably comprising a rabbit polyclonal to His tagged full length human Sproutyl 2, as well as an anti-human Sproutyl antibody comprising a rabbit polyclonal antibody to residues 58-75 of human Sproutyl, and optionally instructions for use.

In preferred embodiments, the diagnostic kit further comprises a means for detection of Sprouty2 expression in the individual or a sample taken from him or her, preferably comprising: a Sprouty2 polynucleotide or a fragment thereof; a complementary nucleotide sequence to Sprouty2 nucleic acid or a fragment thereof; a Sprouty2 polypeptide or a fragment thereof, or an antibody to a Sprouty2, and optionally instructions for use.

Preferably, the diagnostic kit further comprises a therapeutic drug for treatment of breast cancer, preferably comprising Tamoxifen or Herceptin.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, IrI Press; D. M. J. Lilley and J. E. Dahlberg, 1992, Methods ofEnzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; Using Antibodies : A Laboratory Manual : Portable Protocol NO. I by Edward Harlow, David Lane, Ed Harlow (1999, Cold Spring Harbor Laboratory Press, ISBN 0-87969-544-7); Antibodies : A Laboratory Manual by Ed Harlow (Editor), David Lane (Editor) (1988, Cold Spring Harbor Laboratory Press, ISBN 0-87969-314-2), 1855. Handbook of Drug Screening, edited by Ramakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, NY, Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench, Edited Jane Roskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN 0-87969-630-3. Each of these general texts is herein incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows an analysis of microarray database (Global Cancer Map) comparing the expression of Spryl and 2 in human cancers with respect to normal tissues. Tumor tissues, spanning 12 common tumor types, and unmatched normal tissues are subjected to oligonucleotide microarray (Affymetrix GeneChips) gene expression analysis. The gene expression for the individual tumor tissues are compared to the mean values of that of the normal tissue. The panel shows the expression of hSpryl and hSpry2 in human cancers, with respect to normal tissues. hSpry2 is down-regulated in the breast cancer. (Explanation of scale: Red indicates an up-regulation in gene expression in tumors with respect to normal tissue. Green indicates a down-regulation in gene expression in tumors with respect to normal tissue. The scale indicates the fold change in gene expression.)

Figures 2A and 2B. cDNA array studies show a down-regulation of hSpryl and 2 in breast tumors.

Figure 2A The Cancer Profiling Array contains pairs of cDNAs generated from matched tumor and normal (T= tumor; N= normal) tissue samples from individual patients, spotted side by side on a nylon membrane. All samples on these arrays are normalized to two different housekeeping genes: β-actin and ubiquitin. There are 50 matched pairs of normal and tumor cDNA from 50 breast cancer patients. [α-³²P]-labeled N-terminal half of hSpry2 cDNA is hybridized to the Cancer Profiling Array. hSpry2 expression is down-regulated in breast cancer. A high (96%) proportion of breast cancer samples show at least an arbitrary 1.2 fold down-regulation in the expression of hSpry2. Comparatively, the changes in expression of hSpry2 in other tumors are not as significant as that for the breast tumors.

Figure 2B. The group of 3 spots of cDNAs outlined by a box represents a matched pair of normal (top left) and tumor (top right) cDNA from a patient, as well as cDNA from the metastases in the same patient (bottom right). [α-³²P] -labeled N-terminal half of hSpryl cDNA is hybridized to the Cancer Profiling Array. hSpryl expression is down- regulated in breast cancer. A high (78%) proportion of breast cancer samples show at least an arbitrary 1.2 fold down-regulation in the expression of hSpryl. To demonstrate that the sample population of breast cancer tumors in the blot are a true representation of the population of breast cancers, as well as to demonstrate equal loading of cDNA samples on the blot, the blot is stripped and reprobed for ErbB2. 50% of the patients with breast cancer showed at least an arbitrary 2-fold up-regulation in ErbB2. The blot is stripped and re-probed with a gene known to be down-regulated in breast cancer, Maspin. The extent of down-regulation of Maspin in breast cancer is not as profound and widespread as that observed for hSpiy2.

Figures 3 A and 3B show analysis of breast cancer samples by quantitative real¬ time PCR shows a profound down-regulation in levels of hSpryl and hSpry2 in breast cancer.

Figure 3 A. The expression of hSpry2 in 19 malignant breast tumors and paired normal tissue is analyzed. The expression of hSpry2 in the normal tissue is averaged and deemed to be 100% (solid horizontal line) and the SEM is demarcated with the dotted lines. The tumor samples are expressed as a percentage of the mean expression of hSpry2 in normal tissues. Each sample is analyzed three times and a mean value + SEM is calculated. 18 out of 19 (94.7%) of the tumor samples show a significant down-regulation oϊhSpry2 expression, with respect to normal tissue.

Figure 3B. The expression of hSpryl in 19 malignant breast tumors are analyzed and compared to the baseline expression (100%) oϊhSpryl found in normal breast tissue. 18 out of 19 malignant tumors showed a significant down-regulation oϊhSpryl. A 2-100 fold down-regulation oϊhSpryl and hSpry2 is observed in the breast tumors.

Figures 4A-4D show mouse mammary glands at different stages of development show that mSpryl and 2 are localized in the epithelium of the mammary ducts.

Figure 4A. H&E and in situ staining of mammary glands of 2wks, 6wks and 16wks female mice. mSpry2 is highly expressed in developing mammary ducts in 2 and 6 weeks old females and appears to be confined specifically to the epithelial lining of the mammary ducts and is absent in the stroma and adipose tissues. The level of expression starts decreasing as the mice reach sexual maturity, as can be evidenced by the lower levels apparent at 16 weeks.

Figure 4B. H&E and in situ staining of mammary glands of mice undergoing pregnancy, lactation and involution. In pregnant mice the level oϊmSpry2 becomes elevated again when it is highly expressed in the actively developing alveoli. mSpiy2 expression then diminishes in the lactating female and appears to be totally absent during the involution phase.

Figure 4C. hSpry2 sense control probe for mSpry hybridized to a section of the mammary gland from a pregnant mouse, demonstrating a lack of non-specific staining.

Figure 4D shows in situ staining for mSpry2 (left) and mSpryl (right) respectively, showing co-localization of the two Spry isoforms, specifically in the luminal epithelial cells of the mammary ducts of a pregnant mouse. Figures 5A-5D show in situ analysis of human breast tissue shows the down- regulation of Spry isoforms in breast cancer.

Figure 5A. in situ staining for hSpry2 (left) and hFgf8 (right) in a normal section of tissue found in a human ductal carcinoma. Both hSpry2 and hFgfδ are co-localized in the epithelial lining of breast ducts.

Figure 5B. H&E and in situ staining of for hSpryl, hSpry2 and hFgf8 in a human ductal carcinoma (grade 3). There is no detectable hSpry2 or hSpryl staining, although hFgfS (previously shown to be up-regulated in breast cancer) staining is still apparent.

Figure 5C. Immunohistochemical staining of hSpry2 in normal human breast tissue. hSpry2 localizes specifically in the epithelial lining of the breast duct.

Figure 5D. Immunohistochemical staining of hSpry2, ErbB2 (using Neu-2 antibody) and FgfS in an invasive ductal carcinoma (grade 3). hSpry2 expression is down- regulated in breast cancer. In the same tissue, both ErbB2 (stained with Neu-2 antibody) and Fgf8 are present in relatively high amounts. ErbB2 and Fgf8, which have been previously shown to be up-regulated in breast cancer, have been used as positive controls to demonstrate the integrity of the tissue.

Figure 6A-6C. Inhibiting Spry's function causes cells to proliferate faster, exhibit anchorage independent growth and form profoundly larger tumors.

Figure 6A. Proliferation assay. MCF-7 hSpry2Y55F stable and control cells are seeded and cultured in 10% serum for 12hr. The number of living cells is measured using a MTS assay. The quantity of formazan product is measured by the amount of 485nm absorbance is directly proportional to the number of living cells in culture. The values correspond to the average absorbance at 485nm ± SEM derived from triplicates of a representative experiment. hSpry2Y55F MCF-7 cells show a 22% significant increase in proliferation over control MCF-7 cells (* p = 0.0034). The experiment is repeated 3 times with similar results being obtained. Figure 6B. Colony forming assays. MCF-7 hSpry2Y55F stable and control cells are cultured in 6-well plates first covered in soft agar. After 14 days, the colonies are stained with MTT dye. hSpry2Y55-transfected clones formed larger and greater numbers of colonies compared to control MCF-7 cells. The experiment is repeated 3 times with similar results being obtained.

Figure 6C. Xenograft assays in nude mice. To compare the in vivo tumorigenic potential of the MCF-7-hSpry2Y55 cells with that of the control MCF-7 cells, both types of cells are injected separately into each side of the nude mice. The animals are sacrificed at 9 weeks and both tumors are extracted and weighed. The respective tumors from 15 different animals are weighed and tabulated. The values correspond to the average weight of tumors ± SD derived from 15 mice. The hSpry2Y55F cell line formed significantly larger (2.9X) tumor masses compared with the control MCF-7 cell line (* p = 0.0001). The experiment is repeated 2 times with similar results being obtained.

Figure 7. Epigenetic silencing is not responsible for the down-regulation of Spry2 expression in breast tumors. Treatment of T47D breast cancer cells with 2.5μM 5-azaDC and/or 30OnM trichostatin does not up-regulate the expression QΪ hSpryl. In instances where cells are doubly treated, cells are subjected to 96hr of 5-azaDC treatment and trichostatin is added in the last 24hr. Maspin is a gene known to be silenced by hypermethylation and/or histone deacetylation. Treatment with either 5-aza-DC and/or trichostatin is known to up-regulate its expression. It has been used as a positive control to show that both treatments are effective. The human ^-microglobulin gene is used as an internal control.

Figure 8. In order to verify that the [α-³²P] dCTP-labeled, N-terminal hSpry2 cDNA probe is specific for the hSpry2 isoform, a dot blot consisting of full length cDNA of hSpryl, hSpry2, hSpry3 and hSpry4 is prepared. The blots are probed with each radiolabeled N-terminal hSpry2 cDNA. Rows: Full length cDNA. Columns: ³²P labelled N-terminal hSpry cDNA probe. Row 1: hSpryl, Row 2: hSpry2, Row 3: hSpry3, Row 4: hSpry4. Column 1: hSpryl, Column 2: hSpry2, Column 3: hSpry3, Column 4: hSpry4. Figure 9A. cDNAs are immobilized on the commercial Cancer Profiling array as supplied by Clontech. The spots represent normal/tumour cDNAs from various tissues as indicated. Adjacent spots are from the same patient. Where three spots are boxed they are (in clockwise direction) normal, metastatic and tumour tissue. N = normal, T = tumour,. Columns: 1-4 breast, 7-10 uterus, 13-16 colon, 19-20 stomach, 23-24 ovary, 27-28 lung, 31-32 kidney, 35-36 rectum, 39-40 thyroid. Colon, cervix, prostate, pancreas, small intestine as indicated.

Figure 9B. An autoradiograph showing binding of the specific, radiolabeled hSpry2 probe to the Cancer Profiling Array. The X-ray film is exposed to the blot for 10 hours. Columns: breast, uterus, colon, stomach, ovary, lung, kidney, rectum, thyroid. Cervix, prostate, pancreas, small intestine as indicated.

Figure 9C-9E. A scatter-plot diagram of the relationship between the expression of hSpry2 in normal compared with tumour tissue. Each spot is measured on a densitometer, background levels are subtracted and the ratio of change (density of normal spot/density of tumour spot expressed in arbitrary units) is plotted on a scatter diagram. The midpoint line is indicative of no change (ie 1:1 ratio). The dotted lines either side of the midpoint line represents an estimate of experimental error and/or insignificant change based on an analysis of micro-array data. Spots under and over the line represent decreased and increased expression of hSpry2, respectively, in tumour tissues compared with normal tissue from the same patient. The ratio of metastasic tumours are indicated in pink diamonds.

Figure 9C. A scatter diagram comparing the expression ratios (normal/tumour) of hSpry2 in breast = tissues. Breast = blue diamonds, breast (metastasis) = squares. X-axis: normal tissue (arbitrary units); Y-axis: tumour tissue (arbitrary units).

Figure 9D. A scatter diagram comparing the expression ratios (normal/tumour) of hSpry2 in breast versus colon tumour tissues. Breast = blue diamonds, colon = red triangles. X-axis: normal tissue (arbitrary units); Y-axis: tumour tissue (arbitrary units). Figure 9E. A scatter diagram comparing the expression ratios (normal/tumour) of hSpry2 in breast versus uterine tumour tissues. Breast = blue diamonds, uterus = red triangles. X-axis: normal tissue (arbitrary units); Y-axis: tumour tissue (arbitrary units).

Figure 9F. A re-probed Cancer Profiling Array blot to demonstrate equality of cDNA loading. The blot shown in Figure 2B is stripped and incubated with a [α-³²P] dCTP-labeled ubiquitin probe to reveal equality of loading of the individual cDNAs. Columns: breast, uterus, colon, stomach, ovary, lung, kidney, rectum, thyroid. Cervix, prostate, pancreas, small intestine as indicated.

SEQUENCE LISTING

SEQ ID NO: 1 shows the nucleic acid sequence of a Homo sapiens Sproutyl fragment, comprising N-terminal sequences. SEQ ID NO: 2 shows the amino acid sequence of a. Homo sapiens Sproutyl fragment, comprising N-terminal sequences. SEQ ID NO: 3 shows the nucleic acid sequence of full length Homo sapiens Sproutyl. SEQ ID NO: 4 shows the amino acid sequence of full length Homo sapiens Sproutyl.

SEQ ID NO: 5 shows the nucleic acid sequence of a Homo sapiens Sprouty2 fragment, comprising N-terminal sequences. SEQ ID NO: 6 shows the amino acid sequence of a Homo sapiens Sprouty2 fragment, comprising N-terminal sequences. SEQ ID NO: 7 shows the nucleic acid sequence of full length Homo sapiens Sprouty2. SEQ ID NO: 8 shows the amino acid sequence of full length Homo sapiens Sprouty2.

The methods and compositions described here may suitably employ any one or more of the sequences shown in the Sequence Listing.

DETAILED DESCRIPTION

This invention is based on the demonstration that the expression of Sproutyl in breast cancer tissue is down-regulated when compared to normal breast tissue. The Examples show that 94.7% of breast cancers analysed by real-time PCR demonstrate a reduction in Sproutyl and 2 levels.

Accordingly, this enables the level of Sproutyl expression to be used as an indicator of cancer, in particular breast cancer. The level of Sproutyl expression may also be used as an indicator of likelihood of such cancer. We therefore provide for methods of diagnosis or detection of a cancer, preferably breast cancer by detecting modulation of expression of a Sproutyl sequence of an individual.

Sproutyl and Sprouty2 can, for example, be used as potential markers for detection of breast cancer via, for example, analysis of protein levels (e.g., immunohistochemistry) or RNA levels (e.g., by in situ hybridisation).

Restoration of Sproutyl levels to those in normal tissue may also be used as a means of restoring normal function of breast cells. Accordngly, we provide for the use of Sproutyl nucleic acids and polypeptides for the treatment of cancers, including breast cancer.

In highly preferred embodiments, modulation of Sprouty2 expression is also detected in conjunction with Sproutyl for diagnosis. Furthermore, in preferred embodiments, Sprouty2 nucleic acids and polypeptides are also administered in conjunction with Sproutyl for the treatment of cancers, as described in WO 2004/029295, hereby incorporated by reference.

While not wishing to be bound, we believe that Sproutyl acts as a tumour suppressor, along with Sprouty2.

SPROUTYI

Where the terms "Sprouty" and "Spry" are used, these should be taken to refer to any Sprouty sequence, including a Sprouty protein or a Sprouty nucleic acid, including any member of the Sprouty family such as Sproutyl, Sprouty2, Sprouty3 and Sprouty4 and any fragment, variant homologue, derivative, variant thereof.

The terms "Sproutyl" and "Spry2" should preferably be taken to refer to any Sproutyl sequence such as a Sproutyl polypeptide, Sproutyl nucleic acid, fragment, derivative, homologue or variant, "human Sproutyl" and "hSpryl" should be taken to be synonymous with each other, and to refer to any human Sproutyl sequence.

The properties and activities of Sproutyl are described in, for example, in the references.

SPROUTYI POLYPEPTIDES

The methods and compositions described here make use of Sproutyl polypeptides, which are described in detail below.

As used here, the term "Sproutyl polypeptide" is intended to refer to any one or more of AAD56004 sprouty 1 [Mus musculus]; AAD56008 sprouty 1 protein [Gallus gallus]; AAH39139 Spryl protein [Mus musculus]; AAH53428 Spryl protein [Mus musculus]; AAH63856 Sprouty homolog 1, antagonist of FGF signaling [Homo sapiens]; AAH69914 Sprouty homolog 1 [Mus musculus]; AAP21610 sproutyl [Mus musculus]; AAT06102 sprouty 1 [Homo sapiens]; AP21610 sproutyl [Mus musculus]; NP_005832 sprouty homolog 1, antagonist of FGF signaling; sprouty, Drosophila, homolog of, 1 (antagonist of FGF signaling); sprouty (Drosophila) homolog 1 (antagonist of FGF signaling) [Homo sapiens]; NP_005833 sprouty 2; sprouty (Drosophila) homolog 2 [Homo sapiens]; NP_036026 sprouty homolog 1 [Mus musculus]; NP_659032 Wilms tumor homolog [Mus musculus]; NP_955359 sprouty homolog 1, antagonist of FGF signaling; sprouty, Drosophila, homolog of, 1 (antagonist of FGF signaling); sprouty (Drosophila) homolog 1 (antagonist of FGF signaling) [Homo sapiens]; 043609 Sprouty homolog 1 (Spry-1) [Homo sapiens]; Q9PTL1 Sprouty homolog 1 (Spry-1) [Gallus gallus]; or Q9QXV9 Sprouty homolog 1 (Spry-1) [Mus musculus]. Homologues variants and derivatives thereof of any, some or all of these polypeptides are also included. "human Sproutyl", "Sproutyl" and "hSpryl" should be taken to be synonymous, and in the context of polypeptides, to refer to any human Sproutyl sequence, including but not limited to AAH63856 Sprouty homolog 1, antagonist of FGF signaling [Homo sapiens]; AAT06102 sprouty 1 [Homo sapiens]; NP_005832 sprouty homolog 1, antagonist of FGF signaling; sprouty, Drosophila, homolog of, 1 (antagonist of FGF signaling); sprouty (Drosophila) homolog 1 (antagonist of FGF signaling) [Homo sapiens]; NP_955359 sprouty homolog 1, antagonist of FGF signaling; sprouty, Drosophila, homolog of, 1 (antagonist of FGF signaling); sprouty (Drosophila) homolog 1 (antagonist of FGF signaling) [Homo sapiens]; and 043609 Sprouty homolog 1 (Spry-1) [Homo sapiens]

In preferred embodiments, a "Sproutyl polypeptide" comprises or consists of a human Sproutyl polypeptide, preferably, the sequence having accession number 043609 and labelled "Sprouty homolog 1 (Spry-1)". In preferred embodiments, human Sproutyl comprises or preferably has the sequence shown in SEQ ID NO: 4.

Sproutyl polypeptides may be used for a variety of means, for example, administration to an individual suffering from, or suspected to be suffering from, breast cancer, for the treatment thereof. They may also be used for production of specific Sproutyl binding agents, in particular, anti-Sproutyl antibodies. These are described in further detail below. The expression of Sproutyl polypeptides may be detected for diagnosis or detection of cancer, in particular breast cancer.

A "polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.

"Polypeptides" include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well blown in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side- chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications.

Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-inking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-inks, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. See, for instance, Proteins - Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in

Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al., "Analysis for protein modifications and nonprotein cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et aL, "Protein Synthesis: Posttranslational Modifications and Aging", Ann NY Acad Sd (1992) 663:48-62.

The term "polypeptide" includes the various synthetic peptide variations known in the art, such as a retroinverso D peptides. The peptide may be an antigenic determinant and/or a T-cell epitope. The peptide may be immunogenic in vivo. Preferably the peptide is capable of inducing neutralising antibodies in vivo. Preferably, as applied to Sproutyl, the resultant amino acid sequence has one or more activities, preferably, biological activities in common with a Sproutyl polypeptide, preferably a human Sproutyl polypeptide. For example, a Sproutyl homologue may have a lowered expression level in breast cancer cells compared to normal breast cells. In particular, the term "homologue" covers identity with respect to structure and/or function providing the resultant amino acid sequence has Sproutyl activity. With respect to sequence identity (i.e. similarity), preferably there is at least 70%, more preferably at least 75%, more preferably at least 85%, even more preferably at least 90% sequence identity. More preferably there is at least 95%, more preferably at least 98%, sequence identity. These terms also encompass polypeptides derived from amino acids which are allelic variations of the Sproutyl nucleic acid sequence.

Where reference is made to the "activity" or "biological activity" of a polypeptide such as Sproutyl, these terms are intended to refer to the metabolic or physiological function of Sproutyl, including similar activities or improved activities or these activities with decreased undesirable side effects. Also included are antigenic and immunogenic activities of the Sproutyl. Examples of such activities, and methods of assaying and quantifying these activities, are known in the art, and are described in detail elsewhere in this document.

For example, such activities may include any one or more of the following: ability to inhibit one or more events downstream of receptor tyrosine kinases; ability to prevent activation of Ras/Raf; inhibition of the Ras/Raf/MAPK pathway; ability to downregulate ERK phosphorylation when the Ras/MAPK pathway is activated.

Other Sproutyl Polypeptides

Sproutyl variants, homologues, derivatives and fragments are also of use in the methods and compositions described here.

The terms "variant", "homologue", "derivative" or "fragment" in relation to Sproutyl include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid from or to a sequence. Unless the context admits otherwise, references to "Sproutyl" includes references to such variants, homologues, derivatives and fragments of Sproutyl.

As used herein a "deletion" is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent. As used herein an "insertion" or "addition" is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring substance. As used herein "substitution" results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.

Sproutyl polypeptides as described here may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent amino acid sequence. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to the table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

Sproutyl polypeptides may further comprise heterologous amino acid sequences, typically at the N-terminus or C-terminus, preferably the N-terminus. Heterologous sequences may include sequences that affect intra or extracellular protein targeting (such as leader sequences). Heterologous sequences may also include sequences that increase the immunogenicity of the Sproutyl polypeptide and/or which facilitate identification, extraction and/or purification of the polypeptides. Another heterologous sequence that is particularly preferred is a polyamino acid sequence such as polyhistidine which is preferably N-terminal. A polyhistidine sequence of at least 10 amino acids, preferably at least 17 amino acids but fewer than 50 amino acids is especially preferred.

The Sproutyl polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro- sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.

Sproutyl polypeptides as described here are advantageously made by recombinant means, using known techniques. However they may also be made by synthetic means using techniques well known to skilled persons such as solid phase synthesis. Such polypeptides may also be produced as fusion proteins, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and β- galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences, such as a thrombin cleavage site. Preferably the fusion protein will not hinder the function of the protein of interest sequence.

The Sproutyl polypeptides may be in a substantially isolated form. This term is intended to refer to alteration by the hand of man from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide, nucleic acid or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide, nucleic acid or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. It will however be understood that the Sproutyl protein may be mixed with carriers or diluents which will not interfere with the intended purpose of the protein and still be regarded as substantially isolated. A Sproutyl polypeptide may also be in a substantially purified form, in which case it will generally comprise the protein in a preparation in which more than 90%, for example, 95%, 98% or 99% of the protein in the preparation is a Sproutyl polypeptide.

By aligning Sproutyl sequences from different species, it is possible to determine which regions of the amino acid sequence are conserved between different species ("homologous regions"), and which regions vary between the different species ("heterologous regions").

The Sproutyl polypeptides may therefore comprise a sequence which corresponds to at least part of a homologous region. A homologous region shows a high degree of homology between at least two species. For example, the homologous region may show at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% identity at the amino acid level using the tests described above. Peptides which comprise a sequence which corresponds to a homologous region may be used in therapeutic strategies as explained in further detail below. Alternatively, and preferentially, the Sproutyl peptide may comprise a sequence which corresponds to at least part of a heterologous region. A heterologous region shows a low degree of homology between at least two species. For example, the N-terminus of Sprouty is known to be heterologous between Sproutyl, Sprouty2, Sprouty3 and Sprouty4.

Sproutyl Homologues

The Sproutyl polypeptides disclosed for use include homologous sequences obtained from any source, for example related viral/bacterial proteins, cellular homologues and synthetic peptides, as well as variants or derivatives thereof. Thus polypeptides also include those encoding homologues of Sproutyl from other species including animals such as mammals (e.g. mice, rats or rabbits), especially primates, more especially humans. More specifically, homologues include human homologues. In the context of this document, a homologous sequence is taken to include an amino acid sequence which is at least 15, 20, 25, 30, 40, 50, 60, 70, 80 or 90% identical, preferably at least 95 or 98% identical at the amino acid level, preferably over at least 50 or 100, preferably 200, 300, 400 or 500 amino acids with the sequence of a relevant Sproutyl sequence.

In particular, homology should typically be considered with respect to those regions of the sequence known to be essential for protein function rather than non¬ essential neighbouring sequences. This is especially important when considering homologous sequences from distantly related organisms.

Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present document it is preferred to express homology in terms of sequence identity.

Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate % identity between two or more sequences.

% identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).

Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local identity or similarity.

However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package (see below) the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A; Devereux et al, 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al, 1999 ibid- Chapter 18), FASTA (Altschul et al, 1990, J. MoI. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al, 1999 ibid, pages 7-58 to 7-60). However it is preferred to use the GCG Bestfit program.

Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

The terms "variant" or "derivative" in relation to amino acid sequences includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the sequence providing the resultant amino acid sequence retains substantially the same activity as the unmodified sequence, preferably having at least the same activity as the Sproutyl polypeptides (e.g., a sequence having accession number 043609 and labelled "Sprouty homolog 1 (Spry-1)".).

Polypeptides having the Sproutyl amino acid sequence disclosed here, or fragments or homologues thereof may be modified for use in the methods and compositions described here. Typically, modifications are made that maintain the biological activity of the sequence. Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions provided that the modified sequence retains the biological activity of the unmodified sequence. Alternatively, modifications may be made to deliberately inactivate one or more functional domains of the polypeptides described here. Amino acid substitutions may include the use of non-naturally occurring analogues, for example to increase blood plasma half-life of a therapeutically administered polypeptide.

