WO2000015780A1 - Inhibitor of brn-3b and its use for the treatment of breast and ovarian cancer - Google Patents

Abstract

An inhibitor of Brn-3b expression and/or activity is useful in the treatment of breast or ovarian cancer. A method for identifying an inhibitor of Brn-3b expression comprises: (a) providing a test construct comprising a Brn-3b promoter operably linked to a coding sequence; (b) contacting a substance to be tested with the test construct under conditions that would permit the polypeptide encoded by the said coding sequence to be expressed in the absence of the said substance; and (c) determining whether the said substance inhibits the expression of Brn-3b.

Description

INHIBITOR OF BRN-3B AND ITS USE FOR THE TREATMENT OF BREAST AND OVARIAN CANCERS

Field of the invention

This invention relates to inhibitors of Brn-3b expression and/or activity and screening methods for the identification of such inhibitors. It further relates to the use of said 5 inhibitors in the treatment of cancer.

Background to the invention

The POU (Pit-Oct-Unc) family of transcription factors was originally defined on the basis of a common 150-160 amino acid domain which was found in found in the mammalian transcription factor Pit- 1 , Oct-1 and Oct-2, and the nematode regulatory

10 protein Unc-86 (for review see 1, 2). The common POU domain constitutes the DNA binding domain of these proteins and consists of two portions, a POU-specific domain which is unique to the POU factors and a POU-homeodomain which is related to the homeobox found in a number of other transcription factors.

Following the identification of the original POU family transcription factors, a number

15 of other members of this family have been defined (1-3) and shown to play critical roles in the regulation of gene expression. Moreover, mutations in the genes encoding several of these factors have been shown to be responsible for particular human diseases. Thus, for example, mutation in the gene encoding Pit-1 has been shown to result in a failure of pituitary gland development and consequent dwarfϊsm in both mice and humans (for

20 review see 4) whilst a mutation in the gene encoding Bm-4 has been shown to result in X- linked deafness (5). More recently, the gene encoding the POU factor Brn-3c has been shown to be mutated in a family with progressive late onset deafness (6).

Brn-3a, Brn-3b and Bm-3c are closely related members of the POU family which are encoded by different genes (7) and are expressed in distinct but overlapping patterns in the

25 developing and adult nervous system (3, 8-11). In addition however, expression of Brn-3a and Brn-3b has also been detected in some non-neuronal cells such as cervical epithelium (9, 12).

Brn-3a has been shown to be over-expressed in aggressive neuroendocrine tumours (14), whilst both Brn-3a and Brn-3b are expressed at high levels in human neuroblastomas (15, 16) Brn-3a and Brn-3b have previously been detected in the human breast cancer cell line MCF-7 (13) We have recently shown that mean Brn-3a levels are increased over 300 fold in human samples exhibiting cervical intra-epithelial neoplasia grade 3 (CLN3) compared to normal human cervical samples but Brn-3b levels are similar in the two groups of samples (12)

Summary of the invention

The present invention is based on the finding that mammary tumour tissue which has reduced expression levels of the BRCA-1 gene shows elevated expression of the Brn-3b POU family transcπption factor The BRCA-1 gene was identified on the basis of its mutation in a number of cases of familial breast cancer indicating that its mactivation can cause this disease Athough BRCA-1 does not appear to be mutated in cases of sporadic breast cancer, its expression has been shown to be reduced in a number of cases

The elevated expression of Brn-3b is not found in normal mammary cells, benign tumours or malignant tumour samples which do not exhibit reduced levels of the BRCA-1 gene In contrast no correlation between the level of BRCA-1 expression and the expression of the related POU family transcription factor Brn-3a Moreover, Brn-3b but not Brn-3a can strongly repress the BRCA-1 promoter approximately 20 fold in transfections carried out in mammary tumour cells

Thus, Brn-3b may play an important role m regulating expression of BRCA-1 in mammary tumours with enhanced expression of Brn-3b resulting in reduced BRCA-1 expression and thereby being potentially involved in tumour development The repression of Brn-3b expression by either pharmacological or by gene therapy procedures represents a potential method for treating breast cancers

Thus, the invention provides an inhibitor of Brn-3b expression and/or activity for use m a method of treatment of the human of animal body Such inhibitors are useful, in particular, in the treatment of breast cancer and/or ovaπan cancer

Screens may be carried out to identify inhibitors of Bra-3b expression and/or activity Accordingly, the invention also provides a method for identifying an inhibitor of Brn-3b expression comprising (a) providing a test construct comprising a Brn-3b promoter operably linked to a coding sequence, (b) contacting a substance to be tested with the test construct under conditions that would permit the polypeptide encoded by the said coding sequence to be expressed in the absence of the said substance; and

(c) determining whether the said substance inhibits the expression of Brn-3b; and - a method for identifying an inhibitor of Brn-3b activity comprising:

(a) providing a Brn-3b polypeptide or a homologue thereof, or a fragment thereof;

(b) contacting a substance to be tested with the Brn-3b polypeptide under conditions that would permit activity of the polypeptide in the absence of said substance; and

(c) determining whether the said substance inhibits the activity of Brn-3b. The invention further provides a pharmaceutical composition comprising an inhibitor of Brn-3b expression and/or activity and a therapeutically acceptable carrier or diluent.

