WO2008015383A2

WO2008015383A2 - Bmp-9 and bmp-10 and their use in breast and prostate cancer therapies

Info

Publication number: WO2008015383A2
Application number: PCT/GB2007/002755
Authority: WO
Inventors: Wen Guo Jiang
Original assignee: University College Cardiff Consultants Ltd; Cardiff University
Current assignee: University College Cardiff Consultants Ltd; Cardiff University
Priority date: 2006-07-29
Filing date: 2007-07-20
Publication date: 2008-02-07
Anticipated expiration: 2009-01-29
Also published as: GB0615129D0; WO2008015383A8; WO2008015383A3

Abstract

The invention relates to the role of BMP-9 and/or BMP-10 in the treatment of breast or prostate cancer.

Description

ANTI-CANCER ACTIVITY OF BMP-9 AND BMP-10 AND THEIR USE IN CANCER THERAPIES

The invention relates to two novel agents, BMP-9/BMP-10, for treating breast or prostate cancer; vectors adapted to transform or transfect breast or prostate tissue which vectors are designed to express either, or both, BMP-9 and BMP-10; cell lines which have been transformed or transfected so as to express native or recombinant BMP-9 and/or BMP-10; a method for the recombinant production of BMP-9 and/or BMP-10; and methods for treating breast or prostate cancer comprising exposing breast or prostate cancer tissue to BMP-9 and/or BMP-10.

Bone morphgenesis proteins (BMPs) are a protein family that belongs to the TGFβ (transforming growth factor beta) superfamily. BMPs are widely involved in the regulation of cell functions including cell growth and apopotosis. More than 20 BMPs have been reported so far. The proteins are also important in embryo development and tissue regeneration. Some of the BMPs are also known to be important players in cancer. For example, BMP-2, BMP-6 and BMP-7 have been shown to be aberrantly expressed in human cancers and that raised levels of the proteins either in the circulation or in tumours are associated with progression and spread of cancer.

BMPs exert their effects through a heteromeric receptor complex, which consists of two types of serine-threonine kinase transmembrane receptors. The Type-I receptors include BMP receptor type IA (BMPR-IA)₁ type IB (BMPR-IB), Activin receptor-like kinase-1 (ALK-1), ALK-4, ALK-5 and activin A receptor type I (ActRI). The Type-ll receptors include BMP receptor type Il (BMPR-II), activin A receptor type HA (ActRII) and activin A receptor type MB (ActRIIB). Upon binding to BMP ligands, the Type-ll receptors then phosphorylates the Type-I receptors. This leads to the recruitment of the pathway-restricted Smads (R-Smads, Smadsi , 5 and 8) to the complex. The intercellular signaling complex of R- Smads is then translocated into the nucleus, which then regulate the transcription of BMP responsive genes.

We have been investigating the impact of a range of BMPs in human breast and prostate cancer and have found, unexpectedly, that BMP-9 and BMP-10 have a very strong anti-cancer functions. This is in clear contrast to other BMPs of which high levels are most associated with pro-cancer actions.

BMP-9, otherwise known as GDF2 (Growth and Differentiation Factor-2) was first found in liver cells (Celeste et al 1994) and the major source is Kupffer, Stellate and endothelial cells (Miller et al 2000). In embryos, BMP-9 was highly expressed in neural tissues and therefore has been indicated in the development of nervous system. In bones, BMP-9 was able to induce differentiation of osteoclast. BMP-10, discovered in 1999 (Neuhaus et al 1999), is known to be expressed at good levels in heart and lung and has also been indicated in embryo development such as heart. Statements of Invention

Our invention concerns the role of BMP-9 and/or BMP-10 in breast or prostate cancer and more particularly the inhibitory role of BMP-9 and/or BMP- 10 in breast or prostate cancer.

According to a first aspect of the invention there is provided an anti- cancer agent for treating breast or prostate cancer comprising a nucleic acid molecule encoding BMP-9, as shown in Figure 9, or a homologue thereof, or a nucleic acid molecule that hybridises to the sequence shown in Figure 9 under stringent conditions.

According to a second aspect of the invention there is provided an anticancer agent for treating breast or prostate cancer comprising a nucleic acid molecule encoding BMP-10, as shown in Figure 10, or a homologue thereof, or a nucleic acid molecule that hybridises to the sequence shown in Figure 10 under stringent conditions.

Reference herein to stringent conditions includes reference to either increasing the temperature of incubation to above 25⁰C and more preferably above 5O⁰C and more preferably still up to 65⁰C and/or washing the annealed molecules using a salt solution having an ionic strength of 1.0N sodium chloride - 0.02N sodium chloride, and most preferably 0.5N sodium chloride - 0.02N sodium chloride and more preferably still 0.1 N sodium chloride to 0.02N sodium chloride.

According to a further aspect of the invention there is provided a polypeptide that has anti-cancer activity in treating breast or prostate cancer comprising the polypeptide BMP-9 as shown in Figure 9, or a homologue thereof, or a fragment thereof which has BMP-9 anti-cancer activity.

According to a further aspect of the invention there is provided a polypeptide that has anti-cancer activity in treating breast or prostate cancer comprising the polypeptide BMP-10 as shown in Figure 10, or a homologue thereof, or a fragment thereof which has BMP-10 anti-cancer activity. According to a further aspect of the invention there is provided the use of a nucleic acid molecule encoding BMP-9 and/or BMP-10 for treating breast or prostate cancer.

According to a further aspect of the invention there is provided the use of BMP-9 or BMP-10 polypeptide for treating breast or prostate cancer.

According to a further aspect of the invention there is provided a medicament comprising either the nucleic acid molecule encoding BMP-9 and/or BMP-10, and/or the corresponding BMP-9 and/or BMP-10 polypeptide.

The medicament of the invention is most suitable for treating breast or prostate cancer.

In a preferred embodiment of the invention the medicament is formulated with a suitable excipient, carrier or emollient.