Sproutyl Fragments

Polypeptides for use in the methods and compositions described here also include fragments of the full length sequence of any of the Sproutyl polypeptides identified above. Preferably fragments comprise at least one epitope. Methods of identifying epitopes are well known in the art. Fragments will typically comprise at least 6 amino acids, more preferably at least 10, 20, 30, 50 or 100 amino acids. Included are fragments comprising, preferably consisting of, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, or more residues from a relevant Sproutyl amino acid sequence.

We further describe peptides comprising a portion of a Sproutyl polypeptide as described here. Thus, fragments of Sproutyl and its homologues, variants or derivatives are included. The peptides may be between 2 and 200 amino acids, preferably between 4 and 40 amino acids in length. The peptide may be derived from a Sproutyl polypeptide as disclosed here, for example by digestion with a suitable enzyme, such as trypsin. Alternatively the peptide, fragment, etc may be made by recombinant means, or synthesised synthetically.

A preferred fragment of Sproutyl comprises a human Sproutyl fragment, for example, a fragment of a sequence having accession number 043609. A preferred fragment of Sproutyl may preferably comprise an N-terminal sequence of human Sproutyl, such as a sequence shown in SEQ ID NO: 2. Such N-terminal fragments of Sproutyl are particularly useful as the sequence similarity at the N-terminus between

Sproutyl, Sprouty2, Sprouty3 and Sprouty4 is low. Accordingly, fragments comprising N- terminal sequences may be used to generate probes to preferentially detect Sproutyl expression, for example, through antibodies generated against such fragments. These antibodies would be expected to bind specifically to Sproutyl, and are useful in the methods of diagnosis disclosed here.

Particularly preferred fragments include a Sproutyl fragment sequence depicted as SEQ ID NO: 2. It will be appreciated that a number of residues C-terminal side of the SEQ ID NO: 2 sequence may be included without influencing the specificity. Sproutyl and its fragments, homologues, variants and derivatives, may be made by recombinant means. However they may also be made by synthetic means using techniques well known to skilled persons such as solid phase synthesis. The proteins may also be produced as fusion proteins, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and β-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the function of the protein of interest sequence. Proteins may also be obtained by purification of cell extracts from animal cells.

The Sproutyl polypeptides, variants, homologues, fragments and derivatives disclosed here may be in a substantially isolated form. It will be understood that such polypeptides may be mixed with carriers or diluents which will not interfere with the intended purpose of the protein and still be regarded as substantially isolated. A Sproutyl variant, homologue, fragment or derivative may also be in a substantially purified form, in which case it will generally comprise the protein in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the protein in the preparation is a protein.

The Sproutyl polypeptides, variants, homologues, fragments and derivatives disclosed here may be labelled with a revealing label. The revealing label may be any suitable label which allows the polypeptide , etc to be detected. Suitable labels include radioisotopes, e.g. ¹²⁵I, enzymes, antibodies, polynucleotides and linkers such as biotin. Labelled polypeptides may be used in diagnostic procedures such as immunoassays to determine the amount of a polypeptide in a sample. Polypeptides or labelled polypeptides may also be used in serological or cell-mediated immune assays for the detection of immune reactivity to said polypeptides in animals and humans using standard protocols.

A Sproutyl polypeptides, variants, homologues, fragments and derivatives disclosed here, optionally labelled, may also be fixed to a solid phase, for example the surface of an immunoassay well or dipstick. Such labelled and/or immobilised polypeptides may be packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like. Such polypeptides and kits may be used in methods of detection of antibodies to the polypeptides or their allelic or species variants by immunoassay.

Immunoassay methods are well known in the art and will generally comprise: (a) providing a polypeptide comprising an epitope bindable by an antibody against said protein; (b) incubating a biological sample with said polypeptide under conditions which allow for the formation of an antibody-antigen complex; and (c) determining whether antibody-antigen complex comprising said polypeptide is formed.

The Sproutyl polypeptides, variants, homologues, fragments and derivatives disclosed here may be used in in vitro or in vivo cell culture systems to study the role of their corresponding genes and homologues thereof in cell function, including their function in disease. For example, truncated or modified polypeptides may be introduced into a cell to disrupt the normal functions which occur in the cell. The polypeptides may be introduced into the cell by in situ expression of the polypeptide from a recombinant expression vector (see below). The expression vector optionally carries an inducible promoter to control the expression of the polypeptide.

The use of appropriate host cells, such as insect cells or mammalian cells, is expected to provide for such post-translational modifications (e.g. myristolation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products. Such cell culture systems in which the Sproutyl polypeptides, variants, homologues, fragments and derivatives disclosed here are expressed may be used in assay systems to identify candidate substances which interfere with or enhance the functions of the polypeptides in the cell.

The Sproutyl polypeptides, and fragments, homologues, variants or derivatives thereof may be provided with, or used in conjunction with, Sproutyl polypeptides as described in detail in WO 2004/029295 (hereby incorporated by reference). SPROUTYI NUCLEIC ACIDS

The methods and compositions described here may employ, as a means for detecting expression levels of SproutyI, SproutyI polynucleotides, SproutyI nucleotides and SproutyI nucleic acids, as well as variants, homologues, derivatives and fragments of any of these. In addition, we disclose particular SproutyI fragments useful for the methods of diagnosis described here. The SproutyI nucleic acids may also be used for the methods of treatment or prophylaxis described.

The terms "SproutyI polynucleotide", "SproutyI nucleotide" and "SproutyI nucleic acid" may be used interchangeably, and should be understood to specifically include both cDNA and genomic SproutyI sequences. These terms are also intended to include a nucleic acid sequence capable of encoding a SproutyI polypeptide and/or a fragment, derivative, homologue or variant of this.

Where reference is made to a SproutyI nucleic acid, this should be taken as a reference to any member of the SproutyI family of nucleic acids. Of particular interest are SproutyI nucleic acids selected from the group consisting of: AF 176903 Mus musculus sprouty 1 (Spryl) mRNA, complete cds; AF 177875 Gallus gallus sprouty 1 protein (SPRYl) mRNA, partial cds; AY260058 Mus musculus SproutyI (Spryl) gene, complete cds; AY590694 Homo sapiens clone IMAGE: 6028920 sprouty 1 mRNA, partial cds; BC039139 Mus musculus sprouty homolog 1 (Drosophila), mRNA (cDNA clone MGC: 18307 IMAGE.-3672353), complete cds; BC053428 Mus musculus sprouty homolog 1 (Drosophila), mRNA (cDNA clone MGC:59320 IMAGE:6506766), complete cds; BC063856 Homo sapiens sprouty homolog 1, antagonist of FGF signaling (Drosophila), transcript variant 1, mRNA (cDNA clone MGC:75188 IMAGE:6181674), complete cds; BC069914 Mus musculus sprouty homolog 1 (Drosophila), mRNA (cDNA clone MGC:78167 IMAGE:5251876), complete cds; NM_005841 Homo sapiens sprouty homolog 1, antagonist of FGF signaling (Drosophila) (SPRYl), transcript variant 1, mRNA; NM_011896 Mus musculus sprouty homolog 1 (Drosophila) (Spryl), mRNA; NMJ44783 Mus musculus Wilms tumor homolog (WtI), mRNA; NM_199327 Homo sapiens sprouty homolog 1, antagonist of FGF signaling (Drosophila) (SPRYl), transcript variant 2, mRNA; NT_016354 Homo sapiens chromosome 4 genomic contig.

Also included are any one or more of the nucleic acid sequences set out as "Other Sproutyl nucleic acid sequences" below.

In preferred embodiments, a Sproutyl nucleic acid comprises a human Sproutyl

(hSpryl) sequence selected from the group consisting of: AY590694 Homo sapiens clone IMAGE:6028920 sprouty 1 mRNA, partial cds; BC063856 Homo sapiens sprouty homolog 1, antagonist of FGF signaling (Drosophila), transcript variant 1, mRNA (cDNA clone MGC:75188 IMAGE:6181674), complete cds; NM_005841 Homo sapiens sprouty homolog 1, antagonist of FGF signaling (Drosophila) (SPRYl), transcript variant 1, mRNA; NM_199327 Homo sapiens sprouty homolog 1, antagonist of FGF signaling (Drosophila) (SPRYl), transcript variant 2, mRNA; NT_016354 Homo sapiens chromosome 4 genomic contig.

In some embodiments, a Sproutyl nucleic acid comprises or has a sequence shown in SEQ ID NO: 3. In preferred embodiments, a Sproutyl nucleic acid comprises or is a human Sproutyl sequence having GenBank accession number NP_005832 or a sequence having GenBank accession number or AAH63856.

Sproutyl nucleic acids may be used for a variety of means, for example, administration to an individual suffering from, or suspected to be suffering from, breast cancer, for the treatment thereof. The expression of Sproutyl nucleic acids may be detected for diagnosis or detection of cancer, in particular breast cancer. Sproutyl nucleic acids may also be used for the expression or production of Sproutyl polypeptides.

"Polynucleotide" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double- stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.

"Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications has been made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

It will be understood by the skilled person that numerous nucleotide sequences can encode the same polypeptide as a result of the degeneracy of the genetic code.

As used herein, the term "nucleotide sequence" refers to nucleotide sequences, oligonucleotide sequences, polynucleotide sequences and variants, homologues, fragments and derivatives thereof (such as portions thereof). The nucleotide sequence may be DNA or RNA of genomic or synthetic or recombinant origin which may be double-stranded or single-stranded whether representing the sense or antisense strand or combinations thereof. The term nucleotide sequence may be prepared by use of recombinant DNA techniques (for example, recombinant DNA).

Preferably, the term "nucleotide sequence" means DNA. Other Nucleic Acids

We also provide nucleic acids which are fragments, homologues, variants or derivatives of Sproutyl nucleic acids. The terms "variant", "homologue", "derivative" or "fragment" in relation to Sproutyl nucleic acid include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acids from or to the sequence of a Sproutyl nucleotide sequence. Unless the context admits otherwise, references to "Sproutyl" and "Sproutyl" include references to such variants, homologues, derivatives and fragments of Sproutyl.

Preferably, the resultant nucleotide sequence encodes a polypeptide having any one or more Sproutyl activity. Preferably, the term "homologue" is intended to cover identity with respect to structure and/or function such that the resultant nucleotide sequence encodes a polypeptide which has Sproutyl activity. For example, a homologue etc of Sproutyl may have a reduced expression level in breast cancer cells compared to normal breast cells. With respect to sequence identity (i.e. similarity), preferably there is at least 70%, more preferably at least 75%, more preferably at least 85%, more preferably at least 90% sequence identity. More preferably there is at least 95%, more preferably at least 98%, sequence identity to a relevant sequence (e.g., a human Sproutyl sequence having GenBank accession number NP_005832 or a sequence having GenBank accession number or AAH63856). These terms also encompass allelic variations of the sequences.

Variants, Derivatives and Homologues

Sproutyl nucleic acid variants, fragments, derivatives and homologues may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of this document, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.

Where the polynucleotide is double-stranded, both strands of the duplex, either individually or in combination, are encompassed by the methods and compositions described here. Where the polynucleotide is single-stranded, it is to be understood that the complementary sequence of that polynucleotide is also included. The terms "variant", "homologue" or "derivative" in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence. Preferably said variant, homologues or derivatives code for a polypeptide having biological activity. Preferably, such fragments, homologues, variants and derivatives of Sproutyl comprise modulated activity, as set out above.

As indicated above, with respect to sequence identity, a "homologue" has preferably at least 5% identity, at least 10% identity, at least 15% identity, at least 20% identity, at least 25% identity, at least 30% identity, at least 35% identity, at least 40% identity, at least 45% identity, at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to the relevant sequence (e.g., a human Sproutyl sequence having GenBank accession number NP_005832 or a sequence having GenBank accession number or AAH63856).

More preferably there is at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, more preferably at least 99% identity. Nucleotide identity comparisons may be conducted as described above. A preferred sequence comparison program is the GCG Wisconsin Bestfit program described above. The default scoring matrix has a match value of 10 for each identical nucleotide and -9 for each mismatch. The default gap creation penalty is -50 and the default gap extension penalty is -3 for each nucleotide.

Hybridisation

We further describe nucleotide sequences that are capable of hybridising selectively to any of the sequences presented herein, or any variant, fragment or derivative thereof, or to the complement of any of the above. Nucleotide sequences are preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40 or 50 nucleotides in length. The term "hybridization" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerase chain reaction technologies.

Polynucleotides capable of selectively hybridising to the nucleotide sequences presented herein, or to their complement, may be at least 40% homologous, at least 45% homologous, at least 50% homologous, at least 55% homologous, at least 60% homologous, at least 65% homologous, at least 70% homologous, at least 75% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous, or at least 95% homologous to the corresponding nucleotide sequences presented herein (e.g., a human Sproutyl sequence having GenBank accession number NP_005832 or a sequence having GenBank accession number or AAH63856). Preferably, such polynucleotides will be generally at least 70%, preferably at least 80 or 90% and more preferably at least 95% or 98% homologous to the corresponding nucleotide sequences over a region of at least 20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides.

The term "selectively hybridizable" means that the polynucleotide used as a probe is used under conditions where a target polynucleotide is found to hybridize to the probe at a level significantly above background. The background hybridization may occur because of other polynucleotides present, for example, in the cDNA or genomic DNA library being screening. In this event, background implies a level of signal generated by interaction between the probe and a non-specific DNA member of the library which is less than 10 fold, preferably less than 100 fold as intense as the specific interaction observed with the target DNA. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with ³²P or ³³P or with non-radioactive probes (e.g., fluorescent dyes, biotin or digoxigenin).

Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, VoI 152, Academic Press, San Diego CA), and confer a defined "stringency" as explained below. Maximum stringency typically occurs at about Tm-5°C (5⁰C below the Tm of the probe); high stringency at about 5°C to 10⁰C below Tm; intermediate stringency at about 10⁰C to 20⁰C below Tm; and low stringency at about 2O⁰C to 25°C below Tm. As will be understood by those of skill in the art, a maximum stringency hybridization can be used to identify or detect identical polynucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences.

In a preferred aspect, we provide nucleotide sequences that can hybridise to the Sproutyl nucleic acids, fragments, variants, homologues or derivatives under stringent conditions (e.g. 65⁰C and O.lxSSC (lxSSC = 0.15 MNaCl, 0.015 M Na₃ Citrate pH 7.0)).

Sproutyl nucleic acid variants, fragments, derivatives and homologues, as well as nucleic acids capable of hybridising thereto may be provided with, or used in conjunction with, Sprouty2 nucleic acids as described in WO 2004/029295, hereby incorporated by reference, in the methods and compositions described here.

Generation of Homologues, Variants and Derivatives

Polynucleotides which are not 100% identical to the preferred sequences (e.g., a human Sproutyl sequence having GenBank accession number NP_005832 or a sequence having GenBank accession number or AAH63856) but which are also included, as well as homologues, variants and derivatives of Sproutyl can be obtained in a number of ways. Other variants of the sequences may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations. For example, Sproutyl homologues may be identified from other individuals, or other species. Further recombinant Sproutyl nucleic acids and polypeptides may be produced by identifying corresponding positions in the homologues, and synthesising or producing the molecule as described elsewhere in this document.

In addition, other viral/bacterial, or cellular homologues of Sproutyl, particularly cellular homologues found in mammalian cells (e.g. rat, mouse, bovine and primate cells), may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to human Sproutyl. Such homologues may be used to design non- human Sproutyl nucleic acids, fragments, variants and homologues. Mutagenesis may be carried out by means known in the art to produce further variety.

Sequences of Sproutyl homologues may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of any of the Sproutyl nucleic acids, fragments, variants and homologues, or other fragments of Sproutyl under conditions of medium to high stringency.

Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences disclosed here.

Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the Sproutyl nucleic acids. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PiIeUp program is widely used.

The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences. It will be appreciated by the skilled person that overall nucleotide homology between sequences from distantly related organisms is likely to be very low and thus in these situations degenerate PCR may be the method of choice rather than screening libraries with labelled fragments the Sproutyl sequences.

In addition, homologous sequences may be identified by searching nucleotide and/or protein databases using search algorithms such as the BLAST suite of programs. Alternatively, such polynucleotides may be obtained by site directed mutagenesis of characterised sequences, for example, Sproutyl nucleic acids, or variants, homologues, derivatives or fragments thereof. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.

The polynucleotides described here may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 8, 9, 10, or 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term "polynucleotides" as used herein.

Polynucleotides such as a DNA polynucleotides and probes may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.

In general, primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.

Primers comprising fragments of Sproutyl are particularly useful in the methods of detection of Sproutyl expression, preferably down-regulation of Sproutyl expression, for example, as associated with breast cancer. Suitable primers for amplification of Sproutyl may be generated from any suitable stretch of the abuot 2500 nucleotide bases of Sproutyl . Particularly preferred primers are those capable of amplifying a sequence of Sproutyl which is specific, i.e., does not have significant homology to other Sprouty family members such as Sproutyl, Sprouty3 and Sprouty4. Such primers preferably are capable of binding to and amplifying an "N-terminal" region of Sproutyl. In preferred embodiments, a primer for amplification of Sproutyl may comprise a sequence 5' AGGGCTATCTTCCTAGCA 3 ' or a sequence 5'

GTGAGAAGCATGGGGT 3'. Preferably, a primer pair comprising the two preceding sequences is provided.

Although Sproutyl primers may be provided on their own, they are most usefully provided as primer pairs, comprising a forward primer and a reverse primer.

In preferred embodiments, a Sproutyl forward primer comprises a sequence selected from the group consisting of the residues of SEQ ID NO: 1 shown in Table IA below:

Table IA

In preferred embodiments, a Sproutyl reverse primer comprises a sequence selected from the group consisting of the residues of SEQ ED NO: 1 shown in Table IB below:

Table IB

Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides), bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector

Polynucleotides or primers may carry a revealing label. Suitable labels include radioisotopes such as ³²P or ³⁵S, digoxigenin, fluorescent dyes, enzyme labels, or other protein labels such as biotin. Such labels may be added to polynucleotides or primers and may be detected using by techniques known />er se. Polynucleotides or primers or fragments thereof labelled or unlabeled may be used by a person skilled in the art in nucleic acid-based tests for detecting or sequencing polynucleotides in the human or animal body.

Such tests for detecting generally comprise bringing a biological sample containing DNA or RNA into contact with a probe comprising a polynucleotide or primer under hybridising conditions and detecting any duplex formed between the probe and nucleic acid in the sample. Such detection may be achieved using techniques such as PCR or by immobilising the probe on a solid support, removing nucleic acid in the sample which is not hybridised to the probe, and then detecting nucleic acid which has hybridised to the probe. Alternatively, the sample nucleic acid may be immobilised on a solid support, and the amount of probe bound to such a support can be detected. Suitable assay methods of this and other formats can be found in for example WO89/03891 and WO90/13667.

Tests for sequencing nucleotides, for example, the Sproutyl nucleic acids, involve bringing a biological sample containing target DNA or RNA into contact with a probe comprising a polynucleotide or primer under hybridising conditions and determining the sequence by, for example the Sanger dideoxy chain termination method (see Sambrook et al).

Such a method generally comprises elongating, in the presence of suitable reagents, the primer by synthesis of a strand complementary to the target DNA or RNA and selectively terminating the elongation reaction at one or more of an A, C, G or T/U residue; allowing strand elongation and termination reaction to occur; separating out according to size the elongated products to determine the sequence of the nucleotides at which selective termination has occurred. Suitable reagents include a DNA polymerase enzyme, the deoxynucleotides dATP, dCTP, dGTP and dTTP, a buffer and ATP. Dideoxynucleotides are used for selective termination.

Sproutyl Control Regions

For some purposes, it may be necessary to utilise or investigate control regions of Sproutyl. Such control regions include promoters, enhancers and locus control regions. By a control region we mean a nucleic acid sequence or structure which is capable of modulating the expression of a coding sequence which is operatively linked to it.

For example, control regions are useful in generating transgenic animals expressing Sproutyl. Furthermore, control regions may be used to generate expression constructs for Sproutyl. This is described in further detail below.

Identification of control regions of Sproutyl is straightforward, and may be carried out in a number of ways. For example, the coding sequence of Sproutyl may be obtained from an organism, by screening a cDNA library using a human or mouse Sproutyl cDNA sequence as a probe. 5' sequences may be obtained by screening an appropriate genomic library, or by primer extension as known in the art. Database searching of genome databases may also be employed. Such 5' sequences which are particularly of interest include non-coding regions. The 5' regions may be examined by eye, or with the aid of computer programs, to identify sequence motifs which indicate the presence of promoter and/or enhancer regions.

Furthermore, sequence alignments may be conducted of Sproutyl nucleic acid sequences from two or more organisms. By aligning Sproutyl sequences from different species, it is possible to determine which regions of the amino acid sequence are conserved between different species. Such conserved regions are likely to contain control regions for the gene in question (i.e., Sproutyl). The mouse and human genomic sequences as disclosed here, for example, a mouse Sproutyl genomic sequence, may be employed for this purpose. Furthermore, Sproutyl homologues from other organisms may be obtained using standard methods of screening using appropriate probes generated from the mouse and human Sproutyl sequences. The genome of the pufferfish (Takifiigu rubripes) or zebrafish may also be screened to identify a Sproutyl homologue; thus, several zebrafish sequences of Sproutyl have been identified (noted above). Comparison of the 5' non-coding region of the Fugu or zebrafish Sproutyl gene with a mouse or human genomic Sproutyl sequence may be used to identify conserved regions containing control regions.

Deletion studies may also be conducted to identify promoter and/or enhancer regions for Sproutyl.

The identity of putative control regions may be confirmed by molecular biology experiments, in which the candidate sequences are linked to a reporter gene and the expression of the reporter detected. NUCLEIC ACID VECTORS

Sproutyl polynucleotides, for example those described here, can be incorporated into a recombinant replicable vector.

The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, we provide a method of making polynucleotides by introducing a polynucleotide into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells include bacteria such as E. coli, yeast, mammalian cell lines and other eukaryotic cell lines, for example insect Sf9 cells.

Preferably, a polynucleotide in a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term "operably linked" means that the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.

The control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators.

Vectors may be transformed or transfected into a suitable host cell as described below to provide for expression of a protein. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the protein, and optionally recovering the expressed protein. Vectors will be chosen that are compatible with the host cell used. The vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. Vectors may be used, for example, to transfect or transform a host cell.

Control sequences operably linked to sequences encoding the Sproutyl polypeptide include promoters/enhancers and other expression regulation signals. These control sequences may be selected to be compatible with the host cell for which the expression vector is designed to be used in. The term promoter is well-known in the art and encompasses nucleic acid regions ranging in size and complexity from minimal promoters to promoters including upstream elements and enhancers.

The promoter is typically selected from promoters which are functional in mammalian cells, although prokaryotic promoters and promoters functional in other eukaryotic cells, such as insect cells, may be used. The promoter is typically derived from promoter sequences of viral or eukaryotic genes. For example, it may be a promoter derived from the genome of a cell in which expression is to occur. With respect to eukaryotic promoters, they may be promoters that function in a ubiquitous manner (such as promoters of α-actin, β-actin, tubulin) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyruvate kinase). They may also be promoters that respond to specific stimuli, for example promoters that bind steroid hormone receptors. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter, the rous sarcoma virus (RSV) LTR promoter or the human cytomegalovirus (CMV) IE promoter.

It may also be advantageous for the promoters to be inducible so that the levels of expression of the heterologous gene can be regulated during the life-time of the cell. Inducible means that the levels of expression obtained using the promoter can be regulated. In addition, any of these promoters may be modified by the addition of further regulatory sequences, for example enhancer sequences. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.

Polynucleotides may also be inserted into the vectors described above in an antisense orientation to provide for the production of antisense RNA. Antisense RNA or other antisense polynucleotides may also be produced by synthetic means. Such antisense polynucleotides may be used in a method of controlling the levels of RNAs transcribed from genes comprising any one of the polynucleotides described here.

The vectors described above which comprise Sproutyl coding sequences may optionally further comprise Sprouty2 coding sequences. Construction of such Sprouty2 vectors is described in detail in WO 2004/029295, hereby incorporated by reference.

HOST CELLS

Vectors and polynucleotides comprising or encoding Sproutyl nucleic acids, fragments, homologues, variants or derivatives thereof (and/or Sprouty2 nucleic acids, fragments, homologues, variants or derivatives thereof) may be introduced into host cells for the purpose of replicating the vectors/polynucleotides and/or expressing the Sproutyl/2 polypeptides encoded by the polynucleotides. Although the Sproutyl polypeptides may be produced using prokaryotic cells as host cells, it is preferred to use eukaryotic cells, for example yeast, insect or mammalian cells, in particular mammalian cells.

Vectors/polynucleotides may be introduced into suitable host cells using a variety of techniques known in the art, such as transfection, transformation and electroporation. Where vectors/polynucleotides are to be administered to animals, several techniques are known in the art, for example infection with recombinant viral vectors such as retroviruses, herpes simplex viruses and adenoviruses, direct injection of nucleic acids and biolistic transformation. PROTEIN EXPRESSION AND PURIFICATION

Host cells comprising polynucleotides may be used to express polypeptides, such as Sproutyl polypeptides, fragments, homologues, variants or derivatives thereof. Host cells may be cultured under suitable conditions which allow expression of the proteins. Expression of the Sproutyl polypeptides may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG.

Sproutyl polypeptides can be extracted from host cells by a variety of techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption.

Sproutyl polypeptides may also be produced recombinantly in an in vitro cell-free system, such as the TnT™ (Promega) rabbit reticulocyte system.

BREAST CANCER

According to the methods and compositions described here, Sproutyl is useful for diagnosising or treating breast cancer. Additionally, in accordance with the disclosure in WO 2004/029295, hereby incorporated by reference, Sproutyl may be used in conjunction with Sprouty2 described therein for such diagnosis or treatment.