As noted above, the inhibitors of Brn-3b expression and/or activity may be used in treating breast or ovarian cancer and therefore the invention provides a method of treatment of treating a host suffering from breast cancer or ovarian cancer, which method comprises administering to the host a therapeutically effective amount of an inhibitor of Bra-3a expression and/or activity.

Brief description of the Figures

Figure 1 shows levels of Brn-3a mRNA as determined by RT/PCR assay in normal/b enign mammary material or in malignant mammary tumours from pre- menopausal or post-menopausal women. Bar designates the mean with standard deviation

(SD) and population size 'n\

Figure 2 shows levels of Brn-3b mRNA as determined by RT/PCR assay in normal benign mammary material or in malignant mammary tumours from pre- menopausal or post-menopausal women. Bar designates the mean with standard deviation (SD) and population size 'n' .

Figure 3 shows comparison of the mRNA levels of Brn-3b and BRCA-1 in malignant mammary tumour material obtained from pre (panel a) or post (panel b) menopausal women or from benign mammary material (panel c). Brn-3b levels are shown as solid bars, BRCA-1 levels as open bars. Figure 4 shows comparison of the mRNA levels of Bm-3a and BRCA-1 in malignant mammary tumour material obtained from pre (panel a) or post (panel b) menopausal women or from benign mammary material (panel c) Brn-3a levels are shown as solid bars, BRCA-1 levels as open bars

Figure 5 shows comparison of BRCA-1 levels in mammary tumours with low or high levels of Brn-3b Bar designates the mean with standard deviation (SD) and population size 'n'

Figure 6 shows a luciferase reporter assay of MCF7 cells co-transfected with a promoter/reporter construct containing the full length BRCA-1 promoter together with expression vector lacking any insert (V) or the same vector expressing either Brn-3a or Brn-3b Values have been equalized relative to the activity obtained in the co-transfection with the reporter construct and empty expression vector (set at 100%) and are the mean of five determinations whose standard deviation is shown by the bars

Detailed description of the invention

Any suitable inhibitor of Brn-3b expression or activity may be employed in the present invention For example, the expression of Brn-3b in a cell may be reduced by presence in that cell of a polynucleotide which can hybridize to the Brn-3b mRNA Therefore a polynucleotide which is capable of hybπdizing to Brn-3b mRNA can constitute an appropriate inhibitor of Brn-3b expression In this regard, two approaches are as follows

(1) Antisense RNA The delivery of a nucleic acid construct which allows the expression of an RNA which can hybridize to the Brn-3b mRNA This results in the formation of an RNA-RNA duplex which may result in the direct inhibition of translation and/or the destabihzation of the target message, for example, rendering susceptibility to nucleases Therefore, the nucleic acid construct will typically lead to the expression of a polynucleotide which hybridizes to the πbosome binding region or the coding region of the Brn-3b mRNA

(2) Antisense ohgonucleotides

An oligonucleotide is delivered which hybridizes to the Brn-3b mRNA Antisense ohgonucleotides are postulated to inhibit target gene expression by interfering with one or more aspects of RNA metabolism including processing, translation and metabolic turnover Chemically modified ohgonucleotides may be used and may enhance resistance to nucleases and/or cell permeability Antisense

The coding sequence of Brn-3b is given in SEQ LD NO 1 An inhibitor of Brn-3b comprises a polynucleotide which can hybridize to the Brn-3b mRNA Typically such a polynucleotide will be an RNA molecule Such a polynucleotide may hybπdize to all or part of the Bra-3b mRNA Typically the polynucleotide will be complementary to all of or a region of the Brn-3b mRNA For example, the polynucleotide may be the exact complement of all or a part of Brn-3b mRNA However, absolute complementarity is not required and preferred polvnucleotides which have sufficient complementaπty to form a duplex having a melting temperature of greater than 40 °C under physiological conditions are particularly suitable for use in the present invention The polynucleotide may be a polynucleotide which hybridises to the Brn-3b mRNA under conditions of medium to high stπngency such as 0 03 M sodium chloride and 0 03 M sodium citrate at from about 50 to about 60 degrees centigrade

It is preferred that the polynucleotide hybridizes to the region of the mRNA corresponding to the coding sequence defined by nucleotides 213 to 392 of SEQ ID NO 1 The polynucleotide may hybridize to all or part of this region However, a polynucleotide may be employed which hybridises to all or part of the 5'- or 3 '-untranslated region of the mRNA These regions correspond to nucleotides 1 to 212 and 1446 to 31 0 of SEQ LD NO 1 The polynucleotide will typically be at least 40, for example at least 60 or at least 80, nucleotides in length and up to 100, 200, 300, 400 500, 600 or 700 nucleotides in length or even up to a few nucleotides, such as five or ten nucleotides, shorter than SEQ LD NO 1 When the polynucleotide is an antisense RNA it may be expressed in a cell from a recombinant rephcable vector Such a rephcable vector comprises a polynucleotide which when transcπbed gives πse to antisense RNA Preferably the polynucleotide giving πse to the antisense RNA is operably linked to a control sequence which is capable of providing for the transcription of the polynucleotide giving πse to the antisense RNA The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner A control sequence "operably linked" to a sequence giving rise to an antisense RNA is hgated in such a way that transcπption of the sequence is achieved under conditions compatible with the control sequences The vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for transcπption to occur and optionally a regulator of the promoter The vectors may contain one or more selectable marker genes, for example an ampicilhn resistance gene in the case of bacteπal plasmid or a neomycin resistance gene for a mammalian vector Vectors may be used in vitro, for example for the production of antisense RNA or used to transfect or transform a host cell The vector may also be adapted to be used in vivo, for example in a method of gene therapy