According to a further aspect of the invention there is provided a method for treating breast or prostate cancer comprising administering to an individual to be treated a medically effective amount of BMP-9 and/or BMP-10.

According to a further aspect of the invention there is provided a method for treating breast or prostate cancer comprising increasing cellular levels of either exogenous or endogenous BMP-9 and/or BMP-10 in breast or prostate cells.

Reference herein to the exogenous increase in cellular levels of BMP-9 and/or BMP-10 includes reference to: introducing into breast and/or prostate cells either externally manufactured BMP-9 and/or BMP-10 polypeptide; or introducing means for increasing the said cells production of BMP-9 and/or BMP-10 by, for example, introducing a copy of, or increasing the number of copies of, the BMP-9 and/or BMP-10 gene.

Reference herein to the endogenous increase in cellular levels of BMP-9 and/or BMP-10 polypeptide includes introducing into breast and/or prostate cells means that enables the breast or prostate cells to increase native production of BMP-9 and/or BMP-10 polypeptide, for example, suitable means include supplementing the cells' manufacturing machinery to provide for the increased production of BMP-9 and/or BMP-10 such as by increasing the effectiveness or activity of the promoter(s) controlling the expression of BMP-9 and/or BMP-10 gene whereby the overall production of BMP-9 and/or BMP-10 is increased, or, additionally or alternatively, supplementing the cells' other cellular protein production pathways whereby the amount of BMP-9 and/or BMP-10 produced is increased. Those skilled in the art will be familiar with ways by which this can be achieved.

According to a yet further aspect of the invention there is provided a vector adapted to transfect or transform breast or prostate cells wherein said vector includes:

1. at least one copy of the BMP-9 and/or BMP-10 gene; and/or

2. at least one over-expressing or constitutively active promoter which is either coupled to the BMP-9 and/or BMP-10 gene of part 1. and/or which is designed for insertion into a genome upstream of the native BMP-9 and/or BMP- 10 gene of said breast or prostate cells; whereby transfection or transformation of said cells with said vector results in the enhanced expression of BMP-9 and/or BMP-10.

According to a further aspect of the invention there is provided a cell line which has been transfected or transformed with a vector encoding at least one copy of the gene for BMP-9 and/or BMP-10 or as described herein.

According to a further aspect of the invention there is provided a 3T3 cell line, or a PC-3 cell line, or a DU145 cell line, or a MDA231 cell line or a T24 cell line which has been transfected or transformed so as to recombinantly express BMP-9 and/or BMP-10.

According to a further aspect of the invention there is provided recombinant BMP-9 and/or BMP-10 which has been manufactured by a host cell that has been transfected or transformed with a vector described herein.

According to a further aspect of the invention there is provided a method for manufacturing recombinant BMP-9 and/or BMP-10 which method comprises:

1. transfecting or transforming a host cell with a vector described herein;

2. culturing said host cell under conditions that favour expression of BMP-9 and/or BMP-10;

3J harvesting the BMP-9 and/or BMP-10 polypeptide produced by said host cell.

In a preferred method of the invention said vector further includes a suitable secretion signal whereby once BMP-9 and/or BMP-10 polypeptide has been produced it is processed through the cell's machinery for secretion and therefore the secreted polypeptide can be harvested from the extra cellular medium.

According to a further aspect of the invention there is provided the use of BMP-9 and/or BMP-10 to treat breast or prostate cancer.

According to a yet further aspect of the invention there is provided the use of BMP-9 and/or BMP-10 in the manufacture of a medicament to treat breast or prostate cancer.

According to a further aspect of the invention there is provided a BMP-9 or BMP-10 receptor or downstream signalling molecule, such as Par-4 or Smad- 1 , or Smad-5 or Smad-8, or an agonist or enhancer of any of the afore molecules for use in treating breast or prostate cancer.

An embodiment of the invention will now be described by way of examples only with reference to the following Figures wherein:

Figure 1 shows Figure-1. Staining of BMP-9 in mammary (left two panels) and prostate (right two panels) tissues (normal (left) and tumour (right) tissues are shown in the respective tissue type). Normal epithelial cells stained positively for BMP-9. The protein staining, however, was lost in tumour cells (right).

Figure 2 shows Staining of BMP-10 in mammary (left two panels) and prostate (right two panels) tissues (normal (left) and tumour (right) tissues are shown in the respective tissue type). Normal epithelial cells stained positively for BMP-10. The protein staining, however, was lost in tumour cells (right).

Figure 3, bottom panel, shows quantitative analysis of the BMP-9 (left)

and BMP-10 (right) transcripts showed a significant reduction of both transcripts

J in tumour breast tissues compared with normal breast tissues. The top panel shows correlation between BMP transcripts and prognosis, in which NPI-1 indicating patients with good prognosis, NPI-2 with moderate and NPI-3 with a poor prognosis. Patients with a poor prognosis had significantly lower levels of BMP-10; Figure 4 shows expression of BMP-9 and BMP-10 in cancer cells, as revealed by RT-PCR.

Figure 5 shows the construction and generation of BMP-9 (A and B) and BMP-10 (C and D) expression vector. A and C are selected from transformed E. CoIi. C and D are purified, respective, plasmids;

Figure 6 shows the establishment of BMP-9 in PC-3 prostate cancer cells (top) and BMP-9 and BMP-10 (bottom) in MDA MB 231 breast cancer cells. Shown are mRNA from PC-3 cells (top), and mRNA from MDA MB 231 cells (bottom);

Figure 6A shows the amount of BMP-9 protein produced in PC-3 cells;

Figure 7 shows protein expression in transfected cancer and other mammalian lines. Successful cell lines were established in 3T3 (fibroblast) for BMP-9; 3T3 (fibroblast), PC-3 (prostate) and T24 (bladder) cells for BMP-10, (HECV is an endothelial cell line and DU14 is a prostate cell line);

Figure 8 shows over-expression of BMP-9 and BMP-10 in cancer cells (MDA MB-231 breast cancer, PC-3 prostate cancer and DU145 prostate cancer) resulted in a slower growth and reduced invasion;