There are several types of breast cancer. The most common is ductal carcinoma, which begins in the lining of the milk ducts of the breast. Another type, lobular carcinoma, begins in the lobules where breast milk is produced. If a malignant tumor invades nearby tissue, it is known as infiltrating or invasive cancer. When breast cancer spreads outside the breast, cancer cells often are found in the lymph nodes under the arm. Breast cancer cells may spread beyond the breast such as to other lymph nodes, the bones, liver, or lungs. The recognised stages of breast cancer comprise:

Stage 0: Very early breast cancer. This type of cancer has not spread within or outside the breast. It is sometimes called DCIS, LCIS, or breast cancer in situ or non¬ invasive cancer.

Stage I: The cancer is no larger than about 1 inch in size and has not spread outside the breast, (also described as early breast cancer.)

Stage II: The presence of any of the following: the cancer is no larger than 1 inch, but has spread to the lymph nodes under the arm; the cancer is between 1 and 2 inches. It may or may not have spread to the lymph nodes under the arm; the cancer is larger than 2 inches, but has not spread to the lymph nodes under the arm.

Stage III and Stage IIIA: The presence of any of the following: the cancer is smaller than 2 inches and has spread to the lymph nodes under the arm, the cancer also is spreading further to other lymph nodes; the cancer is larger than 2 inches and has spread to the lymph nodes under the arm.

Stage IIIB: The presence of any of the following: the cancer has spread to tissues near the breast (skin, chest wall, including the ribs and the muscles in the chest); the cancer has spread to lymph nodes inside the chest wall along the breast bone.

Stage IV: The cancer has spread to other parts of the body, most often the bones, lungs, liver, or brain. Or, the tumor has spread locally to the skin and lymph nodes inside the neck, near the collarbone.

Inflammatory Breast Cancer: Inflammatory breast cancer is a rare, but very serious, aggressive type of breast cancer. The breast may look red and feel warm. There may be ridges, welts, or hives on the breast; or the skin may look wrinkled. It is sometimes misdiagnosed as a simple infection. Recurrent Breast Cancer: Recurrent disease means that the cancer has come back (recurred) after it has been treated. It may come back in the breast, in the soft tissues of the chest (the chest wall), or in another part of the body.

Breast Cancer in situ - DCIS and LCIS

Many breast cancers being found are very early cancers known as breast cancer in situ or noninvasive cancer. Most of these cancers are found by mammography. These very early cell changes may become invasive breast cancer. Two types of breast cancer in situ include the following:

DCIS (ductal carcinoma in situ), which means that abnormal cells are found only in the lining of a milk duct of the breast. The abnormal cells have not spread outside the duct. They have not spread within the breast, beyond the breast, to the lymph nodes under the arm, or to other parts of the body. There are several types of DCIS. If not removed, some types may change over time and become invasive cancers. Some may never become invasive cancers. (DCIS is sometimes called intraductal carcinoma.)

LCIS (lobular carcinoma in situ), which means that abnormal cells are found in the lining of a milk lobule. Although LCIS is not considered to be actual breast cancer at this noninvasive stage, it is a warning sign of increased risk of developing invasive cancer. LCIS is sometimes found when a biopsy is done for another lump or unusual change that is found on a mammogram. Patients with LCIS have a 25 percent chance of developing breast cancer in either breast during the next 25 years.

Microcalcifications are very small specks of calcium that can't be felt, but can be seen on a mammogram. They are formed by rapidly dividing cells. When they are clustered in one area of the breast, this could be an early sign of breast cancer in situ. About half of the breast cancers found by mammography appear as clusters of microcalcifications. The other half appear as lumps. Diagnosis

Our diagnostic methods may be used in conjunction with any known method of diagnosis of breast cancer, including detecting of mutations in either or both of the known breast cancer genes BRCAl and BRCA2. Specifically, our diagnostic methods may be used in conjunction with methods of diagnosis described in WO 2004/029295, hereby incorporated by reference, which involve detection of modulation of Sprouty2. Alternatively, or in addition, the diagnosis may be carried out by detection of Her2 expression, for example by use of anti-Her2 antibody.

Treatment

Known treatments for breast cancer may consist of any one or more of the following: Surgery, radiation therapy, chemotherapy, high-dose chemotherapy, hormonal therapy and immunotherapy. Accordingly, any of the treatment methods described here may be combined with any one or more of the preceding known therapies. In addition, any one or more of the following general therapies known to be effective for treatment or alleviation of cancer may be used.

Nonspecific Immunomodulating Agents

Nonspecific immunomodulating agents are substances that stimulate or indirectly augment the immune system. Often, these agents target key immune system cells and cause secondary responses such as increased production of cytokines and immunoglobulins. Two nonspecific immunomodulating agents used in cancer treatment are bacillus Calmette-Guerin (BCG) and levamisole.

Biological Response Modifiers

Some antibodies, cytokines, and other immune system substances can be produced in the laboratory for use in cancer treatment. These substances are often called biological response modifiers (BRMs). They alter the interaction between the body's immune defenses and cancer cells to boost, direct, or restore the body's ability to fight the disease. BRMs include interferons, interleukins, colony-stimulating factors, monoclonal antibodies, and vaccines. Interferons (IFN)

There are three major types of interferons — interferon alpha, interferon beta, and interferon gamma; interferon alpha is the type most widely used in cancer treatment.

Interferons can improve the way a cancer patient's immune system acts against cancer cells. In addition, interferons may act directly on cancer cells by slowing their growth or promoting their development into cells with more normal behavior. Some interferons may also stimulate NK cells, T cells, and macrophages, boosting the immune system's anticancer function.

Interleukins (IL)

Like interferons, interleukins are cytokines that occur naturally in the body. Many interleukins have been identified; interleukin-2 (IL-2 or aldesleukin) has been the most widely studied in cancer treatment. IL-2 stimulates the growth and activity of many immune cells, such as lymphocytes, that can destroy cancer cells.

Colony-Stimulating Factors (CSFs)

Colony-stimulating factors (CSFs) (sometimes called hematopoietic growth factors) usually do not directly affect tumor cells; rather, they encourage bone marrow stem cells to divide and develop into white blood cells, platelets, and red blood cells. Bone marrow is critical to the body's immune system because it is the source of all blood cells.

G-CSF (filgrastim) and GM-CSF (sargramostim) can increase the number of white blood cells, thereby reducing the risk of infection in patients receiving chemotherapy. G- CSF and GM-CSF can also stimulate the production of stem cells in preparation for stem cell or bone marrow transplants; Erythropoietin can increase the number of red blood cells and reduce the need for red blood cell transfusions in patients receiving chemotherapy; and Oprelvekin can reduce the need for platelet transfusions in patients receiving chemotherapy. Monoclonal Antibodies (MOABs)

Herceptin is used to treat metastatic breast cancer in patients with tumors that produce excess amounts of a protein called HER-2. (Approximately 25 percent of breast cancer tumors produce excess amounts of HER-2). In particular embodiments, the methods of treatment described here may be used in combination with administration of anti-Her2 antibody, for example, Herceptin, to the individual concerned.

Her2/Neu

The HER-2/neu (erbB-2) gene product is a 185-kDA transmembrane receptor tyrosine kinase that belongs to the family of receptors for epidermal growth factor. It is described in some detail in Reese, D. M., et al., Stem Cells, 15, 1-8 (1997) which is incorporated herein by reference.

Recently, enormous attention has been given to the importance of HER-2/neu in breast cancer. HER-2/neu is overexpressed in 20-30% of human breast cancers and the increased expression has been associated with poor prognosis. The discovery of this has led to the development of HERCEPTIN, an antibody to HER-2/neu, which in tests has been found to lengthen remission time in metastatic breast cancer. HER-2/neu is a cell- surface receptor that transmits growth signals to the cell nucleus. HERCEPTIN appears to block these signals thereby apparently inhibiting proliferation of cells mediated by HER- 2/neu in HER-2/neu positive breast cancer.

Overexpression of HER-2/neu has also been found in a portion of ovarian cancers, gastric cancers, endometrial cancers, salivary cancers, pancreatic cancers, prostate cancers, colorectal cancers, and non-small-cell lung cancers. The other cancers associated with overexpression of HER-2-neu are potentially treatable with HERCEPTIN.

Accordingly, our methods of diagnosis may be combined with detection of over- expression of Her2 in an individual. Likewise, the methods of treatment described here may include administration of Herceptin to an individual, in addition to increasing expression of hSproutyl. We therefore provide a combination of Sproutyl nucleic acid or Sproutyl polypeptide, together with an anti-Her2 antibody. We also provide a combination of an anti-Sproutyl antibody together with an anti-Her2 antibody. In preferred embodiments, the anti-Her2 antibody comprises Herceptin.

Sproutyl

WO 2004/029295, hereby incorporated by reference, describes in detail the use of

Sprouty2 nucleic acids and polypeptides for the treatment of breast cancer. Specifically, the methods and compositions described here may be used in combination with the methods described in that document for the treatment of breast cancer.

DIAGNOSTIC METHODS Detection of Expression of Sproutyl

We show in the Examples that the expression of Sproutyl in breast cancer tissue is down-regulated when compared to normal breast tissue. Accordingly, we provide for a method of diagnosis of breast cancer, comprising detecting modulation of expression of Sproutyl, preferably down-regulation of expression of Sproutyl in a cell or tissue of an individual.

Typically, physical examination fo the breast and X-ray mammography is used for the detection of breast cancer. A biopsy of the tumour is typically taken for histopathological examination for the diagnosis of breast cancer. Detection of Sprouty expression, either one or both of Sproutyl and Sprouty2, can be used to diagnose, or further confirm the diagnosis of, breast cancer, along with the standard histopathological procedures. This may be especially useful when the histopathological analysis does not yield a clear result.

The presence and quantity of Sproutyl polypeptides and nucleic acids may be detected in a sample. Thus, the Sproutyl associated diseases, including breast cancer, can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level, preferably a decreased level, of the Sproutyl polypeptide or Sproutyl mRNA. The sample may comprise a cell or tissue sample from an organism or individual suffering or suspected to be suffering from a disease associated with increased, reduced or otherwise abnormal Sproutyl expression, including spatial or temporal changes in level or pattern of expression. The level or pattern of expression of Sproutyl in an organism suffering from or suspected to be suffering from such a disease may be usefully compared with the level or pattern of expression in a normal organism as a means of diagnosis of disease.

The sample preferably comprises a cell or tissue sample from an individual suffering or suspected to be suffering from breast cancer, preferably a breast tissue or cell sample.

In highly preferred embodiments, a decreased level of expression of Sproutyl is detected in the sample. Preferably, the level of Sproutyl is decreased to a significant extent when compared to normal cells, or cells known not to be cancerous. Such cells may be obtained from the individual being tested, or another individual, preferably matched to the tested individual by age, weight, lifestyle, etc.

In preferred embodiments, the level of expression of Sproutyl is reduced by 10%,

20%, 30% or 40% or more. In highly preferred embodiments, the level of expression of Sproutyl is reduced by 45% or more, preferably 50% or more, as judged by cDNA hybridisation. As described in the Examples, when a Sproutyl probe is hybridized to the cDNA of 50 matched normal and tumour patients, there is a reduction of more than 50% of the radioactive signal from the breast tumour cDNA when compared to normal breast cDNA. This reduction in signal was observed in 78% of the patients (patient sample size is 50).

Expression of Sproutyl may be detected in a number of ways, as known in the art. Typically, the amount of Sproutyl in a sample of tissue from an individual is measured, and compared with a sample from an unaffected individual. Both Sproutyl nucleic acid, as well as Sproutyl polypeptide levels may be measured. In one embodiment therefore, we disclose a method of detecting the presence of a nucleic acid comprising a Sproutyl nucleic acid in a sample, by contacting the sample with at least one nucleic acid probe which is specific for the Sproutyl nucleic acid and monitoring said sample for the presence of the Sproutyl nucleic acid. For example, the nucleic acid probe may specifically bind to the Sproutyl nucleic acid, or a portion of it, and binding between the two detected; the presence of the complex itself may also be detected.

Thus, in one embodiment, the amount of Sproutyl nucleic acid in the form of Sproutyl mRNA may be measured in a sample. Sproutyl mRNA may be assayed by in situ hybridization, Northern blotting and reverse transcriptase— polymerase chain reaction. Nucleic acid sequences may be identified by in situ hybridization, Southern blotting, single strand conformational polymorphism, PCR amplification and DNA-chip analysis using specific primers. (Kawasaki, 1990; Sambrook, 1992; Lichter et al, 1990; Orita et al, 1989; Fodor et al., 1993; Pease et al., 1994)

Each of these methods allows quantitative determinations to be made, and are well known in the art. Decreased or increased Sproutyl expression can therefore be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides. Any suitable probe from a Sproutyl sequence, for example, any portion of a suitable human Sproutyl sequence may be used as a probe. Preferred sequences for designing Sproutyl probes include a sequence having accession number NP_005832 or a sequence having accession number or AAH63856.

Furthermore, the polymerase chain reaction may be employed to detect Sproutyl mRNA.

As used herein the term "polymerase chain reaction" or "PCR" refers to the PCR procedure described in the patents to Mullis, et al., U.S. Pat. Nos. 4,683,195 and

4,683,202. The procedure basically involves: (1) treating extracted DNA to form single- stranded complementary strands; (2) adding a pair of oligonucleotide primers, wherein one primer of the pair is substantially complementary to part of the sequence in the sense strand and the other primer of each pair is substantially complementary to a different part of the same sequence in the complementary antisense strand; (3) annealing the paired primers to the complementary sequence; (4) simultaneously extending the annealed primers from a 3' terminus of each primer to synthesize an extension product complementary to the strands annealed to each primer wherein said extension products after separation from the complement serve as templates for the synthesis of an extension product for the other primer of each pair; (5) separating said extension products from said templates to produce single-stranded molecules; and (6) amplifying said single-stranded molecules by repeating at least once said annealing, extending and separating steps.

Preferably, reverse transcription-polymerase chain reaction (RT-PCR) is employed. Quantitative RT-PCR is particularly preferred. Such PCR techniques are well known in the art, and may employ any suitable primer from a Sproutyl sequence.

In a further embodiment, Sproutyl expression may be detected by detecting the presence or amount of Sproutyl polypeptide in a sample. Thus, we disclose a method of detecting the presence of a Sproutyl polypeptide by contacting a cell sample with an antibody capable of binding the polypeptide and monitoring said sample for the presence of the polypeptide. This may conveniently be achieved by monitoring the presence of a complex formed between the antibody and the polypeptide, or monitoring the binding between the polypeptide and the antibody. Methods of detecting binding between two entities are known in the art, and include FRET (fluorescence resonance energy transfer), surface plasmon resonance, etc.

In a preferred embodiment, the Sproutyl polypeptide is detected using an anti- Sproutyl antibody. Such antibodies may be made by means known in the art (as described in further detail below). For example, an anti-Sproutyl antibody may comprise a rabbit polyclonal antibody to human Sproutyl peptide 58-75 (19 amino acids).

Assay techniques that can be used to determine levels of a protein, such as a Sproutyl, in a sample derived from a host are well-known to those of skill in the art. The specimen may be assayed for polypeptides/proteins by immunohistochemical and immunocytochemical staining (see generally Stites and Terr, Basic and Clinical Immunology, Appleton and Lange, 1994), ELISA, RIA, immunoblots, Western blotting, immunoprecipitation, functional assays and protein truncation test. Other assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.

ELISA assays are well known to those skilled in the art. Both polyclonal and monoclonal antibodies may be used in the assays. Where appropriate other immunoassays, such as radioimmunoassays (RIA) may be used as are known to those in the art. Available immunoassays are extensively described in the patent and scientific literature. See, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987;

3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521 as well as Sambrook et al, 1992.

The Examples demonstrate that expression of Sprouty2, in addition to that of Sproutyl, is down-regulated in breast cancer cells. In preferred embodiments, therefore, the expression of Sprouty2 is detected in conjunction with detection of expression of

Sproutyl described above. Detection of Sprouty2 expression may generally be conducted in an analogous manner to that of Sproutyl expression, with generally the same parameters. Furthermore, methods of detection of Sprouty2 are described in detail in WO 2004/029295, hereby incorporated by reference, and it is specifically declared that each of these methods may be used in conjunction with the methods described in this document for detection of Sproutyl.

We also provide diagnostic kits for detecting breast cancer in an individual, or susceptibility to breast cancer in an individual. The diagnostic kit comprises means for detecting expression of Sproutyl in the individual, by any means as described in this document. The diagnostic kit may therefore comprise any one or more of the following: a Sproutyl polynucleotide or a fragment thereof; a complementary nucleotide sequence to Sproutyl nucleic acid or a fragment thereof; a Sproutyl polypeptide or a fragment thereof, or an antibody to a Sproutyl, preferably comprising an anti-human Sproutyl antibody (rabbit polyclonal, made against human Sproutyl peptide 58-75 (19 amino acids)). The diagnostic kit may comprise instructions for use, or other indicia. The diagnostic kit may further comprise means for treatment or prophylaxis of breast cancer, such as any of the compositions described in this document, or any means known in the art for treating breast cancer. In particular, the diagnostic kit may comprise a therapeutic drug such as Tamoxifen (Nolvadex) or its variants such as tamoxifen tamoxifen citrate or any other antiestrogen or estrogen blocker. The therapeutic drug may also comprise an anti- Sproutyl antibody.

The diagnostic kit may also comprise means for detection of Sprouty2 expression in the individual or a sample taken from him or her. Such Sprouty2 detection means may comprise any of the following: a Sprouty2 polynucleotide or a fragment thereof; a complementary nucleotide sequence to Sprouty2 nucleic acid or a fragment thereof; a Sprouty2 polypeptide or a fragment thereof, or an antibody to a Sprouty2, and optionally instructions for use.

In highly preferred embodiments, the kit contains both an anti-Sproutyl antibody as well as an anti-Sprouty2 antibody.

PROPHYLACTICANDTHERAPEUTICMETHODS

We disclose methods of treating an abnormal conditions, such as breast cancer, related to insufficient amounts of Sproutyl or Sproutyl activity. Methods of preventing breast cancer (i.e., prophylaxis) also suitably employ the same or similar approaches.

Breast cancer may be treated or prevented by administration of Sproutyl in whole or in part, whether in the form of a Sproutyl polypeptide, or a nucleic acid encoding Sproutyl. Treatment may also be effected by administration of a molecule identified as being capable of up-regulating the activity or expression of Sproutyl to an individual. Such a compound may be administered along with a pharmaceutically acceptable carrier in an amount effective to activate expression or activity Sproutyl, or by activating a second signal, and thereby alleviating the abnormal condition. The treatment may in preferred embodiments further comprise the additional administration of Sprouty2 in whole or in part, whether in the form of a Sprouty2 polypeptide, or a nucleic acid encoding Sprouty2, or a molecule identified as being capable of up-regulating the activity or expression of Sprouty2. This is described in further detail in WO 2004/029295, hereby incorporated by reference.

Alternatively, gene therapy may be employed to effect the endogenous production of Sproutyl by the relevant cells such as breast cells in the subject. For example, a polynucleotide encoding Sproutyl or a portion of this may be engineered for expression in a replication defective retroviral vector, as discussed above. The retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a Sproutyl polypeptide such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be administered to a subject for engineering cells in vivo and expression of the Sproutyl polypeptide in vivo. For overview of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996). Analogous means may be used to further effect the endogenous production of Sprouty2 in the subject in preferred embodiments.

In particularly preferred embodiments, the level of Sproutyl is increased in a breast cell. Furthermore, in such preferred embodiments, treatment is targeted to, or specific to, breast cells. The expression of Sproutyl is preferably specifically increased only in diseased breast cells (i.e., those cells which are cancerous), and not substantially in other non-diseased breast cells. In the preferred methods, expression of Sproutyl is not substantially elevated in other cells, i.e., cells which are not breast cells. Thus, in such embodiments, the level of Sproutyl remains substantially the same or similar in non-breast cells in the course of or following treatment.

Breast cell specific elevation of Sproutyl levels may be achieved by targeted administration, i.e., applying Sproutyl polypeptide or nucleic acid only to the breast cells and not other cells. However, in preferred embodiments, up-regulation of Sproutyl expression in breast cells (and not substantially in other cell or tissue types) is employed. Such methods may advantageously make use of breast specific expression vectors, as described in further detail below.

Breast-specific Expression of a Transgene (Sprouty)

Cancer gene therapy has to selectively target tumour tissues so as to reduce undesired side effects in normal tissue. Targeting transgene expression to malignant tissues requires the use of specific regulatory elements including promoters based on tumour biology, tissue-specific promoters and inducible regulatory elements (Al).

Promoters based on Tumour Biology

Certain genes are upregulated in breast cancer. The promoters of these genes can be used to drive tumour-selective expression of a transgene using a recombinant replication-defective retroviral vectors. Examples of such genes include the vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor- 1 (VEGFR- 1) and VEGFR-2, which are known to be upregulated in breast cancer in a tumour-stage dependent manner (A2). c-erbB2 oncogene is selectively upregulated in breast carcinomas (A3, A6). L-plastin, a human actin-bindng protein is constitutively and abundantly expressed in malignant epithelial cells but not in normal tissue, except for low-level expression in mature hematopoietic cells (A4). Anti-apoptotic gene Bcl-2 has been found to be upregulated in breast cancer cells (A5). Human breast tumours express high levels of MUCl compared to normal breast tissues (A7).

Tissue Specific Promoters

Certain genes are expressed specifically in breast tissues. Examples of such genes are the human α-lactalbumin (ALA) and ovine β-lactoglobulin (BLG). The promoters of such genes can be used to drive the expression of transgenes in adenoviral vectors in a breast cancer cell-specific manner (A8). Gene therapy for breast carcinoma may be approached by tailoring a virus with affinity to this tissue, such as the mouse mammary tumour virus (MMTV). The glucorticoid-responsive long terminal repeats (LTR) of this retrovirus can be used as promoter for glucocorticoid-induced the expression of a transgene (A9).

Inducible Promoters

Inducible promoters are used as mediators of transient transgene expression. Various stress genes are upregulated in breast tumours upon irradiation or chemotherapeutic treatment. Examples of such stress genes are heat shock protein (HSP) (AlO) and multidrug resistance gene-1 (MDR-I) (Al l). The promoters of these genes can therefore be used to drive the tumour specific expression of a transgene in breast cancers that have been subjected to irradiation or chemotherapy.

Transcriptionally targeted gene therapy is usually achieved by direct intratumour injection of a replication-defective adenoviral expression vector containing the transgene of interest (A6, A12, A13). The transgene can also be delivered by intratumoural injection as a lipid complex with cationic liposomes (A14, A15).

In particularly preferred embodiments, in addition to increasing the level of Sproutyl in a breast cell, the level of Sprouty2 is also increased. Breast cell specific expression of Sprouty2 may employ the promoters described above, operationally linked to a Sprouty2 coding sequence in a suitable vector (described in further detail in described in detail in WO 2004/029595). Two separate vectors, one comprising a Sproutyl sequence, and another comprising a Sprouty2 sequence, may be employed. Alternatively, or in addition, a single vector comprising the relevant promoter, a Sproutyl coding sequence and a Sprouty2 coding sequence may be used for breast tissue specific expression of Sproutyl and Sprouty2.

ANTIBODIES

We further provide for antibodies which bind to a Sproutyl polypeptide, fragment, homologue, variant or derivative thereof. Such antibodies are useful in detecting Sproutyl expression, and in particular in diagnosing a Sproutyl associated disease such as breast cancer. Other preferred antibodies include those which have therapeutic activity, i.e., which are may be used in a therapeutic manner to treat, manage or prevent any Sproutyl associated disease, including breast cancer.

The anti-Sproutyl antibodies may be provided with, or used together with, an anti- Sprouty2 antibody, described in detail in WO 2004/029595.

Examples of antibodies capable of binding to Sproutyl include an anti-human

Sproutyl antibody, comprising a rabbit polyclonal antibody to human Sproutyl peptide 58-75 (19 amino acids).

Furthermore, antibodies which are specific for Sproutyl may be generated against any suitable epitope, for example, an epitope derived from the N terminus of the protein. The sequence of a suitable N terminal fragment of Sproutyl is depicted as SEQ ID NO: 2, and any epitope from this sequence may be used for the generation of specific Sproutyl antibodies.

For the purposes of this document, the term "antibody", unless specified to the contrary, includes but is not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library. Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab')₂ fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody. The antibodies and fragments thereof may be humanised antibodies, for example as described in EP-A-239400. Furthermore, antibodies with fully human variable regions (or their fragments), for example, as described in US Patent Nos. 5,545,807 and 6,075,181 may also be used. Neutralizing antibodies, i.e., those which inhibit any biological activity of Sproutyl, are especially preferred for diagnostics and therapeutics.

Antibodies may be produced by standard techniques, such as by immunisation or by using a phage display library. Such an antibody may be capable of binding specifically to the Sproutyl protein or homologue, fragment, etc. If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) may be immunised with an immunogenic composition comprising a Sproutyl polypeptide or peptide. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminium hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (Bacilli Calmette-Gueriri) and Coiynebacterium parviim are potentially useful human adjuvants which may be employed if purified the substance amino acid sequence is administered to immunologically compromised individuals for the purpose of stimulating systemic defence.

Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to an epitope obtainable from a Sproutyl polypeptide contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, we also provide Sproutyl amino acid sequences or fragments thereof haptenised to another amino acid sequence for use as immunogens in animals or humans.

Monoclonal antibodies directed against epitopes obtainable from a Sproutyl polypeptide or peptide can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced against orbit epitopes can be screened for various properties; i.e., for isotype and epitope affinity.

Monoclonal antibodies may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030) and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss, Inc., 1985).

In addition, techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison et al (1984) Proc Natl Acad Sci 81:6851-6855; Neuberger et al (1984) Nature 312:604-608; Takeda et al (1985) Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies (US Patent No. 4,946,779) can be adapted to produce the substance specific single chain antibodies.

Antibodies, both monoclonal and polyclonal, which are directed against epitopes obtainable from a Sproutyl polypeptide or peptide are particularly useful in diagnosis. Monoclonal antibodies, in particular, may be used to raise anti-idiotype antibodies. Anti¬ idiotype antibodies are immunoglobulins which carry an "internal image" of the substance and/or agent against which protection is desired. Techniques for raising anti-idiotype antibodies are known in the art. These anti-idiotype antibodies may also be useful in therapy.

Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al (1989, Proc Natl Acad Sci 86: 3833- 3837), and Winter G and Milstein C (1991; Nature 349:293-299).