Promoters/enhancers and other expression regulation signals mav be selected to be compatible with the host cell for which the expression vector is designed For example, mammalian promoters, such as b-actin promoters, may be used Tissues-specific promoters, in particular neuronal cell specific promoters (for example the tyrosme hydroxylase (TH), L7, or neuron specific enolase (NSE) promoters), are especially preferred Viral promoters may also be used, for example the Moloney muπne leukaemia virus long terminal repeat (MMLV LTR), the promoter rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter, herpes simplex virus promoters or adenovirus promoters All these promoters are readily available in the art Preferred promoters are tissue specific promoters such as the casein gene promoter

Vectors may further include sequences flanking the polynucleotide giving πse to antisense RNA which compπse sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences This will allow the introduction of the polynucleotides of the invention into the genome of eukaryotic cells or viruses by homologous recombination In particular, a plasmid vector compπsing the expression cassette flanked by viral sequences, for example HSV1 or HSV2 sequences, can be used to prepare a viral vector, fpr example an HSV vector, suitable for dehveπng the polynucleotides of the invention to a mammalian cell Other examples of suitable viral vectors include retroviruses, including lentiviruses, adenoviruses and adeno-associated viruses Gene transfer techniques using these viruses are will known to those skilled in the art Retrovirus vectors for example may be used to stably integrate the polynucleotide giving rise to the antisense RNA into the host genome Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression

Antisense ohgonucleotides An antisense oligonucleotide will typically have a sequence such that it will bind to the Brn-3b mRNA Therefore it will typically have a sequence which is the complement of a region of the sequence shown in SEQ LD NO 1 An antisense oligonucleotide will generally be from 6 to 40 nucleotides in length Preferably it will be from 12 to 20 nucleotides in length Generally the oligonucleotide used will have a sequence that is absolutely complementary to the target sequence However, absolute complementaπty may not be required and in general any oligonucleotide having sufficient complementaπty to form a stable duplex (or triple helix as the case may be) with the target nucleic acid is considered to be suitable The stability of a duplex (or triplex) will depend /«ter aha on the sequence and length of the hybridizing oligonucleotide and the degree of complementaπty between the antisense oligonucleotide and the target sequence The system can tolerate less complementarity when longer ohgonucleotides are used However ohgonucleotides, especially ohgonucleotides of from 6 to 40 nucleotides in length, which have sufficient complementarity to from a duplex having a melting temperature of greater than 40 °C under physiological conditions are particularly suitable for use in the present invention The polynucleotide may be a polynucleotide which hybridises to under conditions of medium to high stπngency such as 0 03 M sodium chloride and 0 03M sodium citrate at from about 50 to about 60 degrees centigrade

Antisense ohgonucleotides may be chemically modified For example, phosphorothioate ohgonucleotides may be used Other deoxynucleotide analogs include methylphosphonates, phosphoramidates, phosphorodithioates, N3'P5'-phosphoramιdates and oligoπbonucleotide phosphorothioates and their 2'-0-alkyl analogs and 2'-0-methylπbonucleotιde methylphosphonates

Alternatively mixed backbone ohgonucleotides (MBOs) may be used MBOs contain segments of phosphothioate o godeoxynucleotides and appropπately placed segments of modified o godeoxy- or oligoπbonucleotides MBOs have segments of phosphorothioate linkages and other segments of other modified ohgonucleotides, such as methylphosphonate, which is non-ionic, and very resistant to nucleases or 2'-0-alkylohgoπbonucleotιdes Administration The vectors and antisense ohgonucleotides of the invention, optionally with an additional therapeutic polypeptide or nucleic acid/vector encoding said therapeutic polypeptide, may thus be administered to a human or animal in need of treatment Cancers which may be treated using the vectors, viral strains, antisense ohgonucleotides and compositions of the invention mclude breast or ovarian cancer and, in particular, breast or ovarian cancer in which Brn-3b expression is up-regulated such as non-familial breast cancer The condition of a patient suffeπng from such a cancer can thus be improved

The antisense ohgonucleotides and compositions compπsing antisense ohgonucleotides of the invention together may be administered by direct miection into the site to be treated, for example mammary tissue Preferably the antisense ohgonucleotides are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition Suitable earners and diluents include isotonic saline solutions, for example phosphate-buffered saline The composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration

The dose at which an antisense oligonucleotide is administered to a patient will depend upon a variety of factors such as the age weight and general condition of the patient, the cancer that is being treated and the stage which the cancer has reached, and the particular antisense oligonucleotide that is being administered A suitable dose may however be from 0 1 to 100 mg/kg body weight such as 1 to 40 mg/kg body weight

The polynucleotides giving πse to antisense RNA of the invention may be administered directly as a naked nucleic acid construct Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents Example of these agents include catiomc agents (for example calcium phosphate and DEAE-dextran) and pofectants (for example pofectam and transfectam )

