WO2004042080A1

WO2004042080A1 - Cancer therapy determination

Info

Publication number: WO2004042080A1
Application number: PCT/EP2003/012245
Authority: WO
Inventors: Paul Harkin; Jennifer Quinn; Richard Kennedy
Original assignee: The Queen's University Of Belfast
Priority date: 2002-11-05
Filing date: 2003-11-03
Publication date: 2004-05-21
Also published as: AU2003283339A1; GB0226595D0

Abstract

A method of determining a suitable chemotherapeutic agent for an individual comprises the steps of assaying a biological sample from the individual for BRCA1 activity. When the activity is greater than or equal to a normal level, an antimicrotubule chemotherapeutic agent is recommended. When the activity is below a normal level, a DNA damaging chemotherapeutic agent is recommended. BRCA1 activity is determined by assaying for BRCA1 mutation, BRCA1 expression levels, or both. A kit for carrying out the method of the invention is also disclosed.

Description

METHOD FOR RECOMMENDATION OF CANCER THERAPY BASED ON BRCAl ASSAY

1

2

3 Technical Field

4

5 The invention relates to a method of determining a

6 suitable chemotherapeutic agent for use in treating

7 an individual with cancer. The invention also

8 relates to a method of treating cancer, which

9 includes an initial step of determining a suitable 0 chemotherapeutic agent. 1 2 Background Art 3 4 Substantial evidence exists to support a role for 5 BRCAl in mediating the cellular response to DNA 6 damage and in particular double strand DNA breaks. 7 BRCAl becomes hyperphosphorylated in response to 8 various DNA damaging agents including γ-irradiation 9 an effect that is mediated in part by the ATM 0 (Cortez et al . , 1999) and chk2 kinases (Lee et al . , 1 2000) . BRCAl has been shown to co-localise at sites 2 of DNA damage with RAD51, the human homologue of 3 bacterial RecA, which is involved in homologous 4 recombination repair following ionising radiation 5 (Scully et al . , 1997). Furthermore, BRCAl is a component of the RAD50, Mrell, NBS1 complex implicated in homologous recombination and non- homologous end joining (Zhong et al . , 1999). Genetic studies support a role for BRCAl in the repair of double strand breaks. Significantly, ES cells from BRCAl knockout mice exhibit a defect in the repair of double strand breaks by homologous recombination (Moynahan et al . , 1999) . More recently, it has been reported that BRCAl resides within a large DNA repair protein complex called BASC (BRCAl associated genome surveillence complex) that includes various mismatch repair proteins including MLH1, MSH2 , MSH6 suggesting a role for BRCAl in mismatch repair (Wang et al., 2000). Interestingly, BRCAl has also been implicated in the transcription-coupled repair of oxidative induced DNA damage (Gowen et al . , 1998), suggesting that BRCAl is a component of multiple repair pathways, which remain to be fully investigated.

It has also been suggested that BRCAl functions as a sensor of DNA damage relaying signals to either the cell cycle checkpoint or cell death machinery. A number of studies have correlated BRCAl deficiency with defects in cell cycle checkpoint control. Human tumour cells lacking functional BRCAl demonstrate a high frequency of chromosome aneuploidy, characteristic of a defective G2/M checkpoint (Tomlinson et al . , 1998). In addition it has been demonstrated that BRCAl is required for both -phase and G2 arrest following ionizing irradiation an effect that is dependent on differential phosphorylation by ATM (Xu et al . , 2001, Xu B et al . , 2002). Furthermore, genetic instability has been observed in BRCAl exon 11 isoform deficient MEFs resulting from a defective G2/M checkpoint and centrosome amplification (Xu et al., 1999) . It has previously been reported that inducible expression of BRCAl can activate both the G2 and mitotic checkpoints following treatment with paclitaxel, an antimicrotubule agent that functions by inhibiting the depolymerisation of tubulin thereby disrupting the mitotic spindle (Mullan et al . , 2001) . It appears therefore that BRCAl can act in a more general capacity to activate cell cycle checkpoints in response to different types of cellular stress.