Antibody fragments which contain specific binding sites for the polypeptide or peptide may also be generated. For example, such fragments include, but are not limited to, the F(ab')₂ fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')₂ fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD et al (1989) Science 256:1275-128 1). Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can also be adapted to produce single chain antibodies to Sproutyl polypeptides. Also, transgenic mice, or other organisms including other mammals, may be used to express humanized antibodies.

The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.

Anti-Sproutyl antibodies may be used in method of detecting a Sproutyl polypeptide present in biological samples by a method which comprises: (a) providing an anti-Sproutyl antibody; (b) incubating a biological sample with said antibody under conditions which allow for the formation of an antibody-antigen complex; and (c) determining whether antibody-antigen complex comprising said antibody is formed.

Suitable samples include extracts tissues such as brain, breast, ovary, lung, colon, pancreas, testes, liver, muscle and bone tissues or from neoplastic growths derived from such tissues. In particular, a preferred sample comprises a breast tissue, preferably a breast tissue from an individual suspected to be suffering from breast cancer.

Antibodies may be bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like.

SCREENING ASSAYS

The Sproutyl proteins and nucleic acids may be employed in a screening process for compounds which bind the Sproutyl polypeptides and which activate (agonists) its activity or inhibit activation of (antagonists) of Sproutyl. Such molecules are useful in the treatment methods described here. Screening assays may also be conducted on animals transgenic for Sproutyl, as described in further detail in the section below.

The molecules identified by such screens, in particular, antagonists of Sproutyl, are useful in the treatment methods described here. Sproutyl may therefore be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See Coligan et al., Current Protocols in Immunology l(2):Chapter 5 (1991). Furthermore, screens may be conducted to identify factors which influence the expression of Sproutyl, in particular in breast cells.

In general, the assays for agonists and antagonists rely on determining the effect of candidate molecules on one or more activities of Sproutyl. An assay may involve assaying Sproutyl activity in the presence of a candidate molecule, and optionally in the absence of the candidate molecule, or in the presence of a molecule known to inhibit or activate a Sproutyl activity.

Sproutyl is responsible for many biological functions, as described in the references. We have demonstrated that expression of Sproutyl is decreased in breast cancer cells; accordingly, control of Sproutyl expression may be employed to treat breast cancer and other cancers. Therefore, it is desirous to find compounds and drugs which stimulate the expression and/or activity of Sproutyl, or which can inhibit the function of this protein. In general, agonists and antagonists are employed for therapeutic and prophylactic purposes for any known cancer, in particular, breast cancer.

Furthermore, any of the activities of Sproutyl as described above may be assayed in the presence of candidate molecules to determine their effects (if any) on these activities. Thus, an assay for agonists and antagonists of Sproutyl may detect the effect of a candidate molecule on any one or more of the following activities: inhibition of one or more events downstream of receptor tyrosine kinases; inhibition of activation of the Ras/Raf/MAPK pathway; inhibition of the Ras/Raf/MAPK pathway by preventing the activation of Ras/Raf; down-regulation of ERK phosphorylation when the Ras/MAPK pathway is activated.

Rational design of candidate compounds likely to be able to interact with Sproutyl may be based upon structural studies of the molecular shapes of a Sproutyl polypeptide. One means for determining which sites interact with specific other proteins is a physical structure determination, e.g., X-ray crystallography or two-dimensional NMR techniques. These will provide guidance as to which amino acid residues form molecular contact regions. For a detailed description of protein structural determination, see, e.g., Blundell and Johnson (1976) Protein Crystallography, Academic Press, New York.

An alternative to rational design uses a screening procedure which involves in general producing appropriate cells which express the Sproutyl proteins on the surface thereof. Such cells include cells from animals, yeast, Drosophila or E. coli. Cells expressing the Sproutyl polypeptide (or cell membrane containing the expressed Sproutyl polypeptide) are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response. For example, Xenopus oocytes may be injected with mRNA encoding any one or more of the Sproutyl polypeptides.

Where the candidate compounds are proteins, in particular antibodies or peptides, libraries of candidate compounds may be screened using phage display techniques. Phage display is a protocol of molecular screening which utilises recombinant bacteriophage. The technology involves transforming bacteriophage with a gene that encodes one compound from the library of candidate compounds, such that each phage or phagemid expresses a particular candidate compound. The transformed bacteriophage (which preferably is tethered to a solid support) expresses the appropriate candidate compound and displays it on their phage coat. Specific candidate compounds which are capable of binding to a Sproutyl polypeptide or peptide are enriched by selection strategies based on affinity interaction. The successful candidate agents are then characterised. Phage display has advantages over standard affinity ligand screening technologies. The phage surface displays the candidate agent in a three dimensional configuration, more closely resembling its naturally occurring conformation. This allows for more specific and higher affinity binding for screening purposes.

Another method of screening a library of compounds utilises eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing a library of compounds. Such cells, either in viable or fixed form, can be used for standard binding-partner assays. See also Parce et al (1989) Science 246:243-247; and Owicki et al (1990) Proc. Nat'l Acad. Sci. USA 87;4007-4011, which describe sensitive methods to detect cellular responses. Competitive assays are particularly useful, where the cells expressing the library of compounds are contacted or incubated with a labelled antibody known to bind to a Sproutyl polypeptide, such as ¹²⁵I-antibody, and a test sample such as a candidate compound whose binding affinity to the binding composition is being measured. The bound and free labelled binding partners for the Sproutyl polypeptide are then separated to assess the degree of binding. The amount of test sample bound is inversely proportional to the amount of labelled antibody binding to the Sproutyl polypeptide.

Any one of numerous techniques can be used to separate bound from free binding partners to assess the degree of binding. This separation step could typically involve a procedure such as adhesion to filters followed by washing, adhesion to plastic following by washing, or centrifugation of the cell membranes.

Still another approach is to use solubilized, unpurified or solubilized purified polypeptide or peptides, for example extracted from transformed eukaryotic or prokaryotic host cells. This allows for a "molecular" binding assay with the advantages of increased specificity, the ability to automate, and high drug test throughput.

Another technique for candidate compound screening involves an approach which provides high throughput screening for new compounds having suitable binding affinity, e.g., to a Sproutyl polypeptide, and is described in detail in International Patent application no. WO 84/03564 (Commonwealth Serum Labs.), published on September 13 1984. First, large numbers of different small peptide test compounds are synthesized on a solid substrate, e.g., plastic pins or some other appropriate surface; see Fodor et al. (1991). Then all the pins are reacted with solubilized Sproutyl polypeptide and washed. The next step involves detecting bound polypeptide. Compounds which interact specifically with the Sproutyl polypeptide will thus be identified. Ligand binding assays provide a direct method for ascertaining pharmacology and are adaptable to a high throughput format. The purified ligand for a Sproutyl polypeptide may be radiolabeled to high specific activity (50-2000 Ci/mmol) for binding studies. A determination is then made that the process of radiolabeling does not diminish the activity of the ligand towards its binding partner. Assay conditions for buffers, ions, pH and other modulators such as nucleotides are optimized to establish a workable signal to noise ratio for both membrane and whole cell sources. For these assays, specific binding is defined as total associated radioactivity minus the radioactivity measured in the presence of an excess of unlabeled competing ligand. Where possible, more than one competing ligand is used to define residual nonspecific binding.

The assays may simply test binding of a candidate compound wherein adherence to the cells bearing the Sproutyl polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor. Further, these assays may test whether the candidate compound results in a signal generated by binding to the Sproutyl polypeptide, using detection systems appropriate to the cells bearing the polypeptides at their surfaces. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.

Further, the assays may simply comprise the steps of mixing a candidate compound with a solution containing a Sproutyl polypeptide to form a mixture, measuring activity of the relevant protein in the mixture, and comparing the activity of the mixture to a standard.

The assays may involve exposing a candidate molecule to a cell, preferably a breast cell, and assaying expression of Sproutyl by any suitable means. Molecules which up-regulate the expression of Sproutyl in such assays may be optionally chosen for further study, and used as drugs to elevate Sproutyl expression. Such drugs may be usefully employed to treat or prevent breast cancer. cDNA encoding Sproutyl protein and antibodies to the proteins may also be used to configure assays for detecting the effect of added compounds on the production of mRNA and protein in cells. For example, an ELISA may be constructed for measuring secreted or cell associated levels of Sproutyl polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of Sproutyl protein (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues. Standard methods for conducting screening assays are well understood in the art.

Examples of potential antagonists of Sproutyl include antibodies or, in some cases, nucleotides and their analogues, including purines and purine analogues, oligonucleotides or proteins which are closely related to a binding partner of Sproutyl, e.g., a fragment of the binding partner, or small molecules which bind to the Sproutyl polypeptide but do not elicit a response, so that the activity of the polypeptide is prevented.

We therefore also provide a compound capable of binding specifically to a Sproutyl polypeptide and/or peptide. Such a compound may be provided together with a compound capable of binding specifically to a Sprouty2 polypeptide, as described in WO 2004/029595. The combination may be used in the methods and compositions described here for treating or preventing cancer, particularly breast cancer.

The term "compound" refers to a chemical compound (naturally occurring or synthesised), such as a biological macromolecule (e.g., nucleic acid, protein, non-peptide, or organic molecule), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, or even an inorganic element or molecule. Preferably the compound is an antibody.

The materials necessary for such screening to be conducted may be packaged into a screening kit. Such a screening kit is useful for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for Sproutyl polypeptides or compounds which decrease or enhance the production of Sproutyl. The screening kit may comprise: (a) a Sproutyl polypeptide; (b) a recombinant cell expressing a Sproutyl polypeptide; (c) a cell membrane expressing a Sproutyl polypeptide; or (d) an antibody to Sproutyl polypeptide. The screening kit may optionally comprise instructions for use.

Screening kits may also be provided which are capable of detecting Sproutyl expression at the nucleic acid level. Such kits may comprise a primer for amplification of Sproutyl, for example, having or comprising the sequence 5'

AGGGCTATCTTCCTAGCA 3' or 5' GTGAGAAGCATGGGGT 3', or a pair of primers for amplification, for example, comprising the two preceding sequences.

The kits may comprise a nucleic acid probe for Sproutyl expression, as described in this document. The kits may also optionally comprise instructions for use.

TRANSGENIC ANIMALS

We further describe transgenic animals capable of expressing natural or recombinant Sproutyl, or a homologue, variant or derivative, at elevated or reduced levels compared to the normal expression level. The transgenic animals are useful as models of cancer, particularly breast cancer. Furthermore, they may be used for screening compounds and molecules capable of modulating Sproutyl expression or function. Such molecules are useful for the methods of treatment described here.

Included are transgenic animals ("Sproutyl knockouf's) which do not express functional Sproutyl. The Sproutyl knockouts may arise as a result of functional disruption of the Sproutyl gene or any portion of that gene, including one or more loss of function mutations, including a deletion or replacement, of the Sproutyl gene. The mutations include single point mutations, and may target coding or non-coding regions of Sproutyl.

Preferably, such a transgenic animal is a non-human mammal, such as a pig, a sheep or a rodent. Most preferably the transgenic animal is a mouse or a rat. Such transgenic animals may be used in screening procedures to identify agonists and/or antagonists of Sproutyl, as well as to test for their efficacy as treatments for diseases in vivo. Mice which are null for Sproutyl may be used for various purposes. For example, transgenic animals that have been engineered to be deficient in the production of Sproutyl may be used in assays to identify agonists and/or antagonists of Sproutyl. One assay is designed to evaluate a potential drug (a candidate ligand or compound) to determine if it produces a physiological response in the absence of Sproutyl . This may be accomplished by administering the drug to a transgenic animal as discussed above, and then assaying the animal for a particular response. Although any physiological parameter could be measured in this assay, preferred responses include altered tumour susceptability, particularly, altered susceptability to developing breast cancer, altered neovascularization.

Tissues derived from the Sproutyl knockout animals may be used in binding assays to determine whether the potential drug (a candidate ligand or compound) binds to Sproutyl. Such assays can be conducted by obtaining a first preparation from the transgenic animal engineered to be deficient in Sproutyl production and a second preparation from a source known to bind any identified Sproutyl ligands or compounds. In general, the first and second preparations will be similar in all respects except for the source from which they are obtained. For example, if breast tissue from a transgenic animal (such as described above and below) is used in an assay, comparable breast tissue from a normal (wild type) animal is used as the source of the second preparation. Each of the preparations is incubated with a ligand known to bind to Sproutyl, both alone and in the presence of the candidate ligand or compound. Preferably, the candidate ligand or compound will be examined at several different concentrations.

The extent to which binding by the known ligand is displaced by the test compound is determined for both the first and second preparations. Tissues derived from transgenic animals may be used in assays directly or the tissues may be processed to isolate extracts or proteins, which are themselves used in the assays. A preferred transgenic animal is the mouse. The ligand may be labeled using any means compatible with binding assays. This would include, without limitation, radioactive, enzymatic, fluorescent or chemiluminescent labeling (as well as other labelling techniques as described elsewhere). Furthermore, antagonists of Sproutyl may be identified by administering candidate compounds, etc, to wild type animals expressing functional Sproutyl, and animals identified which exhibit any of the phenotypic characteristics associated with reduced or abolished expression of Sproutyl function, such as breast cancer or susceptibility to breast cancer.

In a preferred embodiment, the transgenic animal is one which is more susceptible to developing cancer, preferably breast cancer, as compared to a wild type animal or an animal which expresses Sproutyl. Such an animal may be used as a model for breast cancer, to study the mechanisms which cause or are correlated to development of breast cancer. Furthermore, transgenic Sproutyl animals may be used to screen for molecules which affect the development of cancer such as breast cancer in the transgenic animal.

For example, an assay may comprise exposing the transgenic animal to a candidate molecule, and monitoring the development of breast cancer in the animal. Candidate molecules which reduce or abolish development of breast cancer, or which slow down the progress of the cancer once developed, in the transgenic animal (as compared to a transgenic Sproutyl animal which has not been exposed to the candidate molecule) may be selected as potential drugs for treatment of cancer in mammals, for example humans. It will be appreciated that the assay may be modified by allowing the transgenic animal to develop breast cancer, and then exposing the animal to a candidate molecule. As before, suitable candidate breast cancer therapies may be selected from those molecules which reduce the already developed breast cancer, for example, enable the animal to go into remission.

Detailed methods for generating non-human transgenic animal are described in further detail below. Transgenic gene constructs can be introduced into the germ line of an animal to make a transgenic mammal. For example, one or several copies of the construct may be incorporated into the genome of a mammalian embryo by standard transgenic techniques. In an exemplary embodiment, the transgenic non-human animals are produced by introducing transgenes into the germline of the non-human animal. Embryonal target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell. The specific line(s) of any animal are selected for general good health, good embryo yields, good pronuclear visibility in the embryo, and good reproductive fitness. In addition, the haplotype is a significant factor.

Introduction of the transgene into the embryo can be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection. For example, the Sproutyl transgene can be introduced into a mammal by microinjection of the construct into the pronuclei of the fertilized mammalian egg(s) to cause one or more copies of the construct to be retained in the cells of the developing mammal(s). Following introduction of the transgene construct into the fertilized egg, the egg may be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both. In vitro incubation to maturity is within the scope of the methods and compositions described here. One common method in to incubate the embryos in vitro for about 1-7 days, depending on the species, and then reimplant them into the surrogate host.

The progeny of the transgenically manipulated embryos can be tested for the presence of the construct by Southern blot analysis of the segment of tissue. If one or more copies of the exogenous cloned construct remains stably integrated into the genome of such transgenic embryos, it is possible to establish permanent transgenic mammal lines carrying the transgenically added construct.

The litters of transgenically altered mammals can be assayed after birth for the incorporation of the construct into the genome of the offspring. Preferably, this assay is accomplished by hybridizing a probe corresponding to the DNA sequence coding for the desired recombinant protein product or a segment thereof onto chromosomal material from the progeny. Those mammalian progeny found to contain at least one copy of the construct in their genome are grown to maturity. A zygote is essentially the formation of a diploid cell which is capable of developing into a complete organism. Generally, the zygote will be comprised of an egg containing a nucleus formed, either naturally or artificially, by the fusion of two haploid nuclei from a gamete or gametes. Thus, the gamete nuclei must be ones which are naturally compatible, i.e., ones which result in a viable zygote capable of undergoing differentiation and developing into a functioning organism. Generally, a euploid zygote is preferred. If an aneuploid zygote is obtained, then the number of chromosomes should not vary by more than one with respect to the euploid number of the organism from which either gamete originated.

In addition to similar biological considerations, physical ones also govern the amount (e.g., volume) of exogenous genetic material which can be added to the nucleus of the zygote or to the genetic material which forms a part of the zygote nucleus. If no genetic material is removed, then the amount of exogenous genetic material which can be added is limited by the amount which will be absorbed without being physically disruptive. Generally, the volume of exogenous genetic material inserted will not exceed about 10 picoliters. The physical effects of addition must not be so great as to physically destroy the viability of the zygote. The biological limit of the number and variety of DNA sequences will vary depending upon the particular zygote and functions of the exogenous genetic material and will be readily apparent to one skilled in the art, because the genetic material, including the exogenous genetic material, of the resulting zygote must be biologically capable of initiating and maintaining the differentiation and development of the zygote into a functional organism.

The number of copies of the transgene constructs which are added to the zygote is dependent upon the total amount of exogenous genetic material added and will be the amount which enables the genetic transformation to occur. Theoretically only one copy is required; however, generally, numerous copies are utilized, for example, 1,000-20,000 copies of the transgene construct, in order to insure that one copy is functional. There will often be an advantage to having more than one functioning copy of each of the inserted exogenous DNA sequences to enhance the phenotypic expression of the exogenous DNA sequences. Any technique which allows for the addition of the exogenous genetic material into nucleic genetic material can be utilized so long as it is not destructive to the cell, nuclear membrane or other existing cellular or genetic structures. The exogenous genetic material is preferentially inserted into the nucleic genetic material by microinjection. Microinjection of cells and cellular structures is known and is used in the art.

Reimplantation is accomplished using standard methods. Usually, the surrogate host is anesthetized, and the embryos are inserted into the oviduct. The number of embryos implanted into a particular host will vary by species, but will usually be comparable to the number of off spring the species naturally produces.

Transgenic offspring of the surrogate host may be screened for the presence and/or expression of the transgene by any suitable method. Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for the presence of the transgene product. Typically, DNA is prepared from tail tissue and analyzed by Southern analysis or PCR for the transgene. Alternatively, the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis.

Alternative or additional methods for evaluating the presence of the transgene include, without limitation, suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like. Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.

Progeny of the transgenic animals may be obtained by mating the transgenic animal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic animal. Where mating with a partner is to be performed, the partner may or may not be transgenic and/or a knockout; where it is transgenic, it may contain the same or a different transgene, or both. Alternatively, the partner may be a parental line. Where in vitro fertilization is used, the fertilized embryo may be implanted into a surrogate host or incubated in vitro, or both. Using either method, the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.

The transgenic animals will include exogenous genetic material. As set out above, the exogenous genetic material will, in certain embodiments, be a DNA sequence which results in the production of a Sproutyl polypeptide. Further, in such embodiments the sequence will be attached to a transcriptional control element, e.g., a promoter, which preferably allows the expression of the transgene product in a specific type of cell.

Retroviral infection can also be used to introduce transgene into a non-human animal. The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986). The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985) PNAS 82:6148-6152). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388). Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner et al. (1982) Nature 298:623-628). Most of the founders will be mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic non-human animal. Further, the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al. (1982) supra). A third type of target cell for transgene introduction is the embryonal stem cell (ES). ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. (1981) Nature 292:154-156; Bradley et al. (1984) Nature 309:255- 258; Gossler et al. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature 322:445-448). Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal. For review see Jaenisch, R. (1988) Science 240:1468-1474.

We also provide non-human transgenic animals, where the transgenic animal is characterized by having an altered Sproutyl gene, preferably as described above, as models for Sproutyl function, in particular, models for breast cancer.

Alterations to the gene include deletions or other loss of function mutations, introduction of an exogenous gene having a nucleotide sequence with targeted or random mutations, introduction of an exogenous gene from another species, or a combination thereof. The transgenic animals may be either homozygous or heterozygous for the alteration. The animals and cells derived therefrom are useful for screening biologically active agents that may modulate Sproutyl function. The screening methods are of particular use for determining the specificity and action of potential therapies for Sproutyl associated diseases including cancers such as breast cancer. The animals are useful as a model to investigate the role of Sproutyl in normal breast and other tissues.

Another aspect pertains to a transgenic nonhuman animal having a functionally disrupted endogenous Sproutyl gene but which also carries in its genome, and expresses, a transgene encoding a heterologous Sproutyl protein (i.e., a Sproutyl from another species). Preferably, the animal is a mouse and the heterologous Sproutyl is a human Sproutyl . An animal, or cell lines derived from such an animal, which has been reconstituted with human Sproutyl, can be used to identify agents that inhibit human Sproutyl in vivo and in vitro. For example, a stimulus that induces signalling through human Sproutyl can be administered to the animal, or cell line, in the presence and absence of an agent to be tested and the response in the animal, or cell line, can be measured. An agent that inhibits human Sproutyl in vivo or in vitro can be identified based upon a decreased response in the presence of the agent compared to the response in the absence of the agent.

We also provide for a Sproutyl deficient transgenic non-human animal (a

"Sproutyl knock-out" or a "Sproutyl null"). Such an animal is one which expresses lowered or no Sproutyl activity, preferably as a result of an endogenous Sproutyl genomic sequence being disrupted or deleted. The endogenous Sproutyl genomic sequence may be replaced by a null allele, which may comprise non-functional portions of the wild-type Sproutyl sequence. For example, the endogenous Sproutyl genomic sequence may be replaced by an allele of Sproutyl comprising a disrupting sequence which may comprise heterologous sequences, for example, reporter sequences and/or selectable markers. Preferably, the endogenous Sproutyl genomic sequence in a Sproutyl knock-out mouse is replaced by an allele of Sproutyl in which one or more, preferably all, of the transmembrane sequences is replaced by such a disrupting sequence, preferably a lacZ sequence and a neomycin resistance sequence. Preferably, the genomic Sproutyl sequence which is functionally disrupted comprises a mouse Sproutyl genomic sequence.

Preferably, such an animal expresses no Sproutyl activity. Sproutyl knock-outs may be generated by various means known in the art, as described in further detail below.

We further describe a nucleic acid construct for functionally disrupting a Sproutyl gene in a host cell. The nucleic acid construct comprises: a) a non-homologous replacement portion; b) a first homology region located upstream of the non-homologous replacement portion, the first homology region having a nucleotide sequence with substantial identity to a first Sproutyl gene sequence; and c) a second homology region located downstream of the non-homologous replacement portion, the second homology region having a nucleotide sequence with substantial identity to a second Sproutyl gene sequence, the second Sproutyl gene sequence having a location downstream of the first Sproutyl gene sequence in a naturally occurring endogenous Sproutyl gene. Additionally, the first and second homology regions are of sufficient length for homologous recombination between the nucleic acid construct and an endogenous Sproutyl gene in a host cell when the nucleic acid molecule is introduced into the host cell. In a preferred embodiment, the non-homologous replacement portion comprises an expression reporter, preferably including lacZ and a positive selection expression cassette, preferably including a neomycin phosphotransferase gene operatively linked to a regulatory element(s).

Another aspect pertains to recombinant vectors into which the nucleic acid construct has been incorporated. Yet another aspect pertains to host cells into which the nucleic acid construct has been introduced to thereby allow homologous recombination between the nucleic acid construct and an endogenous Sproutyl gene of the host cell, resulting in functional disruption of the endogenous Sproutyl gene. The host cell can be a mammalian cell that normally expresses Sproutyl from the liver, brain, spleen or heart, or a pluripotent cell, such as a mouse embryonic stem cell. Further development of an embryonic stem cell into which the nucleic acid construct has been introduced and homologously recombined with the endogenous Sproutyl gene produces a transgenic nonhuman animal having cells that are descendant from the embryonic stem cell and thus carry the Sproutyl gene disruption in their genome. Animals that carry the Sproutyl gene disruption in their germline can then be selected and bred to produce animals having the Sproutyl gene disruption in all somatic and germ cells. Such mice can then be bred to homozygosity for the Sproutyl gene disruption.

A Sproutyl deficient transgenic animal may be generated as follows:

Construction of Sproutyl Gene Targeting Vector

Murine Sproutyl genomic clones are isolated from a mouse large insert PAC library obtained from HGMP (Hinxton, UK) using the human open reading frame cDNA sequence as a probe using standard techniques. The isolated murine Sproutyl genomic clones are then restriction mapped in the region of the Sproutyl gene using small oligonucleotide probes and standard techniques.

The murine genomic locus is partially sequenced to enable the design of homologous arms to clone into the targeting vector. A 5' homologous arm and a 3' homologous arm are amplified by PCR and the fragment cloned into the targeting vector. Any suitable size may be chosen for the length of these arms to enable homologous recombination; for example, the 5' arm may be between 1 kb and to 2 kb, for example 1.15 kb, while the 3' arm may be about 4 kb in size.

The position of these arms is chosen to functionally disrupt the Sproutyl gene by deleting a coding portion of the gene. A targeting vector is prepared where the deleted Sproutyl sequence is replaced with non-homologous sequences composed of an endogenous gene expression reporter (an in frame fusion with lacZ) upstream of a selection cassette composed of a self promoted neomycin phosphotransferase (neo) gene in the same orientation as the Sproutyl gene.

Transfection and Analysis of Embryonal Stem Cells

Embryonal stem cells (Evans and Kaufman, 1981) are cultured on a neomycin resistant embryonal fibroblast feeder layer grown in Dulbecco's Modified Eagles medium supplemented with 20% Fetal Calf Serum, 10% new-born calf serum, 2 mM glutamine, non-essential amino acids, 100 μM 2-mercaptoethanol and 500 u/ml leukemia inhibitory factor. Medium is changed daily and ES cells are subcultured every three days. 5xlO⁶ ES cells are transfected with 5 μg of linearized plasmid by electroporation (25 μF capacitance and 400 Volts). 24 hours following electroporation the transfected cells are cultured for 9 days in medium containing 200 μg/ml neomycin. Clones are picked into 96 well plates, replicated and expanded before being screened by PCR to identify clones in which homologous recombination had occurred between the endogenous Sproutyl gene and the targeting construct. From 200 picked clones 7 targets are identified. These clones where expanded to allow replicas to be frozen and sufficient high quality DNA to be prepared for Southern blot confirmation of the targeting event using external 5' and 3' probes, all using standard procedures (Russ et al, 2000)

Generation of Sproutyl Deficient Mice

C57BL/6 female and male mice are mated and blastocysts are isolated at 3.5 days of gestation. 10-12 cells from a chosen clone are injected per blastocyst and 7-8 blastocysts are implanted in the uterus of a pseudopregnant Fl female. Five chimeric pups are born of which one male is 100% agouti (indicating cells descendent from the targeted clone). This male chimera is mated with female and MFl and 129 mice, and germline transmission is determined by the agouti coat color and by PCR genotyping respectively.