Typically, nucleic acid constructs are mixed with the transfection agent to produce a composition Preferably the naked nucleic acid construct, viral vector comprising the polynucleotide or composition is combined with a pharmaceutically acceptable earner or diluent to produce a pharmaceutical composition Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline The composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration

The pharmaceutical composition is administered in such a way that the polynucleotide of the invention, viral vector for gene therapy, can be incorporated into cells at an appropπate area When the polynucleotide of the invention is delivered to cells by a viral vector, the amount of virus administered is m the range of from 10 to 10 pfu, preferably from 10 to 10⁷ pfu, more preferably about 10⁶ pfu for herpes viral vectors and from 10 to 10 pfu, preferably from 10⁷ to 10 pfu, more preferably about 10 pfu for adeno viral vectors. When injected, typically 1-2 ml of virus in a pharmaceutically acceptable suitable carrier or diluent is administered. When the polynucleotide of the invention is administered as a naked nucleic acid, the amount of nucleic acid administered is typically in the range of from 1 μg to 10 mg.

Where the polynucleotide giving rise to the antisense RNA is under the control of an inducible promoter, it may only be necessary to induce gene expression for the duration of the treatment. Once the condition has been treated, the inducer is removed and expression of the polypeptide of the invention ceases. This will clearly have clinical advantages. Such a system may, for example, involve administering the antibiotic tetracycline, to activate gene expression via its effect on the tet repressor/NP16 fusion protein.

The use of tissue-specific promoters will be of assistance in the treatment of disease using the polypeptides, polynucleotide and vectors of the invention. For example, several neurological disorders are due to aberrant expression of particular gene products in only a small subset of cells. It will be advantageous to be able express therapeutic genes in only the relevant affected cell types, especially where such genes are toxic when expressed in other cell types.

The routes of administration and dosages described above are intended only as a guide since a skilled physician will be able to determine readily the optimum route of administration and dosage for any particular patient and condition. Screening for inhibitors of Brn-3b expression and/or activity

The invention provides a method for identifying an inhibitor of Brn-3b expression comprising: (i) providing a test construct comprising a Brn-3b promoter operably linked to a coding sequence; (ii) contacting a substance to be tested with the test construct under conditions that would permit expression of the polypeptide encoded by the said coding sequence to be expressed in the absence of the said substance; and (iii) determining whether the said substance inhibits the expression of Brn-3b.

Any suitable assay format may be used for identifying an inhibitor of Brn-3b expression. Typically, however, the assay is carried out using a cell harbouring a promoter reporter polypeptide construct. A typical assay is as follows: - a defined number of cells are inoculated, in for example lOOμl of growth medium, into the wells of a plastics micro-titre plate in the presence of a substance to be tested

- optical density (OD) at 590nm may be measured as may expression of the reporter polypeptide according to any method appropπate for the reporter polypeptide being used

- the micro-titre plates are covered and incubated at 37 °C in the dark - the OD is read again and expression of the reporter polypeptide assayed at convenient time intervals The change in OD is a measure of cell proliferation Control experiments can be earned out, in which the substance to be tested is omitted

Also the substance may be tested with any other known promoter to exclude the possibility that the test substance is a general inhibitor of gene expression Any reporter polypeptide may be used, for example GUS or GFP are used GUS is assayed by measuπng the hydrolysis of a suitable substrate, for example 5-bromo-4-chloro-3- indolyl-β-D-glucoromc acid (X-gluc)or 4-methylumbelhferyl- β-glucuronide (MUG) The hydrolysis of MUG yields a product which can be measured fluorometncally GFP is quantified by measuring fluorescence at 590nm after excitation at 494nm These methods are well known to those skilled in the art

Alternatively the coding sequence may be the Brn-3b coding sequence itself In such an experiment a mammary cancer cell line which exhibits Brn-3b overexpression could be used The expression of Brn-3b may be followed by for example, Northern/RNA blotting, Western/antibody blotting, RNA in situ hybridization or lmmunolocalisation The invention further provides a method for identifying an inhibitor of Brn-3b activity comprising

(l) providing a Brn-3b polypeptide or a homologue thereof, or a fragment thereof, (n) contacting a substance to be tested with the Brn-3b polypeptide under conditions that would permit activity of the polypeptide in the absence of said substance, and (u) determining whether the said substance inhibits the activity of Brn-3b

Suitable Brn-3b for the assay can be obtained, for example, recombinantly by any method known to those skilled in the art Any suitable format may be used for identifying an inhibitor ofBrn-3b

Also the substance may be tested with any other known transcnption factor to exclude the possibility that the test substance is a general inhibitor of transcπption factors activity

In addition to the Brn-3b polypeptide, the reaction mixture can contain a suitable buffer A suitable buffer includes anv suitable biological buffer that can provide buffeπng capability at a pH conducive to the reaction requirements of the Brn-3b polypeptide

Test substances

A substance which inhibits the expression of Brn-3b may do so by binding directly to the promoter, thus preventing the initiation of transcription Alternatively a substance could bind to a protein which is associated with the promoter and is required for transcπption This may result in reduced levels of transcription