In addition to cell cycle arrest, BRCAl has also been implicated in the regulation of apoptosis. It was initially demonstrated that exogenous expression of BRCAl induced apoptosis; an effect that was dependent on c-Jun N-terminal kinase/stress activated protein kinase ( NK/SAPK) activation (Harkin et al . , 1999). It was subsequently reported that BRCAl modulates stress induced apoptotic signalling through a pathway that sequentially involves the H-ras oncogene, MEKK4 , JNK, Fas and Fas ligand and the activation of caspase-9 (Takekawa et al., 1999; Thangaraju et al . , 2000). It has recently been reported that BRCAl dramatically sensitises breast cancer cell lines to interferon gamma mediated apoptosis, indicating that BRCAl may regulate apoptosis in response to diverse stress signals (Andrews et al . , 2002). In contrast, BRCAl deficient cells exhibit a radiosensitive phenotype following exposure to a range of DNA damaging agents including ionizing radiation (Lee et al . , 2000) and the DNA interstrand crosslinking agents, cisplatin and mitomycin C (Moynahan et al . , 2001; Bhattacharyya et al . , 2000). This radiosensitive phenotype, which can be reversed following exogenous expression of wild-type BRCAl, has been associated with a failure in BRCAl dependent DNA damage repair pathways. Consistent with this, antisense inhibition of BRCAl has been reported to decrease repair proficiency and enhance apoptosis in response to cisplatinum (Husain et al . , 1998). It appears therefore that BRCAl in general confers an anti- apoptotic radioresistant phenotype following exposure to DNA damage.

Statements of Invention

According to the invention, there is provided a method of determining a suitable chemotherapeutic agent for an individual, the method comprising the steps of:-

- assaying a biological sample from the individual for BRCAl activity; and

- when the BRCAl activity is greater than or equel to a normal level, recommending an antimicrotubule agent as a chemotherapeutic agent; and/or - when the BRCAl activity is below a normal level, recommending a DNA damaging agent as a chemotherapeutic agent.

Typically, the method is for use with individuals having established cancers. In this regard, the biological sample is preferably a tumour biopsy. In a particularly preferred embodiment, the method is for use with individuals having breast cancer.

The step of assaying for BRCAl activity comprises either the step of assaying for BRCAl expression levels, or assaying for BRCAl mutations, or both. Methods of assaying for BRCAl expression levels, and methods of assaying for BRCAl mutations will be well known to those skilled in the field of the present invention.

In a particular aspect, BRCAl activity will be initially measured by assaying for BRCAl mutation. If mutation is found in the gene, the activity of the gene will be below a normal level. If no mutation is found in the gene, BRCAl expression levels will be assayed.

When the assay involves measuring BRCAl expression levels in a tumour biopsy from a specific tissue in a patient, the normal level may be, for example, defined as the expression level observed in a non- tumourous biopsy from the same tissue in the same patient. This may be determined by quantitative real time RT-PCR, the details and application of which will be well known to those skilled in the field. Thus, for example, when the assay involves measuring BRCAl expression levels in a tumour biopsy from breast tissue of a patient, the threshold level may be approximately defined as that observed in normal breast epithelial tissue from the same patient.

When the assay involves assaying for BRCAl mutations in a tumour biopsy from a specific tissue in a patient, the BRCAl activity will be considered to be below a threshold level when one or more mutations are detected in the gene as compared to the sequence of a normal, unmutated, BRCAl gene. The normal sequence of BRCAl may be obtained from, for example, the sequence published in PubMed. Alternatively, a normal sequence is obtained from DNA from the patient's blood.

In one embodiment of the invention, the activity of the BRCAl gene is determined by assaying for both BRCAl expression levels and BRCAl mutations. Thus, for example, if the BRCAl expression level is found to be normal, it is feasible that BRCAl activity is below a normal level. This would be the case, for example, if the gene included one or more mutations which, while allowing normal expression of the gene, resulted in production of defective gene products. Thus, in one embodiment of the invention, when BRCAl expressions levels are found to be normal, the method includes an additional step of assaying for one or more mutations in the gene, wherein the presence of one or more mutations in the gene is indicataive of BRCAl activity being below a normal level .