Each of the transgenic animals described above may further be transgenic for

Sprouty2, e.g., may comprise a Sprouty2 knockout. Sprouty2 knockout and transgenic animals are described in detail in WO 2004/029595.

PHARMACEUTICAL COMPOSITIONS

While it is possible for the composition comprising the Sproutyl nucleic acid, polypeptide, fragment, homologue, variant or derivative thereof, or specific binding agent to be administered alone, it is preferable to formulate the active ingredient as a pharmaceutical formulation.

We therefore also disclose pharmaceutical compositions comprising Sproutyl agent. Such pharmaceutical compositions are useful for delivery of Sproutyl agent to an individual for the treatment or alleviation of symptoms as described. Where the term "Sproutyl agent" is used, it should be taken to refer to any one or more of a Sproutyl nucleic acid, a Sproutyl polypeptide, a Sproutyl fragment, a Sproutyl homologue, a Sproutyl variant, a Sproutyl derivative, a Sproutyl specific binding agent, an anti- Sproutyl antibody, a vector comprising Sproutyl as described, etc. In preferred embodiments, any of the cognate Sprouty2 agents described in WO 2004/029595 is also included.

The composition may include the Sproutyl agent, a structurally related compound, or an acidic salt thereof. The pharmaceutical formulations comprise an effective amount of Sproutyl agent, together with one or more pharmaceutically-acceptable carriers. An "effective amount" of an Sproutyl agent is the amount sufficient to alleviate at least one symptom of a disease as described. The effective amount will vary depending upon the particular disease or syndrome to be treated or alleviated, as well as other factors including the age and weight of the patient, how advanced the disease etc state is, the general health of the patient, the severity of the symptoms, and whether the Sproutyl agent is being administered alone or in combination with other therapies.

Suitable pharmaceutically acceptable carriers are well known in the art and vary with the desired form and mode of administration of the pharmaceutical formulation. For example, they can include diluents or excipients such as fillers, binders, wetting agents, disintegrators, surface-active agents, lubricants and the like. Typically, the carrier is a solid, a liquid or a vaporizable carrier, or a combination thereof. Each carrier should be "acceptable" in the sense of being compatible with the other ingredients in the formulation and not injurious to the patient. The carrier should be biologically acceptable without eliciting an adverse reaction (e.g. immune response) when administered to the host.

The pharmaceutical compositions disclosed here include those suitable for topical and oral administration, with topical formulations being preferred where the tissue affected is primarily the skin or epidermis (for example, psoriasis, eczema and other epidermal diseases). The topical formulations include those pharmaceutical forms in which the composition is applied externally by direct contact with the skin surface to be treated. A conventional pharmaceutical form for topical application includes a soak, an ointment, a cream, a lotion, a paste, a gel, a stick, a spray, an aerosol, a bath oil, a solution and the like. Topical therapy is delivered by various vehicles, the choice of vehicle can be important and generally is related to whether an acute or chronic disease is to be treated. Other formulations for topical application include shampoos, soaps, shake lotions, and the like, particularly those formulated to leave a residue on the underlying skin, such as the scalp (Arndt et al, in Dermatology In General Medicine 2:2838 (1993)).

In general, the concentration of the Sproutyl agent composition in the topical formulation is in an amount of about 0.5 to 50% by weight of the composition, preferably about 1 to 30%, more preferably about 2-20%, and most preferably about 5-10%. The concentration used can be in the upper portion of the range initially, as treatment continues, the concentration can be lowered or the application of the formulation may be less frequent. Topical applications are often applied twice daily. However, once-daily application of a larger dose or more frequent applications of a smaller dose may be effective. The stratum corneum may act as a reservoir and allow gradual penetration of a drug into the viable skin layers over a prolonged period of time.

In a topical application, a sufficient amount of active ingredient must penetrate a patient's skin in order to obtain a desired pharmacological effect. It is generally understood that the absorption of drug into the skin is a function of the nature of the drug, the behaviour of the vehicle, and the skin. Three major variables account for differences in the rate of absorption or flux of different topical drugs or the same drug in different vehicles; the concentration of drug in the vehicle, the partition coefficient of drug between the stratum corneum and the vehicle and the diffusion coefficient of drug in the stratum corneum. To be effective for treatment, a drug must cross the stratum corneum which is responsible for the barrier function of the skin. In general, a topical formulation which exerts a high in vitro skin penetration is effective in vivo. Ostrenga et al (J. Pharm. Sci., 60:1175-1179 (1971) demonstrated that in vivo efficacy of topically applied steroids was proportional to the steroid penetration rate into dermatomed human skin in vitro.

A skin penetration enhancer which is dermatologically acceptable and compatible with the agent can be incorporated into the formulation to increase the penetration of the active compound(s) from the skin surface into epidermal keratinocytes. A skin enhancer which increases the absorption of the active compound(s) into the skin reduces the amount of agent needed for an effective treatment and provides for a longer lasting effect of the formulation. Skin penetration enhancers are well known in the art. For example, dimethyl sulfoxide (U.S. Pat. No. 3,711,602); oleic acid, 1,2-butanediol surfactant (Cooper, J. Pharm. Sci., 73:1153-1156 (1984)); a combination of ethanol and oleic acid or oleyl alcohol (EP 267,617), 2-ethyl-l,3-hexanediol (WO 87/03490); decyl methyl sulphoxide and Azone.RTM. (Hadgraft, Eur. J. Drug. Metab. Pharmacokinet, 21:165-173 (1996)); alcohols, sulphoxides, fatty acids, esters, Azone.RTM., pyrrolidones, urea and polyoles (Kalbitz et al, Pharmazie, 51:619-637 (1996)); Terpenes such as 1,8-cineole, menthone, limonene and nerolidol (Yamane, J. Pharmacy & Pharmocology, 47:978-989 (1995)); Azone.RTM. and Transcutol (Harrison et al, Pharmaceutical Res. 13:542-546 (1996)); and oleic acid, polyethylene glycol and propylene glycol (Singh et al, Pharmazie, 51:741-744 (1996)) are known to improve skin penetration of an active ingredient.

Levels of penetration of an agent or composition can be determined by techniques known to those of skill in the art. For example, radiolabeling of the active compound, followed by measurement of the amount of radiolabeled compound absorbed by the skin enables one of skill in the art to determine levels of the composition absorbed using any of several methods of determining skin penetration of the test compound. Publications relating to skin penetration studies include Reinfenrath, W G and G S Hawkins. The Weaning Yorkshire Pig as an Animal Model for Measuring Percutaneous Penetration. In:Swine in Biomedical Research (M. E. Tumbleson, Ed.) Plenum, New York, 1986, and Hawkins, G. S. Methodology for the Execution of In Vitro Skin Penetration Determinations. In: Methods for Skin Absorption, B W Kemppainen and W G Reifenrath, Eds., CRC Press, Boca Raton, 1990, pp.67-80; and W. G. Reifenrath, Cosmetics & Toiletries, 110:3-9 (1995).

For some applications, it is preferable to administer a long acting form of agent or composition using formulations known in the arts, such as polymers. The agent can be incorporated into a dermal patch (Junginger, H. E., in Acta Pharmaceutica Nordica 4:117 (1992); Thacharodi et al, in Biomaterials 16:145-148 (1995); Niedner R., in Hautarzt 39:761-766 (1988)) or a bandage according to methods known in the arts, to increase the efficiency of delivery of the drug to the areas to be treated.

Optionally, the topical formulations can have additional excipients for example; preservatives such as methylparaben, benzyl alcohol, sorbic acid or quaternary ammonium compound; stabilizers such as EDTA, antioxidants such as butylated hydroxytoluene or butylated hydroxanisole, and buffers such as citrate and phosphate. The pharmaceutical composition can be administered in an oral formulation in the form of tablets, capsules or solutions. An effective amount of the oral formulation is administered to patients 1 to 3 times daily until the symptoms of the disease alleviated. The effective amount of agent depends on the age, weight and condition of a patient. In general, the daily oral dose of agent is less than 1200 mg, and more than 100 mg. The preferred daily oral dose is about 300-600 mg. Oral formulations are conveniently presented in a unit dosage form and may be prepared by any method known in the art of pharmacy. The composition may be formulated together with a suitable pharmaceutically acceptable carrier into any desired dosage form. Typical unit dosage forms include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories. In general, the formulations are prepared by uniformly and intimately bringing into association the agent composition with liquid carriers or finely divided solid carriers or both, and as necessary, shaping the product. The active ingredient can be incorporated into a variety of basic materials in the form of a liquid, powder, tablets or capsules to give an effective amount of active ingredient to treat the disease.

Other therapeutic agents suitable for use herein are any compatible drugs that are effective for the intended purpose, or drugs that are complementary to the agent formulation. The formulation utilized in a combination therapy may be administered simultaneously, or sequentially with other treatment, such that a combined effect is achieved.

FURTHER ASPECTS OF THE INVENTION

The invention also relates to Sprouty2, which is described in International Application PCT/SG2003/000228 (WO 2004/029295) designating the United States, herein incorporated by reference. Accordingly, this disclsoure should be taken to include reference to inventions as disclosed, and the claims as set forth, in which the term "Sprouty2" is substituted for "Sproutyl".

The invention is described further, for the purpose of illustration only, in the following examples. EXAMPLES

Example 1. Materials and Methods

Synthesis of [a-³² P] dCTP -labeled cDNA

The N-terminal half of hSpiyl (nucleotide 1-540) and hSpry2 (nucleotide 1-531) cDNA fragment are purified from pBluescript KS(-) (Stratagene, San Diego, St Louis) by BamHI/XhoI and EcoRI/XhoI restriction digestion respectively. The cDNA is labeled by random oligonucleotide priming (High Prime DNA Labeling Kit, Roche, Mannheim, Germany) according to the manufacturer's instructions, in the presence of [α-³²P] dCTP (6000Ci/mmol; Amersham Pharmacia, Germany). The labeled probes are purified by spin- column centrifugation (Probe QuantTM G-50 Micro Columns, Amersham Pharmacia) and met or exceeded the manufacturers recommendation for specific activity.

Hybridization of cDNA probes to Cancer Profiling Array

[α-³²P] dCTP-labeled hSpry DNA probes are hybridized to commercial cDNA blot, the Cancer Profiling Array (Clontech, Palo Alto, CA). The probe is hybridized to each blot according to the manufacturer's instructions and exposed to X-ray film at -70⁰C for various lengths of times (24hr to 3 days). To ensure equality of loading of the cDNA samples on the array, the array is stripped and re-probed with human ubiquitin.

RNA extraction and reverse transcription

Primary breast tumors (including 18 ductal and 1 lobular carcinomas) and adjacent normal -appearing breast tissue are obtained from 19 patients with malignant tumors undergoing surgery. The samples are obtained with approval of the appropriate institutional ethics committee and in accordance with the National Health and Medical Research Council of Australia guidelines for the conduct of research involving humans and included 1 stage I, 14 stage II, 2 stage III breast carcinomas and 2 unclassified samples. Total RNA is extracted from the primary breast tissues. RNA extraction is performed using RNA kits as recommended by the manufacturer (Qiagen, GmbH, Hilden, Germany). RNA is treated with RNase-free DNase I (Qiagen) for 30min at room temperature. Reverse transcription reactions are performed in 5μg of DNAse-treated RNA, 1 μl of oligo(dT)_i2-i s and SUPERSCRIPT II RNase H-, using the First-Strand Synthesis System for RT-PCR kit protocol (Invitrogen Life Technologies, Carlsbad, CA).

Quantitative real-time PCR

For each PCR reaction, 2μl of cDNA is mixed with 50OmM primers and 4mM MgCl₂ using the LightCycler-FastStart DNA Master SYBR Green Kit in a LightCycler (Roche, Mannheim, Germany) according to manufacturer's instructions. PCR reactions are carried out in triplicates with the following PCR primers:

human Spry2 forward 5' GCGATCACGGAGTTCAG 3' and reverse 5' GTGGAGTCTCTCGTGT 3'; human Spryl forward 5' AGGGCTATCTTCCTAGCA 3' and reverse 5' GTGAGAAGCATGGGGT 3'; human GAPDH forward 5' GGTGTGAACCATGAGAAG 3 ' and reverse 5 ' CCACAGTTTCCCGGAG 3 '

The crossing points that are calculated by the LightCycler software take into account the difference in amplification efficiency of the target and the reference genes. Crossing points of Spry (target) amplification products are normalized to the crossing points of GAPDH (reference) amplification products. Fold changes in gene expression of hSpryl and hSpryl between normal and tumor tissue are computed.

Histological analysis

Mammary glands are harvested and immediately fixed in 10% formalin (VWR Scientific). Whole-mount preparations of no. 4 mammary glands or primary breast tissues are paraffin-embedded, sectioned at 4 μm and stained with Mayer's hematoxylin-and-eosin B-phloxine (Sigma, St. Louis, MO). In situ hybridization analysis

For RNA in situ hybridization, the N-terminal of hSpryl and hSpry2 cDNA sequence is subcloned into pBluescript KS(-) (Stratagene, San Diego, CA)₅ the plasmids are linearized, and the sense and antisense riboprobes are synthesized from the T3 or T7 transcription sites by using 5U of T3 or T7 RNA polymerase respectively (Promega, Madison, CA). Cryosections are probed with sense or antisense digoxigenin-labeled riboprobes. Hybridization of the riboprobe is visualized immunohistochemically with an alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche, Mannheim, Germany).

Immimohistochemistry

Cryostat sections (10 μm) are fixed in 4% paraformaldehyde in PBS for 30min at room temperature. Sections are washed in PBS and incubated for lhr with rabbit polyclonal antibodies for hSpry2 (Upstate Cell Signalling). After washing in PBS, the slides are incubated for lhr with biotinylated goat anti-rabbit from Dako LSAB 2 system Peroxidase (Dako Corporation, Carpinteria, CA).

After one wash in PBS, the slides are incubated for 20 min with streptavidin conjugated to horseradish peroxidase (Vector Laboratories, Burlingame, CA) before washing again with PB S for 10 min. Antibody staining is done by treatment with diaminobenzidine. Sections are washed in PBS and counterstained with Mayer's hematoxylin. All the incubation and staining steps are performed at room temperature. A negative control without primary antibody is performed for all samples.

Tissue culture

The MCF-7 and T47D cell lines are obtained from ATCC. The cell lines are cultured in RPMI 1640 medium or DMEM high glucose medium, supplemented with 10% heat-inactivated fetal bovine serum, lOOIU/ml penicillin, lOOμg/ml streptomycin, and 2mM L-glutamine. Cells are cultured at 37⁰C and 5% CO₂. Fetal bovine serum is purchased from Hyclone Laboratories (Logan, UT) and all other tissue culture materials are obtained from Sigma Chemical Co. (St Louis, MO). Treatment of cells with 5-aza-deoxycytidine and trichostatin

Cells are seeded at a density of 5xlO⁵ in 100mm dishes. 5-aza-deoxycytidine (5- azaDC) is freshly prepared in deionized water and filter-sterilized. Trichostatin is freshly prepared in ethanol and filter-sterilized. 24hr later, cells are treated with 2.5μM 5-aza- deoxycytidine (Sigma Chemical Co., St Louis, MO) or treated with 300 nM of trichostatin (Sigma Chemical Co., St Louis, MO). The medium is changed every 24hr. In instances where cells are doubly treated, cells are subjected to 96hr of 5-azaDC treatment and 30OnM of trichostatin is added in the last 24hr. After treatment, cells are washed with PBS and total RNA is isolated for RT-PCR.

RT-PCR analysis

Reverse transcription is performed as mentioned above. PCR reactions are carried out using 2 μl of cDNA with the following PCR primers: The hSpry2 and hSpryl primers for real time quantitative PCR analysis are used. Primers for human Maspin are: forward 5' GCTTTTGCCGTTGATCTGTTC 3' and reverse 5' GATCTGACCTTTCGTTTCTTCCA 3'. The PCR conditions are as follows: 1 cycle at 96°C for 2min and 35 cycles at 96°C for 30s, 55°C for 30s, 72°C for lmin. Samples are kept at 72°C for 10 min after the last cycle. The human β-microglobulin gene are co- amplified as internal controls. Primers for β-microglobulin are: forward 5' CTCGCGCTACTCTCTCTTTCTGG 3' and reverse 5' GCTTACATGTCTCGATCCCACTTAA 3 ' . The PCR products are resolved by electrophoresis on 1% agarose gels.

Stable transfection

The hSpry2^Y55F expression construct is made by subcloning the full length hSpry2^Y55F cDNA into the BamHI-XhoI sites of the constitutive mammalian expression vector pCMVTag2B (Stratagene, San Diego, CA) to generate the pCMV-hSpry2^Y55F (FLAG-tagged) construct. The MCF-7 cell line is transfected with pCMV-hSpry2^Y55F, under the control of a CMV promoter for constitutive expression. For control purposes, the MCF-7 cell line is transfected with a vector control. Cell lines are established as MCF-7- hSpry2^Y55F, and MCF-7 control cells. Proliferation assay

MCF-7 and its derived cell are seeded (lxlO⁴/well) in a 96-well plate in triplicates. After incubation at 37°C in 5% CO₂ for 12hr, the number of living cells is measured using a CellTiter 96^® AQ_ueOus One Solution Cell Proliferation Assay (MTS) (Promega, Madison, WI) according to manufacturer's instructions. Assays are performed with addition of a small amount of the CellTiter 96^® AQ_ueous One Solution Reagent directly to culture wells, incubated for 2hr at 37°C in 5% CO₂ and then recording absorbance at 485nm with a 96- well plate reader. The quantity of formazan product as measured by the amount of 485nm absorbance is directly proportional to the number of living cells in culture.

Soft agar colony formation

MCF-7 and its derived cells are cultured in 6-well plates first covered in soft agar layer (RPMI 1640 with 0.5% agar and 10%FBS). The top layer contained 5 x 10³ cells in RPMI 1640 with 0.35% agar and 10% FBS. Medium is added to the top layer to prevent drying of the agarose gel. After 14 days the colonies are stained purple with 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich).

In vivo tumor formation

A total of 5x10⁵ MCF-7-hSpry2^Y55F or MCF-7 control cells are suspended in 200μl matrigel (BD Biosciences) and injected into the mammary (axillary) fat pad of BALB/c nude mice, which simultaneously received a 60-day release pellet containing 0.72mg of β- estradiol (Innovative Research of America, Toledo, OFT). After 9 weeks, the tumors are excised, weighed and tabulated.

Statistics

All data are presented as mean ± S.E.M. We assessed differences between groups by two-tailed non-paired Student's f-test using the Graphpad Prism statistical software. Example 2. hSpry2 Expression is Down-Regulated in Breast Cancer in a High Percentage of Analyzed Samples

As an initial investigation we interrogated the GCM microarray database as to the levels of expression oϊhSpryl and 2. hSpryl and 2 are chosen because when all hSprys are compared these two isoforms appear most similar to each other in function, protein sequence and expression pattern. We also suspect that there might be some level of redundancy in the physiological function of the two proteins.

In the analysis of gene array data (Figure 1), the gene expression levels in cancer tissues are compared to those levels in normal healthy tissue. In mammals Spry2, in particular, has been demonstrated to be associated particularly with FGF signalling molecules, Fgf8 and FGFRl (refs B 15, B 17, B30). Both of these genes, together with the various FGFRs, have been found to be over-expressed in various cancers (refs B31, B32). We have tabulated them alongside the hSprys for comparison. We have previously shown that c-Cbl is functionally associated with hSpry2 (ref B28) therefore c-Cbl expression levels are also included in the analysis.

In terms of patterns of hSpiγ expression (Figure 1), it is apparent that hSpiy2 levels are diminished in breast cancer. There are other trends observable with other cancers but the highlighted case is the most profound. As these results are only indicative, we proceeded to further investigate the expression and tissue localization of the two Spry genes in breast development and cancer.

Example 3. hSpryl and hSpiy2 Expression is Down-Regulated in Breast Cancer, as Assessed on a Matched Tissue Cancer Profiling Array

We next investigate the expression levels of hSpryl and 2 in a Cancer Profiling Array where cDNA from matched normal and tumor tissue samples are spotted on a blot.

The hSpiy2 probe is prepared and tested for specificity before subjecting it to hybridization to the Cancer Profiling array. On the resultant blot, a high proportion of breast cancer samples are observed to have a repressed expression of hSpry2 (Figure 2A). Using densitometry, the intensity of the radioactive probe signal from each cDNA sample is quantitated. Signals from breast tumor cDNA are compared with respect to that of normal breast tissue cDNA. The breast cancers showing at least an arbitrary 1.2-fold downregulation in signal are considered to exhibit a down-regulation in expression of hSpryl. 48 out of 50 (96%) of the paired samples showed a down-regulation of hSpry2. Similarly when the hSpryl probe is hybridized to the Cancer Profiling Array, the same trend is observed: 39 out of 50 (78%) of the paired samples showed a down-regulation of hSpryl (Figure 2B).

To demonstrate that the sample population of breast cancer tumors in the blot are a true representation of the population of breast cancers, as well as to demonstrate equal loading of cDNA samples on the blot, the blot is stripped and reprobed for ErbB2. ErbB2 is a prognostic marker for breast cancer and collated data indicates it is over-expressed in 25-30% of breast cancer (ref B33). The ErbB2 probe is prepared and hybridised to the Cancer Profiling Array. The breast cancers showing at least an arbitrary 2-fold up- regulation in signal are considered to exhibit an up-regulation in expression of ErbB2. ErbB2 is found to be over-expressed in 25 out of 50 (50%) breast cancer tumor samples in comparison to the normal samples (Figure 2B). This is higher than the 30% typically observed in many breast cancer samples but deemed to be within acceptable limits. To further ensure equality in loading of the cDNA samples on the membrane, the membrane is reprobed with a human ubiquitin control probe (provided by the manufacturer) (data not shown).

Such prevalent patterns of down-regulation of Sprys may herald the later application of these isoforms as tumor markers that are breast cancer specific. We decided to examine other molecular markers that have been shown to be down-regulated in breast cancer. Down-regulation of the Maspin gene is recently reported in a significant percentage of breast tumors (refs B34-B35), which prompted the authors to speculate that it may be useful as a diagnostic marker. For the sake of comparison, the Maspin probe is prepared and hybridized to the Cancer Profiling Array. Maspin is not found to be significantly down-regulated in breast tumor samples (Figure 2B) as reported previously (refs B34-B35). Comparatively, the extent of downregulation observed with the Spiys is more profound and universal.

While the cDNA array used does not encompass a wide range of cancers and some of the paired samples are not present in statistically relevant numbers, the evidence that hSpryl and hSpry2 are comprehensively down-regulated specifically in breast cancers is compelling and we decided to further investigate this observation.

Example 4. Analysis of Breast Cancer Samples by Quantitative Real-Time PCR Show a Profound Down-Regulation in Levels of hSpryl and 2 in Breast Cancer

In order to confirm the data from the GCM and Cancer Profiling Array, we needed to compare matched clinical samples and analyze them by an alternative method. To this end we analyzed 19 matched normal and tumor breast samples (including 18 ductal and 1 lobular carcinomas) using real-time quantitative PCR. These included 1 stage I, 14 stage II, and 2 stage III breast carcinomas (2 samples are not classified).

The expression levels of the Spry genes in each normal tissue sample (control) is deemed to be 100% and the comparative level in the matching tumor sample is expressed as a percentage of the control. Each sample is analyzed three times and a mean value is calculated. 18 out of 19 (94.7%) of the tumor samples show a significant down-regulation of hSpryl (Figure 3A) and hSpryl (Figure 3B) expression, with respect to normal tissue. A 2-100 fold down-regulation of hSpryl and hSpryl is observed in the breast tumors.

As before, the expression levels of ErbB2 are analyzed to show the 19 breast cancer samples used are from a representative patient population. 11 of the 19 samples (57.8%) showed a significant over-expression of ErbB2 (data not shown), which is comparable with the values obtained from the Cancer Profiling Array and previously documented reports.

In summary, a profound down-regulation in levels of hSpryl and hSpiy2 in breast tumors is observed. The results obtained from the three methods of analysis are essentially in accordance with each other, although a higher percentage of samples showing depressed hSpryl levels are observed using real-time quantitative PCR analysis.

Example 5. Expression of mSpryl and mSpry2 in Developing Mouse Mammary Gland

It appears from the accumulated data thus far that Spryl and 2 are expressed in normal human breast. Currently there has been no data published on the disposition of Spryl and 2 during development of the breast.

The breast is a unique organ in that it exhibits mostly post-natal development. At puberty, pubertal hormones stimulate the breast ducts to invade the mammary fat pad, branching by bifurcation until the ducts reach the limits of the fat pad. The final developmental fate of the mammary gland is fulfilled only when pregnancy and lactation occurs. There is a further branching of the breast ducts as well as a proliferation of secretory alveoli during pregnancy that occurs to prepare for lactation. Following weaning, the secretory epithelium involutes by apoptosis until the gland once again resembles that of a virgin (refs B36-B37).

We wanted to investigate the expression levels and tissue localization oϊSprys in the various stages of breast development. While it is appreciated that there are differences between human and murine development, we next analyzed mouse tissue sections to identify the temporal and spatial localization of mSpryl and 2 over the described stages of development.