The Brn-3b promoter reporter polypeptide constructs of the invention may include the untranslated region of the Brn-3b gene Therefore a substance may reduce Brn-3b expression by binding to the untranslated region of the Brn-3b gene This could prevent the initiation of translation Alternatively a substance could bind to a protein associated with the untranslated region and prevent the protein associating with the untranslated region

A substance which inhibits the activity of Brn-3b may do so by binding to one or both of the enzymes Such enzyme inhibition may be reversible or irreversible An lπeversible inhibitor dissociates very slowly from its target enzyme because it becomes very tightly bound to the enzyme, either covalently or non-covalently Reversible inhibition, in contrast with meversible inhibition, is characterised by a rapid dissociation of the enzyme-mhibitor complex

The test substance may be a competitive inhibitor In competitive inhibition, the enzyme can bind substrate (forming an enzyme-substrate complex) or inhibitor (enzyme-inhibitor complex) but not both Many competitive inhibitors resemble the substrate and bind the active site of the enzyme The substrate is therefore prevented from binding to the same active site A competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate

The inhibitor may also be a non-competitive inhibitor In non-competitive inhibition, which is also reversible, the inhibitor and substrate can bind simultaneously to an enzyme molecule This means that their binding sites do not overlap A non-competitive inhibitor acts by decreasing the turnover number of an enzyme rather than by diminishing the proportion of enzyme molecules that are bound to substrate

The inhibitor can also be a mixed inhibitor Mixed inhibition occurs when an inhibitor both effects the binding of substrate and alters the turnover number of the enzyme A substance which inhibits the activity of Brn-3b may also do so by binding to the substrate The substance may itself catalyze a reaction of the substrate, so that the substrate is not available to the enzyme. Alternatively the inhibitor may simply prevent the substrate binding to the enzyme.

Suitable candidate substances include antibody products (for example, monoclonal and polyclonal antibodies, single chain antibodies, chimaeric antibodies and CDR-grafted antibodies) which are specific for Brn-3b Furthermore, combinatorial libraries, defined chemical entities, peptide and peptide mimetics, ohgonucleotides and natural product libraries may be screened for activity as inhibitors of Brn-3b in assays such as those described below. The candidate substances may be chemical compounds. The candidate substances may be used in an initial screen of, for example, ten substances per reaction, and the substance of these batches which show inhibition tested individually. Inhibitors of Brn-3b expression and/or activity

An inhibitor of Brn-3b expression and/or activity is one which produces a measurable reduction in Brn-3b expression and/or activity in the assays described above. Preferred substances are those which inhibit Brn-3b expression and/or activity by at least 10%, at least 20%, at least 30%, at least 40% at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% at a concentration of the inhibitor of 1 μg ml ^"', lOμg ml^"1, lOOμg ml^"', 500μg ml^"', lmg ml^"1, lOmg ml^"', lOOmg ml^"'. The percentage inhibition represents the percentage decrease in expression/activity in a comparison of assays in the presence and absence of the test substance. Any combination of the above mentioned degrees of percentage inhibition and concentration of inhibitor may be used to define an inhibitor of the invention, with greater inhibition at lower concentrations being prefeπed.

Candidate substances which show activity in assays such as those described below can then be tested on mammary cancer cell lines for example. Candidate inhibitors could be tested for their ability to attenuate Bm-3b expression in mammary cancer cell lines in which Brn-3b is up-regulated and also for the effect on BRCA-1 expression in mammary cancer cell lines in which BRCA-1 is down-regulated. Human use

Inhibitors of Brn-3b expression and/or activity identified by the screening procedures described above may be used to treat breast or ovarian cancer and, in particular, breast or ovarian cancer in which Brn-3b expression is up-regulated such as non-familial breast cancer. The condition of a patient suffering from a cancer can therefore be improved by administration of such an inhibitor. A therapeutically effective amount of such an inhibitor may be given to a human patient in need thereof

The formulation of an inhibitor for use in preventing or treating breast or ovaπan cancer will depend upon factors such as the nature of the substance identified, whether a pharmaceutical or veterinary use is intended, etc Typically an inhibitor is formulated for use with a pharmaceutically acceptable carrier or diluent For example it may be formulated for topical, parenteral, intravenous, intramuscular, subcutaneous, intraocular, transdermal or oral administration A physician will be able to determine the required route of administration for each particular patient The pharmaceutical earner or diluent may be, for example, an isotonic solution The dose of inhibitor may be determined according to vaπous parameters, especially according to the substance used, the age, weight and condition of the patient to be treated, the route of administration, and the required regimen Again, a physician will be able to determine the required route of administration and dosage for any particular patient The following Example illustrates the invention EXAMPLE

Matenals and Methods Unless indicated otherwise, the methods used are standard biochemistry and molecular biology techniques Examples of suitable general methodology textbooks include Sambrook et al , Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al , Current Protocols in Molecular Biology (1995), John Wiley and Sons, Inc Normal and breast cancer samples

Total RNA from normal mammary gland and from malignant breast tumours (from pre and post menopausal women) was obtained from the Candis Tissue Bank Research Centre Liverpool University Reverse transcriptase/polymerase chain reaction assay