Figure 9 shows the impact of BMP-9 on the ability of prostate cancer cells to adhere to the Extracellular matrix;

Figure 10 shows the effect of BMP-9 over-expression on cellular motility using an in vitro migration assay;

Figure 11 shows BMP-9 induces apoptosis in PC-3 cells via Par-4. A, overexpression of BMP-9 induces apoptosis in PC-3 cells using a flow cytometry. The percentage of the apoptotic cells in pc-3^BMP"9exp cells is 29.4% (top), which is remarkably higher than the 0.45% of pc-3^pEF/His cells (middle) and 5.00% of PC-S^ (bottom). B, top panel shows an increase of Par-4 transcripts in PC-3BMP-9exp cells using RT-PCR₁ while the lower panel of B shows the elevated level of Par-4 protein in PC-3 by over-expression of BMP-9 using Western blot analysis. C, elevated protein level of Par-4 in pc-3^{BMP 9exp} cells was also detected using immunochemical staining. D, elevated protein level of Par-4 in pc-3^BMP"9exp cells was further examined using immunofluorescent staining. Both immunochemical and immunofluorescent staining revealed an enhanced staining in nuclei.

Figure 12 shows Both BMPR-1 B and BMPR-2 mediate inhibitory effects on cell growth of prostate cancer cells. A, knockdown of BMPR-IB and BMPR-II mRNA in PC-3 cells by the ribozyme transgenes was verified using RT-PCR. The gene transcripts of BMPR-IB were diminished in the pc-3^{ΔBMPR IB} cells by the ribozyme transgenes (top). The PCR reactions were performed 30 cycles. The mRNA level of BMPR-II was also markedly reduced in the pc-3^ΔBMPR"", compared with the PC-S^ and pc-3^pEF/His (bottom). B, the ribozyme transgenes reduced the protein production of BMPR-IB and BMPR-II in PC-3 cells using western blot analysis. The protein production of BMPR-IB was eliminated in the _{pC 3}ΔBMPR-IB _cel|s_ _{compared with tne} p_C.₃WT _an(j p^pEF/His _{(tQp) A sjmj|ar}

reduction of BMPR-II protein was also revealed in pc-3^ΔBMPR ", compared with both 90-3™* and pc-3^pEF/His cells, (bottom). Shown are a resentatives of three independent experiments for each. C₁ knockdown BMPR-IB and BMPR-II promote the cell growth of PC-3 cells using in vitro cell growth assay. The cell growth rate at day 4 is 239.3%±30.7% of PC-3^m cells and 209.9%±26.9% of _pc_₃pEF/His _ce|_{)Si whjch of PC}_₃ΔBMPR-IB _ce|_{|s js 42}3.4%±57.1 %, and 543.8%+119.1 % of pc-3^ΔBMPR~" cells. The' growth rate was calculated as a percentage using the absorbance of day 1 as a baseline. The error bars are standard deviation, n=6. Three independent experiments were performed. D, influence on the invasiveness of PC-3 cells by knockdown of BMPR-IB and BMPR-II was assessed using an in vitro invasion assay. Each cell lines were triplicated, three independent experiments were performed. Error bars are standard deviations.

Figure 13 shows BMP-9 signals through a Smad-dependent pathway in PC-3 cells. A, effects of rh-BMP-9 on in vitro cell growth of PC-3 cells was examined using the in vitro cell growth assay. The cell growth of PC-3 cells were reduced by the exposure of rh-BMP-9 at 50ng/ml. Error bars are standard deviations. * p<0.05 vs control. B, rh-BMP-9 (20ng/ml) induced apoptosis in PC- 3 cells using flow cytometry. The percentage of cells in exposure to rh-BMP-9 undergoing apoptosis is 16.88%, which is higher than the control (7.73%). C, BMP-9 signal through Smad dependent pathway in PC-3 cells. The role of BMPR-IB (top) and BMPR-II (middle) in the BMP-9 signaling in PC-3 cells. Phosphorylated R-Smads involved in signal transduction of BMP-9 using immunoprecipitation and western blot analysis (bottom). D, lmmunofluorescent staining of phosphorylated Smad-1 by BMP-9 in PC-3 cells.

Figure 14 shows the nucleic acid sequence and corresponding amino acid sequence of BMP-9; and

Figure 15 shows the nucleic acid sequence and the corresponding amino acid sequence of BMP-10. Materials and Methods Human tumour tissues

Human fresh frozen mammary and prostate tissues, archival materials were collected immediately after surgery and stored at -8O⁰C. Cell lines were purchased from ATCC and ECACC.

Cell Lines

PC-3 (ECACC, European Collection of Animal Cell Culture, Salisbury, UK), DU-145, LNCapFGC, CA-HPV10 and PZ-HPV-7 (ATCC, American Type Culture Collection, Mannasas, Virginia) were routinely maintained in DMEM-F12 medium supplemented with 10% fetal calf serum and antibiotics. Polyclonal goat anti-BMP-9, monoclonal mouse anti-actin, polyclonal goat anti-phospho Smad 1 and monoclonal mouse anti-Par-4 were obtained from Santa Cruz Biotechnology (Santa Cruz, California, USA). Other reagents or kits were obtained from Sigma- Aldrich, Poole, United Kingdom. Immuno-histochemical Staining of Breast and prostate tissue sections

Frozen sections of normal and tumours were cut at a thickness of 6μm using a cryostat. The sections were mounted on super frost plus microscope slides, air-dried and then fixed in a mixture of 50% Acetone and 50% methanol. The sections were then placed in "Optimax" wash buffer for 5 -10 minutes to rehydrate. Sections were incubated for 20 minutes in a 0.6% BSA blocking solution and probed with the primary antibodies. Following extensive washings, sections were incubated for 30 minutes in the secondary biotinylated antibody (Multilink Swine anti-goat/mouse/rabbit immunoglobulin, Dako Inc.). Following washings, the Avidin Biotin Complex (Vector Laboratories) was then applied to the sections, followed by extensive washing steps. Diamino benzidine chromogen (Vector Labs) was then added to the sections, and incubated in the dark for 5 minutes. Sections were then counter stained in Gill's Haematoxylin and dehydrated in ascending grades of methanol before clearing in xylene and mounting under a cover slip.