In situ hybridizations are carried out as described in 'Materials and Methods' and representative results are shown in Figure 4A. Spry2 is highly expressed in developing mammary ducts in 2 and 6 weeks old females and appears to be confined specifically to the epithelial lining of the mammary ducts and is absent in the stroma and adipose tissues. It is similarly expressed in the male mammary gland tissue at the same stages (data not shown). The level of expression starts decreasing as the mice reach sexual maturity, as evidenced by the lower levels apparent at 16 weeks. In pregnant mice, the level of mSpry2 becomes elevated again when it is highly expressed in the actively developing alveoli (Figure 4B). mSpry2 expression then diminishes in the lactating female and appears to be totally absent during the involution phase. Figure 4C shows the sense control probe for mSpry2 hybridized to a section of the mammary gland from a pregnant mouse, showing a lack of non-specific staining.

In summary it appears that mSpry2 is expressed in development during the stages of tissue modeling when the epithelial ducts are forming and branching (puberty), and later when the alveoli are actively developing (pregnancy). Such a developmental scenario parallels the tissue modeling in the Drosophila trachea and mammalian lung formation, whereby tubular epithelium is remodeled into a branching system under the regulatory influence of the various Sprys.

We performed similar studies for mSpryl to examine the expression and localization in breast tissue. mSpryl is found to be co-localized with mSpry2, albeit at lower expression levels in a pregnant mouse (Figure 4D). This hints that the two isoforms of Spry may have some level of redundancy in their physiological functions during breast development.

Example 6. In Situ Analysis of Human Breast Tissue Shows the Down-Regulation of Spry Isoforms in Breast Cancer

We next investigated the disposition of hSpry isoforms in human breast tissue, both normal and cancerous. It is desirable to locate and demonstrate the situation in an unaffected area within the same tissue selected for cancer state analysis.

The disposition of hSpry2 and hFgβ are shown within healthy tissue removed during cancer surgery (Figure 5A). The staining clearly shows that both hSpry2 and hFgβ are expressed in epithelial cells lining the breast ducts. When the tumor-encompassing tissue (invasive ductal carcinoma, grade 3) from the same sample is analyzed it is apparent that there is no detectable hSpry>2 or hSpryl expression, although hFgβ (previously shown to be up-regulated in breast cancer) (ref B38) is still apparent (Figure 5B). The same observations are noted for another breast cancer specimen, which is an invasive ductal carcinoma (grade 2) (data not shown).

Immunohistochemistry is then performed to correlate the gene expression to protein levels. In a segment of healthy breast tissue, hSpry2 is shown to be present in epithelial cells that line the lumen of mammary ducts (Figure 5C). Correspondingly when the epithelial tissue has become cancerous (in the same individual) (invasive ductal carcinoma-grade 3) there is little or no Spry2 protein present (Figure 5D). In the same cancerous tissue both ErbB2 (stained with Neu-2 antibody) and Fgf8 are present in relatively high amounts (Figure 5D). Both ErbB2 (ref B33) and Fgf8 (ref B38) have been previously shown to be up-regulated in breast cancer.

Taken together, the expression level and disposition of proteins indicate that both hSpryl and more profoundly hSpry2, are present in normal luminal epithelial tissue. However, when this tissue becomes cancerous the expression (and protein) levels decrease substantially in a high percentage of the breast tumors analyzed. The extent of down- regulation oϊSprys is not found to be correlated to the staging of cancer in the patients we analyzed, although the sample size may not be large enough to be conclusive.

Example 7. Down-Regulation of hSpry2 in Breast Cancer is Not Due to Epigenetic Silencing

We have demonstrated that a high percentage (94.7%) of breast cancers demonstrate a down-regulation of Spry 1 and 2. This is unlikely to be caused by genetic changes such as chromosomal rearrangements or deletions. Besides, there is no evidence in the literature documenting alterations in the chromosomal regions where hSpryl and hSpry2 are located. We next examined the possibility of epigenetic causes in contributing to the down-regulation of Spiys. Many genes are known to be silenced by methylation of gene promoter regions in cancer. Methyl-CpG binding domain proteins are recruited to the methylated cytosines and they work with histone deacetylases (HDAC) to repress the transcription of a gene (refs B39-B40). We chose breast cancer cell lines where previous analysis had shown low hSpry2 levels. T47D cells are treated with the DNA methyl-transferase inhibitor, 5-aza- deoxycytidine (5-aza-DC) and the HDAC inhibitor, trichostatin. As a positive control, we have monitored the expression of a Mαspin, which is known to be silenced by methylation and/or histone acetylation in cancers (ref B41). The results demonstrate that whereas Mαspin expression increases when cells are treated with 5-aza-DC, there is no increase in the expression of hSpryl (Suppl. Figure 1). Likewise, Mαspin expression increases when cells are treated with trichostatin but there is no concurrent increase in the expression of hSpry2 (Suppl. Figure 1). To preclude the likelihood of an atypical, cell-specific result the experiment is repeated on another breast cancer cell line, MCF-7, where the same observations are reiterated (data not shown).

To further confirm that methylation is not responsible for the silencing of Spry! expression in breast cancer, we employed bisulphite PCR methods to sequence the CpG- rich islands found 5' upstream of the hSPRY2 gene in a human breast cancer specimen. We noted that the hSPRY2 gene has a CpG region extending from the 5' upstream sequence through the first exon and into the adjacent intron. We sequenced the region 600bp upstream of the first exon, which is noted to be particularly CpG-rich, having a GC content of 74.8% and an observed CpG/expected CpG ratio of 0.936 (using CpG Island Searcher; http://ccnt.hsc.usc.edu/cpgislands/). We observed that the cytosine residues in these CpG-rich dinucleotides are not methylated (data not shown)

These results clearly demonstrate that the decreased expression levels of hSpry2 seen in breast cancers are neither due to DNA methylation nor histone hypoacetylation. Taken together, it is likely that a transcriptional factor(s) upstream of Spry has been aberrated by a mechanism not involving epigenetic silencing. Gross et αl (ref B42) highlighted Spryl as the downstream transcriptional effector of WTl in the developing kidney. The situation of the consistent down-regulation of hSprys in breast cancer is unlikely to be due to the status of WTl expression since it has been established that WTl is up-regulated in breast cancer (ref B43). When the WTl probe is hybridized to the Cancer Profiling Array, no difference in WTl expression is observed between normal and tumor breast tissue samples (data not shown). Further characterization of the transcriptional mechanism of the Spry genes in normal versus cancerous breast tissue is required to shed light on the transcriptional factors involved.

Example 8. Inhibiting Spry's Function in MCF-7 Cells Results in Cells Proliferating Faster and Exhibiting Anchorage-Independent Growth

A central question arises when the gene that plays a pivotal role in a major signal transduction pathway shows consistently aberrant expression in a particular cancer; is the change contributing to the tumorigenic process? We have seen that Spryl and 2 are highly expressed at times in the breast during extensive changes in ductal patterning, and that the expression in normal mature breasts may be sufficient to play a surveillance role against inappropriate cell growth or movement. It may be postulated that down-regulation of Sp/γs in cancer allows for uncontrolled proliferation due to unchecked upstream hyperactivation of the MAP kinase pathway and thereby permits the tumorigenic process to proceed.

We therefore wanted to address whether down-regulation of hSpry2 in breast cancer has a role to play in tumorigenesis. In order to simulate the down-regulation in expression of Sprys in breast cancer, we decided to quench the function of endogenous wild-type Sprys by over-expressing the Y^55F mutant of hSpry2 and compare the tumorigenic potential of these cells with parental cells in nude mice. There have been several reports that the tyrosine phosphorylation of Y⁵⁵ on hSpry2 is necessary for its Ras/MAP kinase inhibitory function (refs B23, B27, B28) Some reports provide compelling evidence that the hSpry2^Y55F mutant functions as a dominant negative factor in inhibiting hSpry2 function (refs B23, B27). Furthermore, there is a distinct possibility that hSpryl and hSpry2 may play redundant roles fulfilling the general function of Spry; however, a cell line over-expressing hSpry2^Y55 will nullify the effect of both wild-type Spryl and Spry2 since the tyrosine is highly conserved in all Spry isoforms, and the function that occurs due to phosphorylation on this residue will be blocked by an over- expression of such a point mutant, irrespective of its isoform. hSpry2^Y55F-transfected stable MCF-7 cell lines are generated as described in 'Materials and Methods' and as an initial analysis, the effect of over-expression of hSpry2^Y55F is assessed based on the proliferation rate of the cells. The hSpry2^Y55F- transfected clones showed a 22% significant increase in proliferation over control MCF-7 cells (p = 0.0034) (Figure 6A), indicating that nullifying the effect of wild-type Sprys impacts on the growth rate of the MCF-7 cells.

Next we assessed the anchorage-independent growth of the stable hSpry2^Y55 cells in the formation of colonies using the soft agar assay, in comparison with the control MCF-7 cells. hSpry2^Y55-transfected clones formed larger and greater numbers of colonies compared to control MCF-7 cells (Figure 6B). This indicates that the abrogation of the function of wild-type Sprys in MCF-7 cells causes cell transformation, resulting in the loss of contact inhibition and anchorage independent growth.

Example 9. Inhibiting Spry's Function in MCF-7 Cells Results in the Formation of Larger Tumors

The in vivo tumorigenic potential of the stable cells are assessed using an in vivo animal model of breast cancer that involves the growth of MCF-7 xenografts in nude mice. To avoid individual variation, hSpry2^Y5⁵ MCF-7 and control MCF-7 cells are injected into each side of the nude mice. The progression of tumors is followed at weekly intervals over 9 weeks after which the animals are sacrificed and the tumors extracted and weighed. It is immediately apparent that the hSpry2^Y55F cells caused a significantly larger tumor mass when compared with the control cells (p = 0.0001). An image of a typical mouse is shown in Figure 6C. The respective tumors from 15 different animals are weighed and the results displayed in the bar chart shown in Figure 6C. The values correspond to the average weight of tumors + SEM derived from 15 mice. The average weight of hSpry2^Y55F tumors is 2.9 times greater than control tumors. To confirm that hSpry2^Y55F is still consistently expressed in the xenograft tumors excised from the nude mice, proteins from both tumors are extracted and hSpry2^Y55F (FLAG-tagged) expression is confirmed using Western blotting techniques (data not shown). These experiments indicate that the down-regulation or elimination of wild-type Spry function may contribute to transformation of cells, resulting in hyperproliferation and the larger tumor masses observed with hSpry2^Y55F tumors. The results suggest a previously unrecognized role for Spry in cancer development.

The following Example 10 describes experiments showing down-regulation of hSpry2 (human Sprouty 2) in breast cancer cells, and corresponds to Examples 1 and 2 of WO 2004//029295. Figures 8 and 9A-9F correspond to Figures 1 and 2A-2F of WO 2004//029295. WO 2004//029295 is hereby incorporated by reference.

Example 10. hSprouty2 Profiling Array and Probe

A commercial cancer profiling array prepared from 241 paired samples of cDNA extracted from normal and tumour tissues from patients is analyzed with the specific probe for hSpry2.

Cancer Profiling Array

A Cancer Profiling Array is purchased from Clontech (Palo Alto, CA). The array consists of 241 pairs of cDNA spots generated from tumour and corresponding normal tissue samples of individual patients spotted side-by-side on a nylon membrane.

The layout of this profiling array is presented at Figure 9A.

hSpry2 Probe and Specificity Synthesis of Ta-³²Pl dCTP-labeled hSpry2 cDNA probe

An N-terminal hSpry2 cDNA fragment (531bp) is purified from PXJ40FLAG vector after restriction digestion. The cDNA is labelled by random oligonucleotide priming (High Prime DNA Labeling Kit, Mannheim, Germany) according to the manufacturer's instructions. The labelled probe is purified by spin-column centrifugation (Probe Quant™ G-50 Micro Columns, Amersham Pharmacia, NJ, USA). The specific activity of the probe is 1x10⁹ cpm/μg and the concentration of probe in the final hybridization buffer is 5- 10x10⁶ cpm/ml.

Specificity of Fa-³²Pl dCTP-labeled hSprv2 cDNA probe

Since the four hSpry homologues hSpryl, hSpry2, hSpry3 and hSpry4 share a highly conserved C-terminal domain, potentially specific probes are prepared to each of the sprouty family from regions of their respective N-termini to minimize non-specific hybridization. The specificity of the hSpry2 probe is affirmed by its ability to hybridize only to hSpry2 cDNA and not to cDNAs from the other human sproutys.

The result of such a preparation, shown in Figure 8, demonstrates the specificity of the hSpry2 probe in the context of the other cDNAs. This specific probe is then used to hybridize to the Cancer Profiling array

Hybridisation and Analysis

Hybridization of cDNA probes to the Cancer Profiling Array

15ml of ExpressHyb (Clontech, Palo Alto, CA) is pre-heated to 68⁰C. To this, 1.5mg of denatured salmon testes is added to reconstitute the pre-hybridization buffer. The membrane is pre-hybridized with 10ml of pre-hybridization buffer at 65°C for 30min in a hybridization bottle subjected to continuous agitation.

The labeled cDNA probe is mixed with 30μg of C_ot-1 DNA, 150μg of sheared salmon testes DNA and 50μl of 2OxSSC. The mixture is first heated at 95-100⁰C for 5min, then at 68°C for 30min, and subsequently added to the remaining 5ml of pre-hybridization buffer to reconstitute the final hybridization buffer.

Following prehybridization, buffer is replaced by 5ml of the final hybridization buffer mixture and the membrane is hybridized at 65°C overnight with continuous agitation. Wash buffers I (2xSSC, 0.5%SDS) and II (0.2xSSC, 0.5%SDS) are preheated to 65⁰C. The membrane is washed three times with 200ml of Wash buffer I and once with 200ml of Wash buffer II, each at 65°C for 30min. The membrane is rinsed with 200ml of 2xSSC at room temperature for 5min with continuous agitation, before exposure to X-ray film at 70⁰C for various lengths of time (24hr to 3 days). To ensure equality of the spotted cDNA samples on the membrane, the membrane is stripped by boiling it in 0.5%SDS solution for 5-10min. Following this, the membrane is re-probed with a human ubiquitin control probe (provided by the manufacturer).

Quantification of hSpry2 Gene Expression and Analysis of Results

The intensity of the signal is quantified by densitometry (Bio-Rad, CA, USA). The background is measured and subtracted off the quantified signal. A scatter plot graph of the adjusted intensities (absolute intensity minus background) of the tumour versus normal tissue is plotted using Microsoft Excel software.

Expression ofhSpiy2 is Repressed in Breast Cancer Tissues

The resultant autoradiograph reveals that hSpry2 is comparatively highly expressed in samples of normal tissue from breast, uterus, rectum and colon, while lower expression levels of are detected in the ovary, stomach, lung, kidney and thyroid (Figure 9B).

The most spectacular result is the high proportion of breast cancer samples where the expression of hSpry2 is significantly repressed. Apparently only 2 of the 50 pairs showed either no difference or opposite expression trends to the general pattern (ie 96% of the paired samples show a significant down-regulation of hSpry2). The optical densities of the paired spots are measured on a densitometer and the ratios of normal/tumour cell expression are plotted graphically (Figure 9C).

One sample pair is deemed to show no significant difference between normal and cancer tissue. Another sample pair is significantly above the line (ie greater expression of hSpry2 in tumour cells compared with normal cells) whereas 48 of the matched pairs (96%) lie beneath the lines signifying much lower expression of hSpry2 in tumour cells compared with normal cells. Three of these samples included metastatic tissues, which all showed insignificant to low levels of hSpry2 expression. Accordingly, hSpry2 expression is lower in breast cancer tissue compared to normal breast tissue.

Tissue Specific Repression of hSpry2 Expression

The distribution of the comparative ratios are also calculated and plotted for colon and uterine cancer in comparison to those of breast cancer (Figures 9D and 9E).

These samples are chosen for comparison because hSpry2 has a relatively high expression in normal tissues of these two organs, and the sample sizes are comparable to the breast tumour sample. It can be seen that the expression of hSpry2 in both of these tumours when compared with normal cell expression shows either no significant change or are only moderately different in relatively few cases.

The reduced level of hSpry2 expression in tumour cells is tissue specific to breast tissue.

Controls

The blot is next stripped and the effectiveness of stripping is verified by exposure on x-ray film overnight. The stripped blot is re-analyzed with an [α-³²P] dCTP-labelled ubiquitin control probe to reveal the equality of cDNA loading on the blot. The resultant autoradiograph is shown in Figure 9F.

The entire blotting procedure described above is repeated using a fresh blot and identical results are obtained. Tissue and Organ Analysis

The data presented in the preceding Examples comes from probing a commercial Cancer Array blot using a Northern blotting technique. Further experiments may be done to provide similar results, for example on tissue samples. Matched samples of tumours and normal from various organs are obtained from the Department of Pathology, National University Hospital, Singapore. A preference is made for breast tissue. RNA is extracted from these tissues using standard techniques and real-time quantitative PCR is carried out using suitably designed hSpry2 primers and probes to analyze the expression level of the hSpry2 transcripts in the various tissues and organs.

An advantage of quantitative real-time PCR is that it is more accurate and quantitative than Northern blotting and requires less tissue sample. It is difficult to obtain large tissue samples of normal breast tissues. Therefore, real-time PCR is an appropriate technique for analyzing the expression level of the hSpry2 transcripts in breast tissues. The expression of Sprouty 2 from 50 breast tumours is analysed and compared to the baseline expression of Sprouty 2 found in normal breast tissue (obtained by analysing 15 normal breast tissues).

The analysis includes comparing and correlating the expression levels of hSpry2 with the levels of expression of other genes that have been demonstrated to be altered in breast cancers. Thus, hSpry2 expression is compared with the expression of EGFR, ErbB2 and Estrogen receptor, which are known to be over expressed in some breast cancers. Furthermore, HIN-I and maspin expression levels are analysed. HIN-I and maspin show repressed expression in a similar manner to hSpry2 but neither is breast specific

HIN-I shows most similarity to the expression pattern of hSpry2. It is therefore advantageous to compare HIN-I and hSpry2 expression in a side-by-side analysis.

Example 11. Role of Spry2 in Inhibition of Tumorigenesis

This Example explores whether Spry2, a negative feedback regulator of MAPK, has a role in the inhibition of tumorigenesis.

An in vitro transformation assay is used to test whether Spry2 possesses tumor suppressor properties. In this system, NIH3T3 cells can be transformed by overexpression of many oncogenes, including viral oncogenes such as SV40 and the polyoma T antigen. Such oncogenes exert their effects by associating with and altering the function of cellular proteins that are important players in the control of cell proliferation and contact inhibition of growth. Transformation would be detected as foci of dense morphologically altered cells among monolayer normal cells.

We chose Middle T antigen (PyMT) which is the principal transforming component of polyomavirus (DNA virus) since it is known to transform NIH3T3 cells in one step and it is easily manipulated and studied. PyMT is not expressed in cancer cells, however, it acts as a potent oncogene because its product binds to and co-opts several signal transduction pathways, including those of the Src family and the ras and PD kinase pathways, which are pathways known to be abnormally activated in some human cancers.

Tissue Culture

NIH3T3 cell lines are obtained from ATCC. The cell lines are cultured in DMEM high glucose medium, supplemented with 10% heat-inactivated fetal calf serum, 100IU/ml penicillin, lOOμg/ml streptomycin, and 2mM L-glutamine. Cells are cultured at 37°C and 5% CO₂. Fetal calf serum is purchased from Hyclone Laboratories (Logan, UT) and all other tissue culture materials are obtained from Sigma Chemical Co. (St Louis, MO).

Stable Transfection

A human Sprouty2 (hSpry2) expression construct is made by subcloning the full length hSpry2 cDNA into the BamHl-Xhol sites of the constitutive mammalian expression vector pCMVTag2B (Stratagene, San Diego, CA) to generate the pCMV- hSpry2 (FLAG-tagged) construct. The NIH3T3 cell line is transfected with pCMV- hSpry2, under the control of a CMV promoter for constitutive expression. For control purposes, the same cell line is transfected with a vector control. Cell lines are established as NIH3T3-hSpry2, and NIH3T3 control cells. Transformation Assays

5 x 10⁵ cells of each cell line are plated onto a 10cm dish. Each plate is transfected with 5μg purified PyMT (A3 strain), derived from J4mT Ong, S. H., et al (2001) ShcA and Grb2 mediate polyoma middle T antigen-induced endothelial transformation and Gobi tyrosine phosphorylation The EMBO Journal (2001) 20, 6327-6336, using the transfection reagent Lipofectamine 2000 reagent (Life Technologies) as recommended by the manufacturer. The cells are scored for formation of foci at the end of 2 weeks.

Staining for Foci Formation

Plates are rinsed with PBS. 5ml of 10% formaldehyde in PBS is added to each plate and left at room temperature for 30 min. The formaldehyde is removed and crystal violet is added to stain for the foci at room temperature for lOmin. The stain is removed and plates are washed with 70% ethanol such that only the foci are selectively stained blue. Results shown are representative of 2 independent experiments.

Results

The results show that NIH3T3-hSpry2 cells are more resistant to transformation by

PymT compared with NIH3T3 control cells. NIH3T3 control cells develop 126 ± 9.5 colonies upon transformation whereas NIH3T3-hSpry2 develop fewer colonies (p<0.5, student's t test). Two clones of NIH3T3-hSpry2 are tested. NIH3T3-hSpry2 (clone 2) and NIH3T3-hSpry2 (clone 10) develop 13.2 ± 9.2 and 29.0 ± 7.1 colonies.

Example 12. Role of Spryl in Inhibition of Tumorigenesis

This Example explores whether Spryl, a negative feedback regulator of MAPK, has a role in the inhibition of tumorigenesis.

The experiments described above in Example 11 for Spry2 are repeated with Spryl, except that a human Sproutyl (hSpryl) expression construct is made by subcloning the full length hSpryl cDNA into the Hindlll-Xhol sites of the constitutive mammalian expression vector pCMVTag2B (Stratagene, San Diego, CA) to generate the pCMV- hSpryl (FLAG-tagged) construct. This construct is transfected into NIH3T3 cells. As a control, a vector is transfected into NIH3T3 cells. Each cell line is transfected with PymT as described.

Results

The number of colonies which develop from NIH3T3 control cells transfected with

PymT is compared to those which develop from NIH3T3-hSpryl cells transfected with PymT. It is expected that the number of colonies from the pCMV-hSpryl transfected cells is less than that of the controls.

Example 13. Tumor Suppressor Role of Spry2

This Example evaluates whether Spry2 inhibits tumorigenesis mediated by the

PyMT in vivo. The Example employs breast cancer transgenic mouse models whose oncogenesis is induced by expression of the polyoma virus middle T oncoprotein (PyMT mice), which are readily available from commercial sources.

In this transgenic mouse model, the expression of the oncoprotein, PyMT, is under the control of mouse mammary tumor virus LTR (MMTV LTR) and is therefore restricted to the mammary epithelium. Mammary hyperplasia can be detected in this model as early as 4 weeks and most importantly, a large percentage of mice developed carcinoma at ~14 weeks and this correlated with the appearance of pulmonary metastases.

Homozygous null for the Spry2 allele (Sp^^"'^"") are crossed with mice that are transgenic for the polyoma virus middle T (PyMT) antigen (Jackson Labs). Female offspring from this cross that are PyMT^+/~ and are wild type for the Spry2 allele (WT) or homozygous null for the Spry2 allele (Spry2^"/~) are saved for analysis.

The tumorigenesis process in PyMT mice is observed. An observation that the loss of Spry2 accelerates the tumorigenesis process in PyMT mice indicates that Spry2 has tumor suppressor properties. Example 14. Tumor Suppressor Role of Spryl

The experiments described above in Example 13 for Spry2 are repeated with Spryl by performing crosses between Spryl^"''^" mice and mice that are transgenic for the polyoma virus middle T (PyMT) antigen.

The progression of tumorigenesis in female offspring from this cross that are

PyMT⁺'^" and are wild type for the Spryl allele (WT) is compared to that of mice which are homozygous null for the Spryl allele (Spry I^"7").

An observation that the loss of Spryl accelerates the tumorigenesis process in PyMT mice indicates that Spryl has tumor suppressor properties.

Example 15. Tumor Suppressor Role of Spry2

Transgenic animals expressing the Spry2 gene specifically in the mammary epithelium are produced and crossed with mice that are transgenic for the polyoma virus middle T (PyMT) antigen.

The tumorigenesis process in the resulting offspring is observed. An observation that the loss of Spry2 accelerates the tumorigenesis process in these mice indicates that Spry2 has tumor suppressor properties.

Example 16. Tumor Suppressor Role of Spryl

The experiments described above in Example 15 for Spry2 are repeated with Spryl. An observation that the loss of Spryl accelerates the tumorigenesis process in the offspring mice indicates that Spryl has tumor suppressor properties. REFERENCES

1. Hacohen N, Kramer S, Sutherland D, Hiromi Y, Krasnow MA. Sprouty encodes a novel antagonist of FGF signaling that patterns apical branching of the Drosophila airways. Cell. 1998 92(2):253-63

2. Kramer S, Okabe M, Hacohen N, Krasnow MA, Hiromi Y. Sprouty: a common antagonist of FGF and EGF signaling pathways in Drosophila. Development. 1999 126(l l):2515-25.

3. Kramer S, Okabe M, Hacohen N, Krasnow MA, Hiromi Y. Sprouty, an intracellular inhibitor of Ras signaling.Cell. 1999 96(5):655-65.

4. Casci T, Vinos J, Freeman M. Sprouty, an intracellular inhibitor of Ras signaling. Cell. 1999 96(5):655-65.

5. Reich A, Sapir A, Shilo B. Sprouty is a general inhibitor of receptor tyrosine kinase signaling. Development. 1999 126(18):4139-47

6. Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, Cobb MH. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev. 2001 22(2): 153-83. Review.

7. Tefft JD, Lee M, Smith S, Leinwand M, Zhao J, Bringas P Jr, Crowe DL, Warburton D. Conserved function of mSpry-2, a murine homologue of Drosophila sprouty, which negatively modulates respiratory organogenesis. Curr Biol. 1999 9(4):219- 22.

8. Gross I, Bassit B, Benezra M, Licht JD. Mammalian sprouty proteins inhibit cell growth and differentiation by preventing ras activation. J Biol Chem. 2001 276(49):46460- 9. Yusoff P, Lao DH, Ong SH, Wong ES, Lim J, Lo TL, Leong HF, Fong CW, Guy GR. Sprouty2 inhibits the Ras/MAP kinase pathway by inhibiting the activation of Raf. J Biol Chem. 2002 277(5):3195-201.