About 0 lμg of RNA from each sample was used as a template for cDNA synthesis Th synthesized cDNA was used in RT-PCR assays as previously descπbed (17) using the following oligonucleotide pnmers Brn-3a 5' GTCGACATGGACTCGGACACG-3', 3'- ACGGTGAATGACTCCCCCGA-5¹, Brn-3b 5'GGAGAAGAAGCGCAAGC-3', 3'CTGAGAACCGGAGAGGTCT-5' Amplification of the lnvaπantly expressed human cyclophihn mRNA used as a control was earned out in parallel using the following primers 5'- TTGGGCCGCGGTACTCCTTTCA-3¹, and 3'-TTTCGTATGGCCCAGGACCG-5' In all cases, 20 μl of each PCR product was fractionated on a 2% agarose gel and blotted onto Hybond-N+ nylon membrane (Amersham International, Little Chalfont, United Kingdom) and hybπdized with homologous complimentary ³²P-lablled probes Membranes were exposed to films, (Eastman Kodak Co , Rochester, NY) and the subsequent autoradiographs were then analyzed using a densitometer (Bio-Rad Laboratones, Hercules,

CA) We have previously shown that this blotting procedure, in con]unctιon with the RT-PCR conditions used, allows accurate quantification of the Brn-3a and Brn-3b mRNAs relative to the constitutively expressed cyclophihn mRNA (9, 12, 17) Plasmid constructs The Bm-3a and Brn-3b expression vectors contain full length cDNA clones under the control of the moloney murme leukaemia virus promoter and have previously been described (7 17) The BRCA-1 promoter/reporter constructs contain 4 kilo-bases or 400 bases of upstream sequence containing the BRCA-1 α and β promoters cloned into the pGL2 luciferase vector Transient transfection

MCF7 cells were routinely grown in Dulbecco's modified Eagle's medium containing L- glutamate and phenol red which was supplemented with 10% foetal calf serum and lOng of insulin per ml Before expenments were earned out subconfluent cells were maintained in phenol red-free Dulbecco's modified Eagle's medium containing 10% dextran coated charcoal- stπped foetal calf serum prepared according to the method descπbed by Mighaccio et al , (18) and lOng of insulin per ml for 72 hours The medium was replaced by 5 ml of fresh medium 12 hours prior to transfection Transfection of plasmid DNA was earned out according to the method of Gorman (19) Routinely 5μg of reporter DNA and 5μg of each expression vector were transfected into 5xl0³ cells and the cells harvested after 72 hours The amount of DNA taken up by the cells in each case was measured by slot blotting of 15μl of the extract and hybridization with a probe derived from the ampicillm resistance gene in the plasmid vector (20) Differences in the intensity of the bands were measured by densitometry and used to equalize the volumes of extracts used for subsequent assay All transfections also included an internal reporter gene encoding the renilla luciferase with a dual luciferase assay being earned out to control for transfection efficiency Luciferase assays were done as descnbed by the manufacturers (Promega) protocol with results measured on a Turner 20-E luminometer Results

To quantitate the level of Brn-3a and Brn-3b mRNAs we used a reverse transcπptase/polymerase chain reaction (RT/PCR) assay which we have previously used to reliably quantitate these mRNAs in small amounts of clinical and other mateπal (12, 15) This assay was used to compare the levels of Brn-3a and Bm-3b mRNAs in normal breast tissue or benign breast tumour material with that obtained from malignant breast tumours from pre- or post- menopausal women The mean levels of Brn-3a (figure 1) and Brn-3b (figure 2) both showed some increase in the matenal prepared from the pre-menopausal malignant tumours compared to that obtained from normal/benign tumour material and a further increase was observed in both cases in the post-menopausal tumour material but this was not statistically significant (p>0 05 in all comparisons of normal versus tumour levels)

Interestingly, however, clear differences were noticed in the distπbution pattern of Brn-3a and Brn-3b expression levels in the different tumour samples Thus, Brn-3a levels appeared to show a continuous distribution between the different samples In contrast, Brn-3b levels exhibited a bipartite distπbution in the different tumour samples with approximately 40% of the samples showing low levels of Brn-3b within the range observed for the normal samples whilst the remaining samples showed much higher levels outside the normal range This bipartite distπbution was observed for tumour samples obtained from both pre-and post-menopausal women To determine whether this bipartite distπbution had any functional significance, we searched for differences between the tumour samples with respectively low or high levels of Brn-3b expression In particular, we examined the level in the different samples of the mRNA encoding BRCA-1 protein which is mutated in many cases of familial breast cancer (21, 22) and which has also been reported to exhibit reduced expression in non-hereditary (sporadic) breast cancer (23) Most interestingly, the levels of BRCA-1 in the different samples showed an inverse expression pattern to that observed with Brn-3b Thus, the samples with low levels of Brn-3b exhibited high levels of BRCA-1 which was observed in the samples deπved from both pre-menopausal (figure 3a) and post-menopausal (figure 3b) women, although the effect was particularly marked in the samples from pre-menopausal women In contrast no significant variation in BRCA-1 mRNA levels was noted in different samples of normal mammary mateπal or material from benign tumours which also had similar levels of Brn-3b (figure 3c) In contrast to the results with Brn-3b, the level of BRCA-1 mRNA in malignant material from pre-menopausal (figure 4a) or post-menopausal (figure 4b) women showed no correlation with the level of Brn-3a mRNA in the different samples. Moreover, similar levels of BRCA-1 mRNA were observed in the normal or benign material despite the different Brn-3a levels in the different samples (figure 4c). The specific association of high Brn-3b levels with low BRCA-1 expression was confirmed by dividing the samples into those with high Brn-3b and low Brn-3b. As illustrated in figure 5, BRCA-1 levels were highly elevated in the sample group with low Brn-3b compared to the level in those with high Brn-3b. This effect was statistically significant (pθ.001 comparing BRCA-1 levels in the high and low Brn-3b expressing groups).