Real-time Quantitative Polymerase Chain Reaction (QPCR) ! The iCycler IQ system (BioRad, Camberley, UK) was employed to quantify the level (shown as copies/μl from internal standard) of BMP-9/10 in the breast specimens. Breast cDNA samples were then examined for each of BMP- 9 and BMP-10, along with the appropriate set of plasmid standards and negative controls. Primer sequences are given in table-1. The QPCR technique utilised the Amplifluor system (Intergen Inc, England) and Q-PCR master mix (ABgene, Surrey, England), in conjunction with a universal probe (UniPrimer™). Real-time QPCR conditions were 95°C for 12 minutes, followed by 65 cycles at 95°C for 15s, 55°C for 60s, 72°C for 20s. The results of the test molecules were normalised against levels of β-actin, using a β-actin quantitation kit from Perkin- Elmers (Perkin-Elmers, Surrey, England, UK). Generation of a BMP-9/10-expressing Cancer Cell Lines

The full coding regions of human BMP-9 and BMP-10 were amplified using the respective primers given in Table-2.

Full length cDNA for the respective BMP were cleaned by gel separated and extraction, T-A cloned into an mammalian expression vector, pEF6/TOPO, which were subsequently used to transform the TOPO10 E. CoI. Discrete bacterial colonies were screened for the presence of the coding sequence and direction of the inserts. Positive colonies were isolated, amplified, and plasmids subsequently extracted using a plasmid extraction kit. Following verification of the plasmids, purified plasmids were used to transfect breast and prostate cancer cell lines, followed by selection with blastidicin containing medium.

Alternatively, the first strand cDNA was synthesized from RNA isolated from normal human tissues using a DuraScript™ RT-PCR kit s. PCR was then used to amplify the coding sequence of full-length human BMP-9 using the Extensor Hi-Fidelity PCR master mix (ABgene Ltd., Epsom, UK). The sequences of primers are shown in Table 3. The verified BMP-9 insert was cloned into a mammalian expression plasmid vector (pEF/His TOPO TA plasmid vector, Invitrogen, Inc., Paisley, UK). The recombinant plasmid vectors were transformed into chemically competent TOP10 E. CoIi (Invitrogen, Inc., Paisley, UK), and the colonies were then analyzed. Colonies carrying correct recombinant plasmids were amplified, and the BMP-9 recombinant plasmids were then extracted

Purified BMP-9 transgenes and control plasmid vectors were then transfected into PC-3 cells individually using an Easjet Plus electroporator (EquiBio Ltd, Kent, UK). After up to 3 weeks of selection with blasticidin the transfectants were used in certain studies.

Both PCR and Western blotting were used to verify the success of transfection and protein expression. Knockdown of BMPR-IB and BMPR-II in PC-3 cells using the respectively prepared ribozvme transgenes

Anti-human BMPR-IB and anti-human BMPR-II hammerhead ribozymes targeting were designed based on the secondary structure of the respective gene generated using Zuker's RNA mFold program (Zuker et al 2003). The ribozymes were individually cloned into a mammalian expression pEF6/V5-His- TOPO plasmid vector (Invitrogen Ltd., Paisley, UK). Ribozyme transgenes and control plasmid vectors were then transfected into PC-3 cells respectively. After up to 3 weeks selection using blasticidin, the transfectants were used in the current study.

RNA Isolation and Reverse Transcription PCR

RNA was isolated using Total RNA Isolation Reagent (ABgene, Epsom, England, UK). Reverse transcription was performed using the DuraScript TMRT-PCR kit, followed by PCR using a REDTaq™ ReadyMix PCR reaction mix (primer sequences shown in Table 3). Cycling conditions were 94 ⁰C for 5min, followed by 36 cycles of 94⁰C for 30 s, 55⁰C for 30 s, and 72⁰C for 40 s. This was followed by a final 10min extension period at 72⁰C. The products were visualized on 2% agarose gel stained with ethidium bromide.

lmmunoprecipitation and Western Blot analysis

The protein concentration in cell lysates were determined using the DC Protein Assay kit (BIO-RAD) and an ELxδOO spectrophotometer (BIO-TEK™). Equal amount of proteins were separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto nitrocellulose sheets. Proteins were then probed with the anti-BMP-9/10 antibody and peroxidase-conjugated secondary antibodies. Protein bands were visualized using the Supersignal™ West Dura system (Pierce Biotechnology, Inc., Rockford, IL₁ USA), and photographed using a UVITech imager (UVITech, Inc., Cambridge, UK).

The plasmids thus generated were referred to as pEF6/BMP9Exp and pEF6/BMP10Exp. The cells generated were referred to as

3T3/CHO/PC3/MDA231^{BMP9EXP/BMP10Exp}. Cell growth Assay

The BMP modified cancer cells were seeded in a 96 well plate at a density of 7,000 cells/well, and incubated at 37°C for up to 5 days. Cells were fixed and stained with Crystal violet. Absorbance was measured on a multiplate reader.