10. Yigzaw Y, Cartin L, Pierre S, Scholich K, Patel TB. The C terminus of sprouty is important for modulation of cellular migration and proliferation. J Biol Chem. 2001

276(25):22742-7.

11. de Maximy AA, Nakatake Y, Moncada S, Itoh N, Thiery JP, Bellusci S. Cloning and expression pattern of a mouse homologue of drosophila sprouty in the mouse embryo. Mech Dev. 1999 81(l-2):213-6.

12. Ozaki K, Kadomoto R, Asato K, Tanimura S, Itoh N, Kohno M.ERK pathway positively regulates the expression of Sprouty genes. Biochem Biophys Res Commun. 2001 285(5):1084-8.

13. Powers CJ, McLeskey SW, Wellstein A. Fibroblast growth factors, their receptors and signaling. Endocr Relat Cancer. 2000 7(3):165-97. Review.

14. Chodosh, L., Gardner, H.P., Rajan, J.V., Stairs, D.B., Marquis, S.T. and Leder,

P. A. Protein kinase expression during murine breast development. Dev. Biol. 2000 219, 259-276.

15. Srivastava S, Verma M, Henson DE. Biomarkers for early detection of colon cancer. Clin Cancer Res. 2001 7(5): 1118-26. Review

16. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000 100(l):57-70.

Review

17. van 't Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AA, Mao M, Peterse HL, van der Kooy K, Marton MJ, Witteveen AT, Schreiber GJ, Kerkhoven RM, Roberts C, Linsley PS, Bernards R, Friend SH. Gene expression profiling predicts clinical outcome of breast cancer. Nature. 2002 415(6871):530-6.

18. Polyak K. On the birth of breast cancer. Biochim Biophys Acta. 2001 1552(1):1-13. Review

19. Singapore Cancer Registry Report No. 5 "Cancer Incidence in Singapore,

1993-1997" published in the Yr 2000

20. Zajchowski D, Band V, Pauzie N, Tager A, Stampfer M, Sager R. Expression of growth factors and oncogenes in normal and tumour-derived human mammary epithelial cells. Cancer Res. 1988 15;48(24 Pt l):7041-7.

21. Schuuring E, Verhoeven E, Mooi WJ, Michalides RJ. Identification and cloning of two overexpressed genes, U21B31/PRAD1 and EMSl, within the amplified chromosome I lql3 region in human carcinomas. Oncogene. 1992 7(2):355-61.

22. Benchimol S, Pirn D, Crawford L. Radioimmunoassay of the cellular protein p53 in mouse and human cell lines. EMBO J. 1982 l(9):1055-62.

23. Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI, Puc J, Miliaresis C, Rodgers L, McCombie R, Bigner SH, Giovanella BC, Ittmann M, Tycko B, Hibshoosh H, Wigler MH, Parsons R. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science. 1997 28;275(5308): 1943-7.

24. Xie J, Johnson RL, Zhang X, Bare JW, Waldman FM, Cogen PH, Menon AG,

Warren RS, Chen LC, Scott MP, Epstein EH Jr. Mutations of the PATCHED gene in several types of sporadic extracutaneous tumors. Cancer Res. 1997 15;57(12):2369-72. 25. Su GH, Hilgers W, Shekher MC, Tang DJ, Yeo CJ, Hruban RH, Kern SE. Alterations in pancreatic, biliary, and breast carcinomas support MKK4 as a genetically targeted tumor suppressor gene. Cancer Res. 1998 l;58(l l):2339-42.

26. Krop IE, Sgroi D, Porter DA₅ Lunetta KL, LeVangie R, Seth P, Kaelin CM, Rhei E, Bosenberg M, Schnitt S, Marks JR, Pagon Z, Belina D, Razumovic J, Polyak K.

HIN-I, a putative cytokine highly expressed in normal but not cancerous mammary epithelial cells. Proc Natl Acad Sci U S A. 2001 98(17):9796-801.

27. Herman JG, Merlo A, Mao L, Lapidus RG, Issa JP, Davidson NE, Sidransky D, Baylin SB. Inactivation of the CDKN2/pl6/MTSl gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res. 1995 55(20):4525-30.

28. Graff JR, Herman JG, Lapidus RG, Chopra H, Xu R, Jarrard DF, Isaacs WB, Pitha PM, Davidson NE, Baylin SB. E-cadherin expression is silenced by DNA hypermethylation in human breast and prostate carcinomas. Cancer Res. 1995 55(22):5195-9.

29. Rodenhiser D, Chakraborty P, Andrews J, Ainsworth P, Mancini D, Lopes E, Singh S. Heterogenous point mutations in the BRCAl breast cancer susceptibility gene occur in high frequency at the site of homonucleotide tracts, short repeats and methylatable CpG/CpNpG motifs. Oncogene. 1996 12(12):2623-9.

30. Falette NS, Fuqua SA, Chamness GC, Cheah MS, Greene GL, McGuire WL.

Estrogen receptor gene methylation in human breast tumors. Cancer Res. 1990 50(13):3974-8.

31. Jhaveri MS, Morrow CS. Methylation-mediated regulation of the glutathione S-transferase Pl gene in human breast cancer cells. Gene. 1998 210(1): 1-7. 32. Huynh H, Alpert L, Pollak M. Silencing of the mammary-derived growth inhibitor (MDGI) gene in breast neoplasms is associated with epigenetic changes. Cancer Res. 1996 56(21):4865-70

33. Raman V, Martensen SA, Reisman D, Evron E, Odenwald WF, Jaffee E, Marks J, Sukumar S. Compromised HOXA5 function can limit p53 expression in human breast tumours. Nature. 2000 405(6789):974-8.

34. Ferguson AT, Evron E, Umbricht CB, Pandita TK, Chan TA, Hermeking H, Marks JR, Lambers AR, Futreal PA, Stampfer MR, Sukumar S. High frequency of hypermethylation at the 14-3-3 sigma locus leads to gene silencing in breast cancer. Proc Natl Acad Sci U S A. 2000 97(11):6049-54

35. Davis CD, Snyderwine EG. Analysis of EGFR, TGF-alpha, neu and c-myc in 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine-induced mammary tumors using RT- PCR. Carcinogenesis. 1995 12:3087-92

36. Sarkar FH, Visscher DW, Crissman JD. Quantitative analysis of Her-2/neu (ERBB2) gene expression using reverse transcriptase polymerase chain reaction.

Diagn MoI Pathol. 1993 3:210-8.

37. Sasa M, Kondo K, Komaki K, Morimoto T, Monden Y. p53 alteration correlates with negative ER, negative PgR, and high histologic grade in breast cancer. J Surg Oncol. 1994 1:46-50.

38. Luppi M, Morselli M, Bandieri E, Federico M, Marasca R, Barozzi P, Ferrari

MG, Savarino M, Frassoldati A, Torelli G. Sensitive detection of circulating breast cancer cells by reverse-transcriptase polymerase chain reaction of maspin gene. Ann. Oncol. 1996 6:619-24.

Al. Haviv YS, Curiel DT. Conditional gene targeting for cancer gene therapy, Advanced Drug Delivery Reviews 53 (2001) 135-154. A2. Xie B et al Co-expression of vascular endothelial growth factor (VEGF) and its receptors (flk-1 and flt-1) in a hormone-induced mammary cancer in the Noble rat, Br. J Cancer. 81 (1999) 1335-1343.

A3. Harris JD et al. Gene therapy for cancer using tumour-specific prodrug activation, Gene Ther. 1 (1994) 170-175.

A4. Chung I et al. Use of L-plastin promoter to develop an adenoviral system that confers transgene expression in ovarian cancer cells but not in normal mesothelial cells, Cancer Gene Ther. 6 (1999) 99-106.

A5. Dong L et al. Mechanisms of transcriptional activation oϊbcl-2 gene expression by 17β-estradiol in breast cancer cells, J Biol. Chem. 274 (1999) 32099-32107.

A6. Pandha HS et al. Genetic prodrug activation therapy for breast cancer: a phase I clinical trial of erbB-2 directed suicide gene expression, J Clin. Oncol. 17 (1999) 2180- 2189.

A7. Kurihara T et al. Selectivity of a replication-competent adenovirus for human breast carcinoma cells expressing the MUCl antigen, J Clin. Invest. 106 (2000) 763-771.

A8. Anderson LM et al. Breast cancer specific expression of the Candida albicans cytosine deaminase gene using a transcriptional targeting approach, Cancer Gene Ther. 7 (2000) 845-852.

A9. Gunzberg WH et al. Factors controlling the expression of mouse mammary tumour virus, Biochem. J. 283 (1992) 625-632.

AlO. Braiden V et al. Eradication of breast cancer xenografts by hyperthermic suicide gene therapy under the control of the heat shock protein promoter, Hum Gene Ther. 11 (2000) 2453-2463. All. Walther W et al. Mdrl promoter-driven tumor necrosis factor-alpha expression for a chemotherapy-controllable combined in vivo gene therapy and chemotherapy of tumors, Cancer Gene Titer. 7 (2000) 893-900.

A12. Stewart AK et al. Adenovector-mediated gene delivery of interleukin-2 in metastatic breast cancer and melanoma: results of a phase I clinical trial. Gene Ther. 6 (1999) 350-363.

A13. Dummer R et al. Biological activity and safety of adenoviral vector- expressed wild-type p53 after intratumoral injection in melanoma and breast cancer patients with p53-overexpressing tumours. Cancer Gene Ther. 1 (2000) 1069-1076.

A14. Yoo GH et al. Phase I trial of intratumoral liposome ElA Gene therapy in patients with recurrent breast and head and neck cancer, Clinical Cancer Research 1 (2001) 1237-1245.

A15. Hortobagyi GN et al. Cationic liposome-mediated EIaA gene transfer to human breast and ovarian cancer cells and its biologic effects: a phase I clinical trial, J Clin. Oncol. 19 (2001) 3422-3433.

B 1. Pawson T, Saxton TM. Signaling networks-do all roads lead to the same genes? Cell 1999; 97: 675-8.

B2. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 2000; 103: 211-25.

B3. Pearson G, Robinson F, Beers Gibson T et al. Mitogen-activated protein

(MAP) kinase pathways: regulation and physiological functions. Endocr Rev 2001; 22: 153-83.

B4. Pouyssegur J, Volmat V, Lenormand P. Fidelity and spatio-temporal control in MAP kinase (ERKs) signalling. Biochem Pharmacol 2002; 64: 755-63. B5. Blume- Jensen P, Hunter T. Oncogenic kinase signalling. Nature 2001; 11: 355-65.

B6. Martin GS. Cell signaling and cancer. Cancer Cell 2003; 4: 167-74.

B7. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100: 57-70.

B8. Downward J. Targeting RAS signalling pathways in cancer therapy. Nat

Rev Cancer 2003; 3: 11-22.

B9. Hacohen Ν, Kramer S, Sutherland D, Hiromi Y, Krasnow MA. Sprouty encodes a novel antagonist of FGF signaling that patterns apical branching of the Drosophila airways. Cell 1998; 92: 253-63.

BlO. Casci T, Vinos J, Freeman M. Sprouty, an intracellular inhibitor of Ras signaling. Cell 1999; 96: 655-65.

BI l. Reich A, Sapir A, Shilo B. Sprouty is a general inhibitor of receptor tyrosine kinase signaling. Development 1999; 126: 4139-47.

B12. Kramer S, Okabe M, Hacohen Ν, Krasnow MA, Hiromi Y. Sprouty: a common antagonist of FGF and EGF signaling pathways in Drosophila. Development 1999; 126: 2515-25.

B 13. de Maximy AA, Νakatake Y, Moncada S, Itoh Ν, Thiery JP, Bellusci S . Cloning and expression pattern of a mouse homologue of drosophila sprouty in the mouse embryo. Mech Dev 1999; 81: 213-6.

B 14. Tefft JD, Lee M, Smith S et al. Conserved function of mSpry-2, a murine homolog of Drosophila sprouty, which negatively modulates respiratory organogenesis. Curr Biol 1999; 9: 219-22. B 15. Minowada G, Jarvis LA, Chi CL et al Vertebrate Sprouty genes are induced by FGF signaling and can cause chondrodysplasia when overexpressed. Development 1999; 126: 4465-75.

B 16. Chambers D, Mason I. Expression of sprouty2 during early development of the chick embryo is coincident with known sites of FGF signalling. Mech Dev 2000; 91: 361-4.

B 17. Chambers D, Medhurst AD, Walsh FS, Price J, Mason I. Differential display of genes expressed at the midbrain - hindbrain junction identifies sprouty2: an FGF8-inducible member of a family of intracellular FGF antagonists. MoI Cell Neurosci 2000; 15: 22-35.

B 18. Mailleux AA, Tefft D, Ndiaye D et al. Evidence that SPROUTY2 functions as an inhibitor of mouse embryonic lung growth and morphogenesis. Mech Dev 2001; 102: 81-94.

B 19. Perl AK, Hokuto I, Impagnatiello MA, Christofori G, Whitsett JA. Temporal effects of Sprouty on lung morphogenesis. Dev Biol 2003; 258, 154-68.

B20. Lee SH, Schloss DJ, Jarvis L, Krasnow MA, Swain JL. Inhibition of angiogenesis by a mouse sprouty protein. J Biol Chem 2001; 276: 4128-33.

B21. Impagnatiello MA, Weitzer S, Gannon G, Compagni A, Cotton M, Christofori G. Mammalian sprouty- 1 and -2 are membrane-anchored phosphoprotein inhibitors of growth factor signaling in endothelial cells. J Cell Biol 2001; 152: 1087-98.

B22. Gross I, Bassit B, Benezra M, Licht JD. Mammalian sprouty proteins inhibit cell growth and differentiation by preventing ras activation. J Biol Chem 2001; 276: 46460-8. B23. Sasaki A, Taketomi T, Wakioka T, Kato R, Yoshimura A. Identification of a dominant negative mutant of Sprouty that potentiates fibroblast growth factor- but not epidermal growth factor-induced ERK activation. J Biol Chem 2001; 276: 36804-8.

B24. Guy GR, Wong ES, Yusoff P et al. Sprouty: how does the branch manager work? JCe// SW 2003; 116: 3061-8.

B25. Tefft D, Lee M, Smith S, Crowe DL, Bellusci S, Warburton D. mSprouty2 inhibits FGFlO-activated MAP kinase by differentially binding to upstream target proteins. Am J Physiol Lung Cell MoI Physiol 2002; 283: L700-6.

B26. Lim J, Yusoff P, Wong ES et al. The cysteine-rich sprouty translocation domain targets mitogen-activated protein kinase inhibitory proteins to phosphatidylinositol 4,5-bisphosphate in plasma membranes. MoI Cell Biol 2002; 22: 7953-66.

B27. Hanafusa H, Torii S, Yasunaga T, Nishida E. Sproutyl and Sprouty2 provide a control mechanism for the Ras/MAPK signaling pathway. Nat Cell Biol 2002; 4: 850-8.

B28. Fong CW, Leong HF, Wong ES, Lim J, Yusoff P, Guy GR. Tyrosine phosphorylation of Sprouty2 enhances its interaction with c-Cbl and is crucial for its function. J Biol Chem 2003; 278: 33456-64.

B29. Ramaswamy S, Tamayo P, Rifkin R et al. Multiclass cancer diagnosis using tumor gene expression signatures. Proc Natl Acad Sd USA 2001; 98: 15149-54.

B30. Yusoff P, Lao DH, Ong SH et al. Sprouty2 inhibits the Ras/MAP kinase pathway by inhibiting the activation of Raf. J Biol Chem 2002; 277: 3195-201.

B31. Morrison RS, Yamaguchi F, Saya H et al. Basic fibroblast growth factor and fibroblast growth factor receptor I are implicated in the growth of human astrocytomas. JNeurooncol 1994; 18: 207-16 B32. Lappi DA. Tumor targeting through fibroblast growth factor receptors. Semin Cancer Biol 1995; 6: 279-88.

B33. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER- 2/neu oncogene. Science 1987, 235: 177-82.

B34. Maass N, Teffner M, Rosel F et al. Decline in the expression of the serine proteinase inhibitor Maspin is associated with tumour progression in ductal carcinomas of the breast. J Pathol 2001; 195: 321-6.

B35. Umekita Y, Ohi Y, Sagara Y, Yoshida H. Expression of maspin predicts poor prognosis in breast cancer patients. Int J Cancer 2002; 100: 452-5.

B36. Wiseman BS, Werb Z. Stromal effects on mammary gland development and breast cancer. Science 2002; 296: 1046-9.

B37. Smalley M, Ashworth A. Stem cells and breast cancer: A field in transit. Nat Rev Cancer 2003; 3: 832-44.

B38. Marsh SK, Bansal GS, Zammit C et al. Increased expression of fibroblast growth factor 8 in human breast cancer. Oncogene 1999; 18: 1053-60.

B39. Robertson KD. DNA methylation and chromatin - unraveling the tangled web. Oncogene 2002; 21: 5361-79.

B40. Rountree MR, Bachman KE, Herman JG, Baylin SB. DNA methylation, chromatin inheritance, and cancer. Oncogene 2001; 20: 3156-65.

B41. Maass N, Biallek M, Rosel F et al. Hypermethylation and histone deacetylation lead to silencing of the maspin gene in human breast cancer. Biochem Biophys Res Commun 2002; 297: 125-8. B42. Gross I, Morrison DJ, Hyink DP et al. The receptor tyrosine kinase regulator Sproutyl is a target of the tumor suppressor WTl and important for kidney development. J Biol Chem 2003; 278: 41420-30.

B43. Loeb DM, Evron E, Patel CB et al. Wilms' tumor suppressor gene (WTl) is expressed in primary breast tumors despite tumor-specific promoter methylation. Cancer Res 2001; 61: 921-5.

B44. Cook WD, McCaw BJ. Accommodating haploinsufficient tumor suppressor genes in Knudson's model. Oncogene 2000; 19: 3434-8.

Each of the applications and patents mentioned in this document, and each document cited or referenced in each of the above applications and patents, including during the prosecution of each of the applications and patents ("application cited documents") and any manufacturer's instructions or catalogues for any products cited or mentioned in each of the applications and patents and in any of the application cited documents, are hereby incorporated herein by reference. Furthermore, all documents cited in this text, and all documents cited or referenced in documents cited in this text, and any manufacturer's instructions or catalogues for any products cited or mentioned in this text, are hereby incorporated herein by reference.

Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the claims. SEQUENCE LISTING

SEQIDNO: 1

HomosapiensSproutylN-terminalcDNAsequence(derivedfromGenBank AccessionNumberBC063856) tgcagagggcgcagctccaccgtggtaacttgcaatgtggccgcccctgcgctggcttctttctcc tcggccctcggtgaacccagcttttcctttaaagcatagacaaatccttgcttagggtcagccaga ccgtgggcttgctttgggctcccgcgtggacgctgaaggctcttcatgatttctcagaaaccttgg tgggccacccaaaaagtgtgttggaaatccacggtgatccttaatatggtgatgggattgtccgaa aaggatttcagatgcatgccaggtttccactgattgccagaactcgagatcactacacatggatcc ccaaaatcaacatggcagtggcagttcgttagttgtgatccagcagccttctttggatagccgtca gagattagactatgagagagagattcagcctactgctattttgtccttagaccagatcaaggccat aagaggcagcaatgaatacacagaagggccttcggtggtgaaaagacctgctcctcggacagcacc aagacaagaaaa

SEQIDNO:2 Homo sapiens Sproutyl N-terminal protein sequence (derived from GenBank

Accession Number BC063856)

MDPQNQHGSGSSLVVIQQPSLDSRQRLDYEREIQPTAILSLDQIKAIRGSNEYTEGPSVVKRPAPR TAPRQE

SEQ ID NO: 3

Homo sapiens Sproutyl Full Length cDNA sequence (GenBank Accession Number BC063856) tgcagagggc gcagctccac cgtggtaact tgcaatgtgg ccgcccctgc gctggcttct ttctcctcgg ccctcggtga acccagcttt tcctttaaag catagacaaa tccttgctta gggtcagcca gaccgtgggc ttgctttggg ctcccgcgtg gacgctgaag gctcttcatg atttctcaga aaccttggtg ggccacccaa aaagtgtgtt ggaaatccac ggtgatcctt aatatggtga tgggattgtc cgaaaaggat ttcagatgca tgccaggttt ccactgattg ccagaactcg agatcactac acatggatcc ccaaaatcaa catggcagtg gcagttcgtt agttgtgatc cagcagcctt ctttggatag ccgtcagaga ttagactatg agagagagat tcagcctact gctattttgt ccttagacca gatcaaggcc ataagaggca gcaatgaata cacagaaggg ccttcggtgg tgaaaagacc tgctcctcgg acagcaccaa gacaagaaaa gcatgaaagg actcatgaaa tcataccaat taatgtgaat aataactacg agcacagaca cacaagccac ctgggacatg cagtactccc aagtaatgcc aggggcccca ttttgagcag atcaaccagc actggaagtg cagccagctc tgggagcaac agcagtgcct cttctgaaca gggactgtta ggaaggtcac caccaaccag accagtccct ggtcataggt ctgaaagggc aatccggacc cagcccaagc aactgattgt ggatgacttg aagggttcct tgaaagagga cctgacacag cacaagttca tttgtgaaca gtgtgggaag tgcaagtgtg gagaatgcac tgctcccagg accctaccat cctgtttggc ctgtaaccgg cagtgccttt gctctgctga gagcatggtg gaatatggaa cctgcatgtg cttagtcaag ggcatcttct accactgctc caatgacgac gaaggggatt cctattcaga taatccttgc tcctgttcac aatcacactg ctgctctaga tacctgtgta tgggagccat gtctttattt ttaccttgct tactctgtta tcctcctgct aaaggatgcc tgaagctgtg caggaggtgt tatgactgga tccatcgccc agggtgcaga tgtaagaact ccaacactgt ctattgtaag ctggagagct gcccctcccg gggtcagggt aaaccatcat gatttttgga ggtgggttgt acctcctgaa cttttagctt tcaagttgtg gctgtttttt gtttttgttt ttgtttttgt tttctttaga atttttccct gtttcccacc ttctcttccc ctgttgccaa ggtctaactc atggattttt ctctttcctc atggatgatc ttcagcaaga gtggactggg aagctgcacc tggctcccac tttcaacaag agcctctgcc atccacttga gggtattgag agccagtggg cttttgtgta gcctttttgt tctgcaagca actttctaaa gttgtgtaca tgaacataca cccacatcca gactacagtg atttagagtt gttttgattg ggtaccgtgg gagcagggaa attggttttt taaaaagcaa ctgtttaatt gcttaaataa gctatgtatt aaatctgtct ccagttaggg ctatcttcct agcataggcc ccttaagtag catgggggat atattttttg ctataacgta aaaattttcc tttaaccact gccctctcct tctttctcct tcaaggttct ttccccctca gttttgttgt tgtcttactc tggagatgcc aagtgtattt tttctttcta tgtaatttta gattcgcctt acaatgtaaa tcttcacatt ggagataata ttggttggac cttgcccatc ttcactctag ccttcgtatt tgtgaaggac tcagccacct tccttcttca ccccatgctt ctcaccaaat ttttgttgtc attgagggca cttggataac tcaagttgat atttatagct gatcaatcta tatgtgtcac agaactatgc tgcctaaagt gatcttggct ccttaatggt ccttttggcc ccttggatag ttaacagctg agtaattcta atctcttctg tgttttcctt gccttaacca caaattgtgg tgctttttgt atattttatg tataaatcac aaagttgaat tctgactatt tttaagacaa aagtctgtta aactttttta ttgtaaagaa tatttattat gcgaatctct attattttat ggtatttatt gcaaaagact gttgaaatgt actcatgttt gaatataaca aaatatcaat acttaacgga aaataaggtg acacgaagaa agtacatatg ttaactataa tgcagaaaat atattaatta atgaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa

SEQIDNO: 4

HomosapiensSproutylFullLengthproteinsequence(GenBankAccession NumberBC063856)

MDPQNQHGSGSSLVVIQQPSLDSRQRLDYEREIQPTAILSLDQIKAIRGSNEYTEGPSVVKRPAPR TAPRQEKHERTHEIIPINVNNNYEHRHTSHLGHAVLPSNARGPILSRSTSTGSAASSGSNSSASSE QGLLGRSPPTRPVPGHRSERAIRTQPKQLIVDDLKGSLKEDLTQHKFICEQCGKCKCGECTAPRTL PSCLACNRQCLCSAESMVEYGTCMCLVKGIFYHCSNDDEGDSYSDNPCSCSQSHCCSRYLCMGAMS LFLPCLLCYPPA KGCLKLCRRCYDWIHRPGCRCKNSNTVYCKLESCPSRGQGKPS

SEQIDNO:5 HomosapiensSprouty2N-terminalcDNAsequence atggaggccagagctcagagtggcaacgggtcgcagcccttgctgcagacgccccgtgacggtggc agacagcgtggggagcccgaccccagagacgccctcacccagcaggtacatgtcttgtctctggat cagatcagagccatccgaaacaccaatgagtacacagaggggcctactgtcgtcccaagacctggg ctcaagcctgctcctcgcccctccactcagcacaaacacgagagactccacggtctgcctgagcac cgccagcctcctaggctccagcactcgcaggtccattcttctgcacgagcccctctgtccagatcc ataagcacggtcagctcagggtcgcggagcagtacgaggacaagtaccagcagcagctcctctgaa cagagactgctaggatcatccttctcctccgggcctgttgctgatggcataatccgggtgcaaccc aaatctgagctcaagccaggtgagcttaagccactgagcaaggaagatttgggcctgcacgcctac agg SEQ ID NO: 6

Homo sapiens Sprouty2 N-terminal protein sequence

MEARAQSGNGSQPLLQTPRDGGRQRGEPDPRDALTQQVHVLSLDQIRAIRNTNEYTEGPTVVPRPG LKPAPRPSTQHKHERLHGLPEHRQPPRLQHSQVHSSARAPLSRSISTVSSGSRSSTRTSTSSSSSE QRLLGSSFSSGPVADGIIRVQPKSELKPGELKPLSKEDLGLHAYR

SEQIDNO: 7

HomosapiensSprouty2FullLengthcDNAsequence atggaggccagagctcagagtggcaacgggtcgcagcccttgctgcagacgccccgtgacggtggc agacagcgtggggagcccgaccccagagacgccctcacccagcaggtacatgtcttgtctctggat cagatcagagccatccgaaacaccaatgagtacacagaggggcctactgtcgtcccaagacctggg ctcaagcctgctcctcgcccctccactcagcacaaacacgagagactccacggtctgcctgagcac cgccagcctcctaggctccagcactcgcaggtccattcttctgcacgagcccctctgtccagatcc ataagcacggtcagctcagggtcgcggagcagtacgaggacaagtaccagcagcagctcctctgaa cagagactgctaggatcatccttctcctccgggcctgttgctgatggcataatccgggtgcaaccc aaatctgagctcaagccaggtgagcttaagccactgagcaaggaagatttgggcctgcacgcctac aggtgtgaggactgtggcaagtgcaaatgtaaggagtgcacctacccaaggcctctgccatcagac tggatctgcgacaagcagtgcctttgctcggcccagaacgtgattgactatgggacttgtgtatgc tgtgtgaaaggtctcttctatcactgttctaatgatgatgaggacaactgtgctgacaacccatgt tcttgcagccagtctcactgttgtacacgatggtcagccatgggtgtcatgtccctctttttgcct tgtttatggtgttaccttccagccaagggttgccttaaattgtgccaggggtgttatgaccgggtt aacaggcctggttgccgctgtaaaaactcaaacacagtttgctgcaaagttcccactgtcccccct aggaactttgaaaaaccaacatag

SEQIDNO: 8

Homo sapiens Sprouty2 Full Length protein sequence MEARAQSGNGSQPLLQTPRDGGRQRGEPDPRDALTQQVHVLSLDQIRAIRNTNEYTEGPTVVPRPG LKPAPRPSTQHKHERLHGLPEHRQPPRLQHSQVHSSARAPLSRSISTVSSGSRSSTRTSTSSSSSE QRLLGSSFSSGPVADGIIRVQPKSELKPGELKPLSKEDLGLHAYRCEDCGKCKCKECTYPRPLPSD WICDKQCLCSAQNVIDYGTCVCCVKGLFYHCSNDDEDNCADNPCSCSQSHCCTRWSAMGVMSLFLP CLWCYLPAKGCLKLCQGCYDRVNRPGCRCKNSNTVCCKVPTVPPRNFEKPT

OTHER SPROUTYI NUCLEIC ACID SEQUENCES

Each of the following sequences may be used in addition to, or in place of, a SproutyI sequence in the methods and compositions as described.