These findings therefore raise the possibility that the Brn-3b transcription factor may have an inhibitory effect on expression of BRCA-1, with the high levels of Bm-3b observed in some malignant mammary tumour samples being associated with a low level of BRCA-1. To investigate this possibility further, we co-transfected MCF7 cells with expression vectors encoding Brn-3a or Brn-3b and a construct in which a four kilo-base fragment of the BRCA-1 promoter drives expression of a luciferase reporter gene.

In this experiment (figure 6) the activity of the promoter was reduced by approximately 50% in the sample co-transfected with Brn-3a compared to the level observed in the sample co- transfected with the corresponding expression vector lacking any insert. However, a much more dramatic inhibitory effect was observed in the sample co-transfected with the Brn-3b expression vector where promoter activity was reduced to approximately 5% of that observed in the sample transfected with empty expression vector. Similar results were also obtained with a reporter construct containing only 400 base pairs of the BRCA-1 promoter indicating that this effect was dependent on this short promoter region (data not shown). This effect was specific to the BRCA-1 promoter, since transfection of the same Brn-3b expression vector with reporters containing an oestrogen response element resulted in a strong stimulatory effect on the promoter in accordance with our previous results (13) (data not shown). Hence Brn-3b can indeed directly inhibit the BRCA-1 promoter in co-transfections into breast cancer cells. References

1 Vernjzer, C P and van der Nhet, P C POU domian transcnption factors Btoc mica et Bwphysica Acta 1173 1-21, 1993

2 Ryan, A K and Rosenfeld, M G POU domain family values - flexibility, partnerships and developmental codes Genes and Development 1 1 1207-1225, 1997

3 He, X , Treacy, M Ν , Simmons, D M , Ingraham, H A , Swanson, L S and Rosenfeld, M G Expression of a large family of POU-domain regulatory genes in mammalian brain development Nature 340 35-42, 1989

4 Anderson, B and Rosenfeld, M G Pιt-1 determines cell types duπng development of the antenor pituitary gland Journal of Biological Chemistiy 269 29335-29338, 1994

5 de Kok, Y J M , van der Maarel, S M , Bitner-Ghndzicz M , Huber, I , Monaco, A P , Malcolm, S , Pembrey, M E , Ropers, H-H and Cremers, F P M Association between X-hnked deafness and mutations in the POU domain gene POU3F4 Science 267 685-688, 1995 6 Vahaua, O , Morell, R , Lynch, E D „ Weiss, S , Kagen, M F , Ahitau, Ν , Morrow, J E ,

Lee, M K „ Skvorak, A B , Morton, C C , Blumenfeld, A , Frydman, M , Fπedman, T B , King, M-C and Avraham, K B Mutation in transcription factor POU4F3 associated with progressive hearing loss in humans Science 279 1950-1954, 1998

7 Theil, T , McLean-Hunter, S , Zomig, M and Moray, T Mouse Brn-3 family of POU transcπption factors a new ammo terminal domain is crucial for the oncogenic activity of

Brn-3A Nucleic Acids Research 21 5921-5929, 1993

8 Geπero, M R , McEvilly, R J , Tuner, E , Lin, C R , O'Connell, S , Jenne, K J , Hobbs, M N and Rosenfeld, M G Bm 3 0 A POU domain protein expressed in the sensory immune and endocπne systems that functions on elements distinct from known octamer motifs Proc Natl AcadSci USA 90 10841-10845, 1993

9 Lillycrop, K A , Budhram-Mahadeo, N S , Lakin, Ν D , Teπenghi, G , Wood, J Ν , Polak, J M and Latchman, D S A novel POU family transcπption factor is closely related to Brn-3 but has a distinct expression pattern m neuronal cells Nucliec Acids Research

20 5093-5096, 1992 10 Turner, E E , Jenne, K J and Rosenfeld, M G Brn-3 2 A Brn-3-related transcπption factor with distinctive central nervous system expression and regulation by retinoic acid Neuron 12 205-218, 1994

11 Ninkina, N N , Stevens, G E M , Wood, J N and Richardson, W D A novel Brn-3 like POU transcnption factor expressed in subsets of rat sensory and spinal cord neurons Nucleic Acids Research 21 3175-3182, 1993 12 Ndisdang, D , Moms, P J , Chapman, C , Ho, L , Singer, A and Latchman, D S The

HPV activating transcription factor Brn-3 a is over expressed in CLN3 lesions Journal of Clinical Investigation 101 1687-1692, 1998

13 Budhram-Mahadeo, V , Parker, M and Latchman, D S

The POU domain factors Brn-3 a and Brn-3b interact with the estrogen receptor and differentially regulate transcπptional activity via an ERE Molecular and Cellular Biology 18 1029-1041, 1997