In the case of immunoprecipitation and western blotting of phosphorylated R-Smads, following 2 hours incubation in serum free DMEM, the cells were then exposed to rh-BMP-9 (20ng/ml), or serum free DMEM alone for 1 hour. The cells were lysed, and protein extracted. Proteins containing phosphorylated serine/threonine were then immunoprecipitated using an anti- phosphoserine/phosphothreonine antibody (Abeam pic, Cambridge, UK). The resulting immunoprecipitates and whole cell lysates were loaded on to the SDS_r PAGE gel. Following Western blotting, proteins were probed using goat polyclonal anti-BMPR-IB IgG, goat polyclonal anti-BMPR-ll IgG, mouse monoclonal anti-Smad-1 IgG, goat polyclonal anti-Smad-5 IgG and Rabbit polyclonal anti-Smad-8 IgG (Santa Cruz Biotechnology, California, USA). Tumour Cell Invasion Assay

Transwell chambers, equipped with a 6.5mm diameter polycarbonate filter insert (pore size 8μm)(Becton Dickinson, Labware, Oxford, UK)₁ were pre- coated with 50μg/insert of solubilised tissue basement membrane, Matrigel (Collaborative Research Products, Bedford, Massachusetts, USA). 40,000 cancer cells were seeded into each insert and allowed to invade for 3 days. Following incubation, cells that had invaded through the basement membrane were fixed (4% formaldehyde), and then stained with crystal violet. For analysis, the cells were counted in 10 fields/insert (x40 magnification); to determine the mean number of invaded cancer cells.

In vitro cell growth assay

Cells were plated into a 96-well plate (2,500cells/well). Cell growth was assessed after a period of incubation (upto 5 days). Crystal violet was used to stain cells, and absorbance was determined at a wavelength of 540nm using a spectrophotometer (BIO-TEK, Elx800, UK).

In vitro invasion assay

Transwell inserts with 8 μm pore size were coated with 50 _μg Matrigel (BD Matrigel™ Basement Membrane Matrix) and air dried. The Matrigel was rehydrated before use. 20,000 cells were added to each well. After 96 hours cells that had migrated through the matrix to the other side of the insert were fixed in 4% formalin, stained with 0.5% (weight/volume) crystal violet and counted under a microscope.

Wounding assay

50,000 Cells were seeded into each well of a chamber slide and allowed to reach near confluence. The layer of cells was then scraped with a fine gauge needle. The movement of cells to close the wound was recorded using a time lapse video recorder and analyzed using the Optimas 6.0 motion analysis.

Cell-matrix adhesion assay

40,000 cells were added in each well of 96-well plate, previously coated with the Matrigel (5μg/well). After 40 minutes of incubation, non adherent cells were washed off using^' BSS buffer. The remaining adherent cells were then fixed and stained with crystal violet. The number of adherent cells was then counted.

Flow cytometric analysis of apoptosis

All cells including those floating in the culture medium, were harvested after a period of incubation. Cells were washed in cold PBS and resuspended in 1X annexin-binding buffer at a density of 1 * 10⁶ cells/ml after centrifugation. 5 μl of FITC annexin V and 1 μl of the Pl working solution (100μg/ml) were added to 100 μl of the cell suspension. After a 15min incubation at room temperature, 400 μl of 1X annexin-binding buffer was added, mixed gently and the samples were kept on ice.The stained cells were immediately analyzed using the flow cytometer and FlowMax software package.

Immunocvtochemical Staining of Par-4

Cells were fixed and permeabilized with 0.1% Triton for 5 minutes in tris buffered saline. Following blocking with horse serum the cells were probed with anti-Par-4 antibody, labeled with bionylated secondary antibody and visualized using the VECTASTAIN® ABC system (Vector Laboratories, Inc., Nottingham, UK).

lmmunofluorescent Staining of Par-4

Cells were fixed in ice-cold ethanol and then rehydrated and permeabilized before staining, lmmunofluorescent staining of Par-4 was performed using anti-Par-4 antibody (1 :100 from original), and the FITC labelled anti-mouse IgG was used at 1 :100. The staining was visualised under Olympus™ BX51 fluorescent microscope, and photographed using a cooled digital C4742-80 camera (Hamamatsu Photonics Ltd., Hertfordshire, UK).

To stain the phosphorylated Smad-1 , PC-3 cells were seeded on glass chamber slide in a duplicated manner. Following 2 hours of incubation in serum free DMEM the cells were exposed to rh-BMP-9 (20ng), or serum free medium alone for 1 hour. The immunofluorescent staining of phosphorylated Smad-1 was performed using anti-Phosph-Smad1 antibody is 1 :50 from original, and the TRITC labelled anti-goat IgG was used at 1 :200, and then photographed. Results

1. Human breast cancer lost protein expression of both BMP-9 and BMP- 10 We first evaluated the staining pattern of both BMPs in human mammary tissues. As shown in Figure 1 and Figure-2, normal mammary A or prostate C epithelial cells expressed moderate level of BMP-9 (Figure -1 , normal or left panels A+C) and moderate level of BMP-10 protein (Figure-2, normal or left panels A+C). However, staining of both BMP-9 and BMP-10 was virtually lost in breast cancer cells (Figure-1 and Figure-2 tumour or B panels, respectively). A similar pattern of staining was seen in prostate tissues for both BMP-9 and BMP- 10 (Figure 1 for BMP-9 and Figure 2 for BMP-10) tumour or D panels.

2. Expression of BMP-9 and BMP-10 mRNA using quantitative analysis Using Q-RT-PCR₁ we found that both BMP-9 and BMP-10 transcripts were expressed at a significantly lower level in breast tumour tissues compared with normal tissues (Figure 3 bottom panel). The data was analysed against the prognosis, by using the Nottingham Prognostic Index (NPI), in which NPI-1 represents patients with NPI<3.4 and had good prognosis, NPI-2 with NPI 3.4- 5.4 and moderate prognosis, and NPI-3 with NPI >5.4 and poor prognosis. As shown in Figure-3 top panel, patients with poor prognosis had dramatically reduced BMP-10.

3. Expression of BMP-9 and BMP- 10 in a range of cancer cell lines

As shown in Figure-4, cancer cell lines had different pattern of expression of both BMP-9 and BMP-10.

4. Constructing the expression vector of BMP-9 and BMP1- 10 BMP-9 and BMP-10 were amplified from normal human cDNA bank and cloned into a mammalian expression vector. Following screening (Figure 5 A and C), plasmids were extracted and purified (Figure-5 B and D) and used for electroporation.