BB202558 full-length enriched, 0 day neonate thymus Mus musculus cDNA clone A430046P07 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; NT_039228 Mus musculus chromosome 3 genomic contig, strain C57BL/6J BB546290 full-length enriched, 0 day neonate eyeball Mus musculus cDNA clone E130309F14 3' similar to AF176903 Mus rausculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB 120991 full-length enriched, adult male urinary bladder Mus musculus cDNA clone 9530080A14 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB 107270 full-length enriched, adult male urinary bladder Mus musculus cDNA clone 9530005FQ2 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB104138 full-length enriched, 12 days embryo, embryonic body between diaphragm region and neck Mus musculus cDNA clone 9430090G21 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB469926 full-length enriched, 12 days embryo eyeball Mus musculus cDNA clone D230030A21 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB389912 full-length enriched, 0 day neonate cerebellum Mus musculus cDNA clone C230066A10 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB381417 full-length enriched, 0 day neonate cerebellum Mus musculus cDNA clone C230012L16 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB379645 full-length enriched, 16 days embryo head Mus musculus cDNA clone C130099O05 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB373428 full-length enriched, 16 days embryo head Mus musculus cDNA clone C130070F03 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB352693 full-length enriched, 10 days neonate cerebellum Mus musculus cDNA clone B930090F24 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB351533 full-length enriched, 10 days neonate cerebellum Mus musculus cDNA clone B930085B20 3' similar to AFl 76903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB347056 full- length enriched, 10 days neonate cerebellum Mus musculus cDNA clone B930064A11 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence;

BB346111 full-length enriched, 10 days neonate cerebellum Mus musculus cDNA clone B930060D12 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB343333 full-length enriched, 10 days neonate cerebellum Mus musculus cDNA clone B930044E21 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB343101 full-length enriched, 10 days neonate cerebellum Mus musculus cDNA clone B930042F09 3' similar to AFl 76903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB342645 full-length enriched, 10 days neonate cerebellum Mus musculus cDNA clone B930038L01 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB341612 full-length enriched, 10 days neonate cerebellum Mus musculus cDNA clone B930032P07 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB340059 full-length enriched, 10 days neonate cerebellum Mus musculus cDNA clone B930022C24 3' similar to AFl 76903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB334839 full- length enriched, 10 days neonate medulla oblongata Mus musculus cDNA clone B830028E18 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB334705 full-length enriched, 10 days neonate medulla oblongata Mus musculus cDNA clone B830026C20 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB334693 full-length enriched, 10 days neonate medulla oblongata Mus musculus cDNA clone B830025P11 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB334590 full-length enriched, 10 days neonate medulla oblongata Mus musculus cDNA clone B830024H02 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB334501 full- length enriched, 10 days neonate medulla oblongata Mus musculus cDNA clone B830023A08 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB334314 full-length enriched, 10 days neonate medulla oblongata Mus musculus cDNA clone B830020N23 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB332686 full-length enriched, 6 days neonate medulla oblongata Mus musculus cDNA clone B730050N22 3' similar to AFl 76903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB332651 full-length enriched, 6 days neonate medulla oblongata Mus musculus cDNA clone B730050H08 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB332289 full- length enriched, 6 days neonate medulla oblongata Mus musculus cDNA clone

B730046C01 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB332087 full-length enriched, 6 days neonate medulla oblongata Mus musculus cDNA clone B730043M24 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB332049 full-length enriched, 6 days neonate medulla oblongata Mus musculus cDNA clone B730043I09 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB332027 full-length enriched, 6 days neonate medulla oblongata Mus musculus cDNA clone B730043F14 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB332005 full- length enriched, 6 days neonate medulla oblongata Mus musculus cDNA clone B730043B22 3' similar to AFl 76903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB331656 full-length enriched, 6 days neonate medulla oblongata Mus musculus cDNA clone B730037K02 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB330589 full-length enriched, 6 days neonate medulla oblongata Mus musculus cDNA clone B730016B07 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB330089 full-length enriched, 6 days neonate medulla oblongata Mus musculus cDNA clone B730007B06 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB329890 full- length enriched, 6 days neonate medulla oblongata Mus musculus cDNA clone B730002I17 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB329876 full-length enriched, 6 days neonate medulla oblongata Mus musculus cDNA clone B730002G06 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB329789 full-length enriched, 4 days neonate thymus Mus musculus cDNA clone B630019M14 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB329661 full-length enriched, 4 days neonate thymus Mus musculus cDNA clone B630016K11 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB329622 full-length enriched, 4 days neonate thymus Mus musculus cDNA clone B630016A09 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB329592 full-length enriched, 4 days neonate thymus Mus musculus cDNA clone B630013C03 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB329392 full-length enriched, 4 days neonate thymus Mus musculus cDNA clone B630006P04 3' similar to AFl 76903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB329347 full- length enriched, 4 days neonate thymus Mus musculus cDNA clone B630006I20 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB329340 full- length enriched, 4 days neonate thymus Mus musculus cDNA clone B630006H24 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB329299 full-length enriched, 4 days neonate thymus Mus musculus cDNA clone B630006D18 3¹ similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB329267 full-length enriched, 4 days neonate thymus Mus musculus cDNA clone B630006A03 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB329017 full-length enriched, adult male cortex Mus musculus cDNA clone B530005O22 3' similar to AFl 76903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB328936 full-length enriched, adult male cortex Mus musculus cDNA clone B530005A12 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA,

MRNA sequence; BB328848 full-length enriched, adult male cortex Mus musculus cDNA clone B530003B12 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB328698 full-length enriched, adult male cortex Mus musculus cDNA clone B530001D18 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB328625 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430320F21 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB328605 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430320D02 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB327929 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430304K15 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB327788 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430302I23 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB326818 full- length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430212H21 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence;

BB326744 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430212A19 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB326721 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430211N18 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB326630 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430210P18 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB326485 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430210A21 3¹ similar to AF 176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB326472 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430209O12 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB326145 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430204K09 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB326123 full- length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430204E07 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB326069 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430203H14 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB325712 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430119E23 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB325598 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430118G04 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB325548 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430117N15 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB325508 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430117109 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB325473 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430117D13 3' similar to AFl 76903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB325465 full- length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430117C16 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB325068 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430113K22 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB325049 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430113122 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB325011 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430113F06 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB324867 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430112E23 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB324857 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430112D17 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB324843 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430112B03 3' similar to AFl 76903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB324835 full- length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430111P21 3' similar to AFl 76903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB324818 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430111O05 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB324621 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430110E08 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB324575 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430109M01 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB324521 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430109F02 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB324459 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430108N04 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB324384 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430107O19 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB324359 full- length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430107K03 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB 324243 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430105M15 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB324220 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430105J23 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB324152 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430104P16 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB324095 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430104E13 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB323991 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430102K05 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB323715 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430011D16 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB323590 full- length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430009I22 3' similar to AFl 76903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB323449 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430006A01 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB323355 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430003G04 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB323259 full-length enriched, 4 days neonate male adipose Mus musculus cDNA clone B430001I05 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB323100 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330033I03 3^r similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB323092 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330033H12 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB323014 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330021K16 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB323002 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330021J11 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB323000 full- length enriched, adult male adrenal gland Mus musculus cDNA clone B330021J03 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB322859 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330020A06 3¹ similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB322836 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330019N14 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA

MRNA sequence; BB322823 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330019L23 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB322694 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330018B21 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB322693 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330018B12 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB322690 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330018A20 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB322655 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330017L24 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB322595 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330017C11 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB322472 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330016A06 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB322467 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330015P16 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB322371 full- length enriched, adult male adrenal gland Mus musculus cDNA clone B330015B20 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB322359 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330015A03 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB322335 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330014L12 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB322326 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330014J23 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB322319 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330014I24 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB322189 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330013F07 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB322148 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330012P11 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB322061 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330007I01 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB322016 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330006I17 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB321990 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330005P16 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB321973 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330005K19 3¹ similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB321869 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330003K11 3^r similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB321847 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330003E20 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB321762 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330002B20 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB321758 full-length enriched, adult male adrenal gland Mus musculus cDNA clone B330002B08 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB321741 full- length enriched, adult male adrenal gland Mus musculus cDNA clone B330001D24 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB321189 full-length enriched, adult male corpora quadrigemina Mus musculus cDNA clone B230397G15 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB319833 full-length enriched, adult male corpora quadrigemina Mus musculus cDNA clone B230382N09 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB318370 full-length enriched, adult male corpora quadrigemina Mus musculus cDNA clone B230374J01 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB313843 full-length enriched, adult male corpora quadrigemina Mus musculus cDNA clone B230343P21 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB313702 full- length enriched, adult male corpora quadrigemina Mus musculus cDNA clone B230343F15 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB310680 full-length enriched, adult male corpora quadrigemina Mus musculus cDNA clone B230316M06 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB299664 full-length enriched, adult male corpora quadrigemina Mus musculus cDNA clone B230009O15 3¹ similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB298094 full-length enriched, 9.5 days embryo parthenogenote Mus musculus cDNA clone B130065J05 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB293564 full- length enriched, 9.5 days embryo parthenogenote Mus musculus cDNA clone

B130019G11 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB274266 full-length enriched, 10 days neonate cortex Mus musculus cDNA clone A830087F04 3' similar to AFl 76903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB265703 full-length enriched, 10 days neonate cortex Mus musculus cDNA clone A830021L19 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB260058 full-length enriched, 7 days neonate cerebellum Mus musculus cDNA clone A730092M14 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB254094 full-length enriched, 7 days neonate cerebellum Mus musculus cDNA clone A730060G13 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB250328 full-length enriched, 7 days neonate cerebellum Mus musculus cDNA clone A730040J04 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB248601 full-length enriched, 7 days neonate cerebellum Mus musculus cDNA clone A730027A11 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB246543 full- length enriched, 7 days neonate cerebellum Mus musculus cDNA clone A730014O17 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB 246151 full-length enriched, 7 days neonate cerebellum Mus musculus cDNA clone A730013G09 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB232653 full-length enriched, 3 days neonate thymus Mus musculus cDNA clone A630040B11 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB232523 full-length enriched, 3 days neonate thymus Mus musculus cDNA clone A630039G03 3' similar to AF 176903 Mus musculus sprouty 1

(Spryl)mRNA MRNA sequence; BB231511 full-length enriched, 3 days neonate thymus Mus musculus cDNA clone A630032O06 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB229656 full-length enriched, 3 days neonate thymus Mus musculus cDNA clone A630022P21 3' similar to AFl 76903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB226745 full-length enriched, adult male aorta and vein Mus musculus cDNA clone A530098G02 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB221101 full-length enriched, adult male aorta and vein Mus musculus cDNA clone A530064D14 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB214825 full-length enriched, adult male aorta and vein Mus musculus cDNA clone A530023C18 3' similar to

AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB213935 full- length enriched, adult male aorta and vein Mus musculus cDNA clone A530015P16 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB211154 full-length enriched, 0 day neonate thymus Mus musculus cDNA clone A430103I12 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB209787 full-length enriched, 0 day neonate thymus Mus musculus cDNA clone A430094D15 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB208963 full-length enriched, 0 day neonate thymus Mus musculus cDNA clone A430090F24 3^? similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB202225 full-length enriched, 0 day neonate thymus Mus musculus cDNA clone A430045D16 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB202195 full-length enriched, 0 day neonate thymus Mus musculus cDNA clone A430045B08 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB 197996 full-length enriched, adult male spinal cord Mus musculus cDNA clone A330105K08 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB 183769 full-length enriched, adult male hypothalamus Mus musculus cDNA clone A230109O21 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB 183288 full-length enriched, adult male hypothalamus Mus musculus cDNA clone A230106B09 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB182507 full- length enriched, adult male hypothalamus Mus musculus cDNA clone A230098M17 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence;

BB 180925 full-length enriched, adult male hypothalamus Mus musculus cDNA clone A230087P03 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB 178521 full-length enriched, adult male hypothalamus Mus musculus cDNA clone A230074F21 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB 172227 full-length enriched, adult male hypothalamus Mus musculus cDNA clone A230036106 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB172226 full-length enriched, adult male hypothalamus Mus musculus cDNA clone A230036I04 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB151225 full-length enriched, 6 days neonate skin Mus musculus cDNA clone A030011P14 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB 135489 full-length enriched, adult male bone Mus musculus cDNA clone 9830126L12 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB128941 full-length enriched, 16 days neonate cerebellum Mus musculus cDNA clone 9630035111 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB095277 full-length enriched, 12 days embryo, embryonic body between diaphragm region and neck Mus musculus cDNA clone 9430050G12 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB084824 full-length enriched, adult male diencephalon Mus musculus cDNA clone 9330195P17 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB065333 full-length enriched, 15 days embryo male testis Mus musculus cDNA clone 8030443M03 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB054911 full-length enriched, 12 days embryo male wolffian duct Mus musculus cDNA clone 6720474K08 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB051942 full-length enriched, 12 days embryo male wolffian duct Mus musculus cDNA clone 6720436L08 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB049750 full-length enriched, adult male cerebellum Mus musculus cDNA clone 6530418J19 3' similar to AF176903 Mus musculus sprouty 1 (Spryl)mRNA MRNA sequence; BB043367 full-length enriched, 13 days embryo male testis Mus musculus cDNA clone 6030473A10 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB028489 full-length enriched, 6 days neonate head Mus musculus cDNA clone 5430407H22 3' similar to AF 176903 Mus musculus sprouty 1 (Spryl), MRNA sequence; BB026458 full-length enriched, adult male pituitary gland Mus musculus cDNA clone 5330433E13 3' similar to AF176903 Mus musculus sprouty 1 (Spryl) mRNA, MRNA sequence; BB008474 full-length enriched, 10 day neonate skin Mus musculus cDNA clone 4732482019 3' similar to NM_011896 Mus musculus sprouty homolog 1 (Drosophila) (Spryl), MRNA sequence; BB006108 full-length enriched, 10 day neonate skin Mus musculus cDNA clone 4732469009 3' similar to NM_011896 Mus musculus sprouty homolog 1 (Drosophila) (Spryl), MRNA sequence; BB001702 full- length enriched, 0 day neonate skin Mus musculus cDNA clone 4631407H20 3' similar to NM_011896 Mus musculus sprouty homolog 1 (Drosophila) (Spryl), MRNA sequence.

Claims

1. A method of diagnosis of a cancer in an individual, the method comprising detecting modulation of expression of a Sproutyl sequence in the individual, or any part of the individual.

2. A method according to Claim 1, in which the cancer comprises breast cancer.

3. A method according to Claim 1 or 2, in which the method comprises detecting down-regulation of Sproutyl expression in a breast cell or tissue of or from the individual.

4. A method according to any preceding claim, in which a Sproutyl nucleic acid is detected by means of a probe comprising at least a portion of a nucleic acid having the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

5. A method according to any preceding claim, which comprises detecting a Sproutyl polypeptide, preferably by means of an antibody to Sproutyl, in a sample comprising a breast cell or tissue from the individual.

6. A method according to any preceding claim, in which the expression of Sproutyl in the sample is compared to the expression of Sproutyl in a control breast cell known to be non-cancerous.

7. A method according to Claim 6, in which a down-regulation of Sproutyl expression in the sample compared to the control breast cell is diagnostic of breast cancer, or susceptibility to breast cancer.

8. A method for identifying a pre-cancerous breast cell, comprising detecting a reduced level of a Sproutyl polypeptide and/or a Sproutyl nucleic acid in the cell, or an extract thereof.

9. A method according to any preceding claim, which further comprises detection of modulation, preferably down-regulation, of expression of a Sprouty2 sequence, preferably by means of a probe comprising at least a portion of a nucleic acid having the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 7, or an antibody to Sprouty2.

10. A specific binding agent for Sproutyl for use in a method of diagnosis of a cancer, preferably breast cancer.

11. A specific binding agent according to Claim 10 for a use as specified therein, which is selected from the group consisting of:

(a) a nucleic acid probe comprising a Sproutyl nucleic acid, or a fragment thereof capable of hybridisation to a Sproutyl sequence;

(b) a nucleic acid probe having the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a fragment thereof;

(c) a primer comprising between 10 to 15 residues from a sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, preferably having a sequence selected from the sequences set out in Tables IA or IB;

(d) a pair of primers comprising a forward primer selected from sequences depicted in Table IA together with a reverse primer selected from sequences depicted in Table IB; and

(e) an anti- Sproutyl antibody.

12. A combination of a specific binding agent according to Claim 10 or 11 and a specific binding agent for Sprouty2, for use in a method of diagnosis of a cancer, preferably breast cancer.

13. A method of treatment or prophylaxis of a cancer in an individual, the method comprising modulating the amount of a Sproutyl polypeptide or nucleic acid in a cell of an individual.

14. A method according to Claim 13, in which the cancer comprises a breast cancer.

15. A method according to Claim 13 or 14, in which the amount of a Sproutyl polypeptide or nucleic acid is increased, preferably specifically, in a breast cell of the individual.

16. A method according to Claim 13, 14 or 15, in which the amount of a Sproutyl polypeptide or nucleic acid is not substantially increased in any other cell or tissue type.

17. A method of manipulating a cell, the method comprising the steps of:

(a) detecting a reduced level of a Sproutyl polypeptide or nucleic acid in a cell, or an extract thereof;

(b) increasing the level of a Sproutyl polypeptide or nucleic acid in the cell.

18. A method according to Claim 17, in which the cell is derived from or present in an individual at risk of developing breast cancer.

19. A method according to any of Claims 13 to 18, in which the expression or activity of an endogenous Sproutyl sequence is up-regulated.

20. A method according to any of Claims 13 to 19, in which a control sequence, preferably a promoter and/or an enhancer sequence of Sproutyl is replaced with an endogenous control sequence.

21. A method according to any of Claims 13 to 20, in which the expression of a Sproutyl sequence is up-regulated in a breast cell of the individual, but not substantially in any other cell or tissue type.

22. A method according to any of Claims 13 to 21, in which an expression construct capable of delivering breast cell specific expression of Sproutyl is introduced into a cell of the individual.

23. A method according to any of Claims 13 to 22, in which the method further comprises modulating, preferably increasing, the amount of a Sprouty2 polypeptide or nucleic acid in a cell, preferably a breast cell, of an individual, preferably such that the amount of a Sprouty2 polypeptide or nucleic acid is not substantially increased in any other cell or tissue type, preferably by means of an expression construct capable of delivering breast cell specific expression of Sprouty2.

24. A nucleic acid construct comprising a Sproutyl gene or a coding portion thereof, together with one or more control elements selected from the group consisting of:

(a) a tumour specific promoter selected from the group consisting of: vascular endothelial growth factor (VEGF) promoter, vascular endothelial growth factor receptor- 1 (VEGFR-I) promoter, VEGFR-2 promoter, c-erbB2 promoter, L-plastin promoter, Bcl-2 promoter and MUCl promoter;

(b) a breast tissue specific promoter selected from the group consisting of: human α-lactalbumin (ALA) promoter, ovine β-lactoglobulin (BLG) promoter and a long terminal repeat (LTR) of a mouse mammary tumour virus (MMTV);

(c) an inducible promoter selected from the group consisting of: a stress gene promoter, a heat shock protein (HSP) promoter and a multidrug resistance gene-1

(MDR-I) promoter.

25. A nucleic acid construct according to Claim 24 which further comprises a Sprouty2 gene or a coding portion thereof.

26. A method according to any of Claims 13 to 23, in which an expression construct comprising a nucleic acid according to Claim 24 or 25 is introduced into a cell of the individual.

27. A method according to any of Claims 13 to 23 and 26, which comprises administering a Sproutyl polypeptide or nucleic acid to the individual.

28. A method according to Claim 27, which further comprises administering a Sprouty2 polypeptide or nucleic acid to the individual.

29. A host cell comprising a nucleic acid construct according to Claim 24 or 25.

30. A breast cell transformed with a nucleic acid construct according to Claim 24 or

25.

31. A transgenic non-human animal comprising a transgene which does not express, or expresses a reduced level, of Sproutyl.

32. A transgenic non-human animal according to Claim 31 which further comprises a transgene which does not express, or expresses a reduced level, of Sprouty2.

33. Use of a transgenic non-human animal according to Claim 31 or 32 as a model for breast cancer.

34. A method of identifying a molecule capable of binding to a Sproutyl polypeptide, the method comprising contacting a Sproutyl polypeptide with a candidate molecule and determining whether the candidate molecule binds to the Sproutyl polypeptide.

35. A method of identifying a modulator of Sproutyl expression, the method comprising contacting a cell with a candidate molecule, and detecting elevated expression of Sproutyl in or of the cell.

36. A method of identifying a drug, the method comprising exposing a transgenic animal according to Claim 31 or 32 to a candidate molecule, and detecting or monitoring the development or lack thereof of a breast cancer in the transgenic animal.

37. A method according to Claim 34, 35 or 36, which further comprises isolating and/or synthesising the molecule.

38. A molecule identified, isolated or synthesised by a method according to any of Claims 34 to 37.

39. Use of a molecule capable of modulating, preferably down-regulating, the expression of a Sproutyl sequence, preferably a molecule according to Claim 38, in a method of treatment of a breast cancer.

40. A molecule capable of modulating, preferably down-regulating, the expression of a Sproutyl sequence, preferably a molecule according to Claim 38, for use in a method of treatment of a breast cancer.

41. A nucleic acid having the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a fragment thereof capable of specifically hybridising to a Sproutyl sequence, preferably in combination with a nucleic acid having the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 7 or a fragment thereof capable of specifically hybridising to a Sprouty2 sequence.

42. A primer comprising between 10 to 15 residues from a sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, preferably having a sequence selected from the sequences set out in Tables IA or IB, preferably in combination with a primer comprising between 10 to 15 residues from a sequence shown in SEQ ID NO: 4 or SEQ ID NO: 7.

43. A Sproutyl polypeptide or nucleic acid, or a combination of such with a Sprouty2 polypeptide or nucleic acid, for use in a method of treatment of a cancer, preferably breast cancer, in an individual.

44. An antibody capable of specific binding to Sproutyl, or a combination of such with an antibody capable of specific binding to Sprouty2, for use in a method of treatment of a cancer, preferably breast cancer, in an individual.

45. A method or thing according to any preceding claim, in which the Sproutyl comprises human Sproutyl, preferably comprising a human Sproutyl nucleic acid having a GenBank accession number NP_005832 or AAH63856, or a human Sproutyl polypeptide having a GenBank accession number 043609.

46. A diagnostic kit for detecting breast cancer in an individual, or susceptibility of the individual to breast cancer, comprising means for detection of Sproutyl expression in the individual or a sample taken from him or her.

47. A diagnostic kit according to Claim 46, in which the means for detection is selected from the group consisting of: a Sproutyl polynucleotide or a fragment thereof; a complementary nucleotide sequence to Sproutyl nucleic acid or a fragment thereof; a Sproutyl polypeptide or a fragment thereof, or an antibody to a Sproutyl, preferably comprising an anti-human Sproutyl antibody, more preferably comprising a rabbit polyclonal to His tagged full length human Sproutyl 2, as well as an anti-human Sproutyl antibody comprising a rabbit polyclonal antibody to residues 58-75 of human Sproutyl, and optionally instructions for use.

48. A diagnostic kit according to Claim 46 or 47, which further comprises a means for detection of Sprouty2 expression in the individual or a sample taken from him or her, preferably comprising: a Sprouty2 polynucleotide or a fragment thereof; a complementary nucleotide sequence to Sprouty2 nucleic acid or a fragment thereof; a Sprouty2 polypeptide or a fragment thereof, or an antibody to a Sprouty2, and optionally instructions for use.

49. A diagnostic kit according to Claim 46, 47 or 48, which further comprises a therapeutic drug for treatment of breast cancer, preferably comprising Tamoxifen or Herceptin.

50. A method of treatment or diagnosis substantially as hereinbefore described with reference to and as shown in Figures 1 to 9F of the accompanying drawings.

51. A specific binding agent substantially as hereinbefore described with reference to and as shown in Figures 1 to 9F of the accompanying drawings.