14 Leblond-Francillard, M , Picon, A , Bertagna, X and Keyzer, Y High expression of the POU factor Brn-3a in aggressive neuroendocπne tumors Journal of Clinical Endocrinology and Metabolism 82 89-94, 1997 15 Smith, M D and Latchman, D S The functionally antagonistic POU family transcription factors Brn-3a and Brn-3b show opposite changes in expression during the growth anest and differentiation of human neuroblastoma cells International Journal of Cancer 67 653-660, 1996

16 Deans, Z C , Dawson, S J , Kihman, M W , Wallace, D , Wilson, M C and Latchman, D S Differential regulation of genes encoding synaptic proteins by the Oct-2 transcnption factor Mυl Brain Res 51 1-7, 1997

17 Morris, P J , Theil, T , Ring, C J A , Lillycrop, K A , Moray, T and Latchman, D S The opposite and antagonistic effects of the closely related POU family transcπption factors on the activity of a target promoter are dependent upon differences in the POU domain Molecular and Cellular Biology 14 6907-6914, 1994

18 Mighaccio, A , Pagano, M and Auπcchio, F Immediate and transient stimulation of protein tyrosine phosphorylation by estradiol in MCF-7 cells Oncogene 8 2183-2191, 1993

19 Gorman, C M High efficency gene transfer into mammalian cells In DNA cloning, a pratical approach, edited by Glover, D M LRL Press, 1985, p 143-190 20 Abken, H and Reifenrath, B A procedure to standardize CAT reporter gene assay

Nucleic Acids Research 20 3527, 1992

Claims

1 Use of an inhibitor of Brn-3b expression and/or activity for the manufacture of a medicament for use in the treatment of breast cancer and/or ovaπan cancer

2 Use according to claim 1, wherein the inhibitor compπses a polynucleotide which is capable of hybπdizmg to the Brn-3b mRNA

3 Use according to claim 1, wherein the inhibitor compnses a nucleic acid vector which is capable of expressing a polynucleotide as defined in claim 2 in a host cell

4 Use according to claim 3 wherem the vector is a viral vector

5 Use according to claim 4, wherein the viral vector is a an adenovirus vector, a retroviral vector, an adeno-associated virus vector or herpes simplex virus strain

6 An inhibitor of Brn-3 b expression and or activity for use in a method of treatment of the human or animal body by therapy

7 An inhibitor according to claim 6 for use in a method of treatment of breast cancer or ovarian cancer 8 An inhibitor according to claim 6 or 7 compπsing a polynucleotide which is capable of hybridizing to the Brn-3b mRNA

9 An inhibitor according to claim 6 or 7 compπsing a nucleic acid vector which is capable of expressing a polynucleotide as defined in claim 8 in a host cell

10 An inhibitor according to claim 9, wherein the vector is a viral vector 11 An inhibitor according to claim 10, wherein the viral vector is a an adenovirus vector, a retroviral vector, an adeno-associated virus vector or herpes simplex virus strain

12 A method for identifying an inhibitor of Bm-3b expression comprising

(a) providing a test construct compπsing a Bm-3b promoter operably linked to a coding sequence, (b) contacting a substance to be tested with the test construct under conditions that would permit the polypeptide encoded by the said coding sequence to be expressed in the absence of the said substance, and (c) determining whether the said substance inhibits the expression of Brn-3b

13 A method according to claim 12 wherein the coding sequence encodes a Brn-3b polypeptide 14 A method according to claim 12, wherein the coding sequence encodes a reporter polypeptide

15 A method according to any one of claims 12 to 14, wherein a host cell harbours the test construct 16 A method according to any one of claims 15, wherein the host cell is a mammalian cell

17 A method for identifying an inhibitor of Brn-3 b activity compπsing

(a) providing a Brn-3 b polypeptide or a homologue thereof, or a fragment thereof,

(b) contacting a substance to be tested with the Brn-3b polypeptide under conditions that would permit activity of the polypeptide in the absence of said substance, and

(c) determining whether the said substance inhibits the activity of Bm-3b

18 A method according to any one of claims 12 to 17 further compπsing (d,) administeπng a said substance which has been determined to inhibit Brn-3b to mammalian cells, and (e,) determining the effect of the said substance on the expression of BRCA- 1

19 A method according to claim 18, wherein the mammalian cells are breast cancer cells or a ovaπan cancer cells

20 A method according to claim 19, wherein the ability of the said substance to inhibit the proliferation of the cell is determined 21 An inhibitor identified by a method according to any one of claims 12 to 20

22 An inhibitor according to claim 21 for use in a method of treatment of the human or animal body by therapy

23 An inhibitor according to claim 22 for use in a method of treatment of breast cancer or ovaπan cancer 24 Use of an inhibitor as defined in claim 21 for the manufacture of a medicament for use the treatment of breast cancer and/or ovarian cancer

25 A pharmaceutical composition compπsing an inhibitor according to any one of claims 1 to 6 and 21 and a pharmaceutically acceptable earner or diluent

26 A method of treatment of treating a host suffering from breast cancer or ovarian cancer, which method compπses administering to the host a therapeutically effective amount of an inhibitor of Brn-3 a expression and/or activity

27. A method of treatment according to claim 26, wherein the inhibitor is as defined in any one of claims 1 to 6 and 21.