5. Over-expression of BMP-9 and BMP-10

As shown in Figure 6, we have successfully established cell lines that stably expressed BMP-9 and BMP-10. These were used for subsequent analysis (functional tests). To investigate whether the transfected cells also produce more BMP-9 protein, we determined the protein level of BMP-9 in the cells using Western blot analysis. Over-expression of the BMP-9 protein in PC- β^BMP-^9ex _{P ce}||_{s was con}fj_rmec| _wfl_n increasing protein production of BMP-9 precursor protein (approximate 6OkDa), dimers (28kDa) and monomers (14kDa) of BMP-9 mature ligands (Fig. 6A).

6. Establishing BMP-9/10 over-expressing cells for generating recombinant BMP-9 and BMP-10

A range of cell lines were transfected, however, the fate of the transfected cells was very different. As shown in Figure-7 top panel, it was only possible to establish a 3T3 cell line for the purpose of producing recombinant protein. BMP- 9 transfected PC-3 prostate cancer cells soon developed senescence.

For BMP-10 transfected cells, we were able to establish 3T3 (fibroblast), PC-3 (prostate) and T24 (bladder) cells which over-produced rBMP-10 (Figure7 bottom panel).

7. BMP-9 and BMP-10 over-expression resulted in death/senescence of cancer cells as well as CHO cells It was during the selection stage that we realised that over-expressing BMP-9 in CHO and PC-3 (prostate) and DU-145 (breast) results in cells rapidly developing cell senescence, and so we were unable to establish stably transfected cells. This suggests that BMP-9 triggers the cancer cells to terminal development, which eventually lead to death.

With BMP-10 transfection, we were able, with difficulty, to establish a PC3 prostate cancer cell line as well as 3T3 fibroblast cell line. Again, other cells, together with CHO cells, developed senescence and eventually death following electroporation.

Figure-8 (top three panels) shows the growth of cancer cells that have stable integration for BMP-9 and BMP-10. These cells were selected under extreme conditions, as most cells were dead in most experiments. ^

8. BMP-9 and BMP-10 over-expression resulted in. changes in invasion. As shown in figure-8 (bottom two panels), BMP-9 and BMP-10 over-expression resulted in breast cancer cells being less invasive compared with controls (p=0.02 and p=0.005, respectively).

9. Adhesion to the extracellular matrix (ECM) is an important property and process of cancer cells during metastasis. To investigate the impact of BMP-9 on ability of prostate cancer cells to adhere to the ECM, we employed a previously established in vitro cell-matrix adhesion assay. Over-expression of BMP-9 markedly reduced cell-matrix adhesion of PC-3 cells. The number of adherent pc-3^{BMP 9exp} cells is 7±2.4, p<0.01 vs both PC^ (30±7.1) and PC- ₃ ^pEF/His (37_±10.3)_j as shown in Fig. 9. We also examined the effect of BMP-9 over-expression on cellular motility using an in vitro migration/wounding assay. pc3^BMP9exp cells showed a significantly reduced cellular migration, compared with the controls. A remarkable reduction of distance migrated was seen in these cells, 60 minutes after wounding. The average distance migrated over 90 minutes for pc-3^BMP'9exp cells was 17.22±3.61 μm, p<0.05 vs both PC-β^ (32.47±2.98μm) and pc-3^pEF/His (30.26±2.43μm), as shown in Fig. 10.

10. To investigate whether the apoptosis is involved in the inhibitory effect of BMP-9 on the growth of prostate cancer cells, we determined the proportion of apoptotic cells. As demonstrated in Fig. 11A, there was a marked shift of cell population to apoptosis in the BMP-9 over-expressing cells, which is 29.4% in the pc-3^BMP~9exp cells, in comparison with PC^ (5.0%)and PC-

Prostate apoptosis response-4 (Par-4) has been demonstrated as being up-regulated in androgen-independent prostate cancer cells which were induced to undergo apoptosis (28-30). The PC-3 cells used in this study is androgen insensitive, and derived from bone metastasis of prostate cancer. Therefore, we hypothesised that Par-4 may be involved in the apoptosis induced by BMP-9. We examined the levels of Par-4 mRNA in the pc-3^BMP~9exp cells using conventional RT-PCR, in comparison with the wild type and control plasmid transfectants. Fig. 11B shows an up-regulation in the mRNA level of Par-4 in p_C_₃ ^BMP-^9ex^p _{ce||s We furthe|}- _verjfj_ecj BMP-9 induced expression of Par-4 at the protein level in PC-3 cells using western blot analysis and immunochemical staining. An elevated protein level of Par-4 was shown in the pc-3^BMP"9exp cells (Fig. 11 B-lower panel). The immunocytochemical staining also demonstrated an increased Par-4 protein in pc-3^BMP"9exp cells which appeared to be more condensed in the nuclei of the cells (Fig. 11C). This phenomenon is further elucidated in the immunofluorescent staining of Par-4 (Fig. 11 D).

11. Both BMPR-IB and BMPR-II were detectable in PC-3 cells (data not shown). In our current study, we transfected the ribozyme transgenes into PC-3 cells which targeted either BMPR-IB or BMPR-II. The expression of BMPR-IB mRNA was completely eliminated from PC-3 cells by the ribozyme transgene (pc-3^ΔBMPR~IB) in comparison to the expression levels seen in wild type (PC^) cells and PC-3 control plasmid (PC-3^pEF/Hls) cells (Fig. 12A upper panel). A substantial decrease of the BMPR-II transcripts was demonstrated in _PC_₃ΔBMPR-II _{ce||Sj compared t0 botn} pc^^ and PC-3^pEF/His cells (Fig. 12A lower panel). Knockdown in the mRNA of both receptors was reflected at protein level. As shown in Figure 12, there was a significant reduction of BMPR-IB protein (45kDa) in PC-3^ΔBMPR"IB cells, in comparison to the PC-S¹ and pc-3^pEF/Hls cells (Fig. 12B upper panel). In fact, the BMPR-IB protein was not detectable in PC- oΔBMPR-IB O cells. The protein yield of BMPR-II (70-8OkDa) was also decreased after the knockdown of BMPR-II transcripts in pc-3^ΔBMPR"" cells (Fig. 12B lower panel).

We further examined the effect on cell growth after loss of BMPR-IB and BMPR-II in PC-3 cells. The cell growth was facilitated by knockdown of BMPR-II, the rate of cell growth at day 4 was significantly increased in pc-3^ΔBMPR~" (543.8%±119.1 %), p<0.01 vs. PC^ (239.3%±30.7%), and p<0.05 vs. PC- ₃ ^pEF/His (209.9%±26.9%). There was also a marked increase of cell growth in _PC_₃ ^ΔBM^PR-IB _ce||s (423.4%±57.1%), p<0.01 vs. PC-3^OT and p<0.05 vs. PC- 3^pEF/His (Fig. 12C).

To investigate the signal transduction of BMP-9, we generated recombinant human BMP-9 protein. Firstly we examined the biological activities of the rh-BMP-9 by determining its effects on cell growth and apoptosis in vitro. Cell growth was inhibited after a 3 day exposure to rh-BMP-9 (50ng/ml), p<0.05 in comparison with the control (Fig. 13A). The apoptosis-inducing effect of BMP- 9 in PC-3 cells was examined using the rh-BMP-9. According to the aforementioned concentration in the in vitro cell growth assay and the functional concentrations of both BMP-9 reported recently (16, 31-33), we chose 20ng/ml for recombinant BMP-9 in the current study. rh-BMP-9 of this concentration could induce apoptosis in PC-3 cells (Fig. 13B).

To investigate the involvement of BMPR-IB and BMPR-II in the signal transduction of BMP-9 in PC-3, we utilized the aforementioned PC-3 cells which had lost or reduced the expression of these receptors. Both receptors were phosphorylated in the PC-3 wild type cells (PC-S™¹) on exposure to rh-BMP-9 (Fig. 13C top and middle). The activation of BMPR-II was more apparent in comparison with that of BMPR-IB. The phosphorylation of BMPR-IB by BMP-9 was prevented by the knockdown of BMPR-II. Similarly, a blockage of the phosphorylation of BMPR-H was seen in the BMPR-IB knockdown cells. This suggests the loss of either receptor could reduce the activation of another in PC- 3 cells. To elucidate the Smad dependent signaling by BMP-9 in prostate cancer cells, we examined the activation of Smad-1 , 5 and 8 (Fig. 13C bottom), which are the R-Smads involved in the the signaling of the BMPs. BMP-9 induced a remarkable phosphorylation of Smad-1 , and weak phosphorylation of Smad-5 and Smad-8. The loss of BMPR-II significantly diminished the phosphorylation of the Smad-1 , 5 and 8. In contrast, the loss of BMPR-IB only reduced the phosphorylation of BMPR-II₁ but had no effect on the activation of Smads except Smad-5. This indicated that BMP-9 was unable to induce the phosphorylation of BMPR-II without BMPR-IB. lmmunofluorescent staining of phosphorylated Smad-1 further revealed an activation and nuclear translocation of Smad-1 , as the result of exposure to rh-BMP-9 in PC-3 cells (Fig. 13D). The phosphorylation and translocation of Smad-1 was remarkably reduced by the knockdown of BMPR-II, but not the loss of BMPR-IB.

Following the activation of both BMPR-II and BMPR-IB, the R-Smads were stimulated and translocated into the nuclei, leading to the transcriptional regulation of the responsive genes. Although Smad-1 , Smad-5 and Smad-8 were phosphorylated by BMP-9 in PC-3 cells, activation of Smad-1 was the strongest one.

Conclusion

Both BMP-9 and BMP-10 are expressed at lower levels in breast and prostate tumour tissues compared with normal tissues. The levels are particularly low in aggressive tumours. This suggests that loss of both BMPs are associated with an aggressive clinical condition in clinical cancer. We have cloned full length human BMP-9 and BMP-10 and constructed expression vectors for both BMPs. We have generated mammalian cell lines (i.e. 3T3 cells) suitable for producing recombinant proteins. The study has further shown that over-expressing BMP-9 and BMP-10 in cancer cells lead to unexpected slower growth; reduced migration, motility, invasion and adhesion; and finally senescence and apoptosis in cancer cells.

Apoptosis is the key event for physiological growth control and regulation of tissue homeostasis. In the present study, we have demonstrated that BMP-9 inhibited in vitro cell growth of prostate cancer cells. This was not related to necrotic cell death, as observed during the study. This has lead us to discover that apoptosis is potentially the underlying mechanism for the reduced rate of cell growth induced by BMP9. Here, we have provided evidence for the very first time that one of the critical mechanisms underlying BMP-9 induced apoptosis in prostate cancer cells is the induction of prostate apoptosis response-4 (Par-4). Par-4 is a proapoptotic protein, which was originally identified in prostate cancer cells undergoing apoptosis in response to ionomycin. Par-4 is involved in the apoptosis induced by tumor necrosis factor (TNF), doxorubicin, etόposide, UV irradiation, growth factor deprivation, and ionizing radiation. In the current study we found that the apoptosis induced by BMP-9 was due to an up-regulation of Par-4. An elevated and condensed immunofluorescent nuclear staining of Par-4 was seen in BMP-9 over-expressed PC-3 cells. The nuclear translocation of Par-4 was also revealed in the BMP-9 over-expressed PC-3 cells using immunofluorescent staining of Par-4. It is concluded therefore that Par-4 is involved in the apoptosis induced by BMP-9 in the PC-3 androgen insensitive prostate cancer cells (PC-3). Loss of the expressions of BMP receptors, type I (BMPR-IA₁ BMPR-IB)₁ and type Il (BMPR-JI), especially BMPR-II in prostate cancer, resulted in enhanced cell growth. This suggests that both BMPR-IB and BMPR-II play crucial roles in the control of the cellular behavior of prostate cancer cells. Taken together, all this data suggests BMP-9 and BMP-10 are therapeutic agents in cancers including breast and prostate cancer.

TABLE 1 : Primers Designed for RT-PCR or Quantitative PCR

TABLE 2: Primers designed for amplifying coding sequence of BMPs

TABLE 3: Primer sequences for PCR

Primer sequences for PCR

PCR

Λnnealiug

Forward Products Temperature (⁰C) (bps) hIlMP-9 5'-CAGTCACGAGGAGGACAC 5'-GATGTCCTCGMGTTTACCC 124 55 hBiMPR-IB 5'-ATGGAACTTGCTGTAπGCT ^• 5'-CMCTCGAGTGnAGGTGGT 90 55 bBMPR-II 5'-TTTGGGAAAGAAACAMTCT S'-TGGATMGGACCMTTTTTG 113 55 liSmad-1 S'-TCACTGATCCTTCCMCMT 5'-CCTGGTGTTTTCMTAGTGG 86 55 hSπιaϋ-S S'-AGCTCACCAAMTGTGTACC 5'-TGMGATGMTCTCMTCCA 109 55 h.Sιnad-8 5'-CGTGTATGMCTGACCMGA 5'-GATGMTCTCMTCCAGCAG 113 55 hPm-4 5'-GATCTTACGCTTCCCπACC 5'-ATGCCAGGAGACGACCTC 104 55 hB.MP-9 5'-ATGTGTCCTGGGGCACTGT 5'-CCTGCACCCACACTCTG 1287 66 hji-actin 5'-ATGATATCGCCGCGCTCG S'-CGCTCGGTGAGGATCTTCA 5SO 55

REFERENCES:

Celeste AJ, Song JJ₁ Cox K, Rosen V, Wozney JM. Bone morphogenetic protein-9, a new member of the TGF-beta superfamily. J Bone Min Res 1994;Suppl 1 (9):S136.

Miller AF, Harvey SA, Thies RS, Olson MS. Bone morphogenetic protein- 9. An autocrine/paracrine cytokine in the liver. The Journal of biological chemistry 2000;275(24): 17937-45

Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 2003;31 (13):3406-15.

Claims

1. An anti-cancer agent for treating breast or prostate cancer comprising a nucleic acid molecule encoding BMP-9, as shown in Figure 9, SEQ ID NO: 1 , or a homologue thereof, or a nucleic acid molecule that hybridises to the sequence shown in Figure 9, SEQ ID NO: 1 under stringent conditions.

2. An anti-cancer agent for treating breast or prostate cancer comprising a nucleic acid molecule encoding BMP-10, as shown in Figure 10, SEQ ID NO: 2, or a homologue thereof, or a nucleic acid molecule that hybridises to the sequence shown in Figure 10, SEQ ID NO: 2, under stringent conditions.

3. A polypeptide that has anti-cancer activity in treating breast or prostate cancer comprising the polypeptide BMP-9, as shown in Figure 9, SEQ ID NO: 3, or a homologue thereof, or a fragment thereof which has BMP-9 anti-cancer activity.

4. A polypeptide that has anti-cancer activity in treating breast or prostate cancer comprising the polypeptide BMP-10 as shown in Figure 10, SEQ ID NO:

4. or a homologue thereof, or a fragment thereof which has BMP-10 anti-cancer activity.

5. Use of a nucleic acid molecule according to claims 1 or 2 for treating breast or prostate cancer.

6. Use of a polypeptide according to claims 3 and 4 for treating breast or prostate cancer.

7. A medicament comprising a nucleic acid molecule according to claims 1 or 2; and/or the corresponding polypeptide according to claims 3 and 4.

8. A medicament according to claim 7 wherein the medicament is formulated with a suitable excipient, carrier or emollient.

9. A method for treating breast or prostate cancer comprising administering to an individual to be treated a medically effective amount of the polypeptide according to claim 3 and/or claim 4.

10. A method for treating breast or prostate cancer comprising increasing breast or prostate cellular levels of either exogenous or endogenous polypeptide according to claim 3 or claim 4.

11. A vector adapted to transfect or transform breast or prostate cells wherein said vector includes:

1. at least one copy of the BMP-9 gene according to claim 1 and/or the BMP-10 gene according to claim 2; and/or

2. at least one over-expressing or constitutively active promoter which is either coupled to the BMP-9 gene according to claim 1 and/or the BMP-10 gene according to claim 2; and/or which is designed for insertion into a genome upstream of the native BMP-9 gene according to claim 1 and/or the BMP-10 gene according to claim 2 of breast or prostate cells; whereby transfection or transformation of said cells with said vector results in the enhanced production of BMP-9 and/or BMP-10.

12. A cell line which has been transfected or transformed with a vector encoding at least one copy of the gene for BMP-9 and/or BMP-10 according to claims 1 and/or 2.

13. A cell line according to claim 12 comprising 3T3, PC-3, Du145, MDA231 or T24.

14. Recombinant BMP-9 and/or BMP-10 which has been manufactured by a host cell according to claims 12 or 13.

15. A method for manufacturing recombinant BMP-9 and/or BMP-10 which method comprises:

1. transfecting and/or transforming a host cell with a vector according to claim 11

2. culturing said host cell under conditions that favour expression of BMP-9 and/or BMP-10; and

3. harvesting the BMP-9 and/or BMP-10 polypeptide produced by said host cell.

16. A method according to claim 15 wherein said vector further includes a suitable secretion signal which is functionally coupled to BMP-9 and/or BMP-10 whereby once BMP-9 and/or BMP-10 polypeptide has been produced it is processed through the cell's machinery for secretion and therefore the secreted polypeptide can be harvested from the extra cellular medium.

17. The use of a nucleic acid molecule according to claims 1 or 2 in the manufacture of a medicament for treating breast or prostate cancer.

18. The use of a polypeptide according to claims 3 and 4 in the manufacture of a medicament for treating breast or prostate cancer.