Suitably, the antimicrobule agent is selected from the group comprising: taxol, paclitaxel, taxatere, docetaxel and vinorelbine. Typically, the DNA damaging chemotherapeutic agent is selected from the group comprising: cisplatin, etoposide, doxorubicin, bleomycin and cyclophosphamide .

The invention also relates to a method of treating cancer in an individual, the method comprising the steps of : -

- assaying a biological sample from the individual for BRCAl activity; and

- when the BRCAl activity is greater than or equel to a normal level, treating the individual with an antimicrotubule chemotherapeutic agent; or

- when the BRCAl activity is below a normal level, treating the individual with a DNA damaging chemotherapeutic agent.

Typically, the method is for use with an individual having an established cancer. Ideally, the cancer is breast cancer. In this regard, the biological sample is preferably a tumour biopsy. In one embodiment, the treatment involves applying the chemotherapeutic agent directly to the tumour or systemic administration.

The step of assaying for BRCAl activity may include either the step of assaying for BRCAl expression levels, or assaying for BRCAl mutations, or both. Methods of assaying for BRCAl expression levels, and methods of assaying for BRCAl mutations will be well known to those skilled in the field of the present invention.

The normal activity level is determined as described previously. When the assay involves assaying for BRCAl mutations in a tumour biopsy from a specific tissue in a patient, the BRCAl activity will be considered to be below a normal level when one or more mutations are detected.

Suitably, the antimicrobule agent is selected from the group comprising: taxol, paclitaxel, taxatere, docetaxel and vinorelbine. Typically, the DNA damaging chemotherapeutic agent is selected from the group comprising: cisplatin, etoposide, doxorubicin and bleomycin.

Treatment protocols for treating an individual with a chemotherapeutic antimicrotubule agent, or with a DNA damaging chemotherapeutic agent, will be well known to those skilled in the art of chemotherapy and will not be discussed further. The invention also relates to the use of a BRCAl activity assay to determine a suitable chemotherapeutic agent for an individual. Suitably, the BRCAl activity assay may comprise either an assay for BRCAl expression, an assay for one or more mutations in BRCAl, or both.

The invention also relates to a kit for determining a suitable chemotherapeutic agent for an individual, the kit comprising: - means for assaying a biological sample obtained from the individual for BRCAl activity; and - instructions for carrying out the method of the invention. Suitably, the means for assaying for BRCAl activity will comprise assaying for BRCAl mutation or expression levels, or both.

New evidence to clarify the role played by BRCAl as a mediator of apoptosis and to demonstrate that BRCAl functions as a molecular determinant of response to a range of cytotoxic chemotherapeutic agents is described. Specifically, it has been demonstrated that BRCAl abrogates the apoptotic phenotype induced by a range of DNA damaging agents including cisplatin, etoposide and doxorubucin while inducing dramatic sensitivity to a range of antimicrotubule agents including paclitaxel and vinorelbine. The data therefore suggests that BRCAl can regulate both pro and anti-apoptotic pathways depending on the nature of the cellular stress and therefore may impact on how BRCAl mutation carriers may respond to specific chemotherapeutic agents. Thus, the present invention provides a means whereby a determination can be made as to what is the most appropriate and effective cancer therapy for an individual. The determination involves either assaying BRCAl expression levels in a tumour sample from an individual or determining whether the individual carries a BRCAl mutant. Once the determination has been made, it is possible to determine the appropriate treatment for the individual. Thus, if the tumour sample has normal or raised levels of BRCAl expression, the appropriate cancer therapy is treatment with an anitmicrotubule agent. Otherwise, if the tumour sample has less than normal expression levels of BRCAl, or if the BRCAl gene in the tumour tissue is mutated, the appropriate cancer therapy is treatment with a DNA damaging agent.

Brief Description of the Figures

The invention will be more clearly understood from the following description of experiments, with reference to the accompanying Figures in which:

Fig. 1 illustrates that inducible expression of BRCAl sensitizes MBR62-bcl2 cells to paclitaxel- induced apoptosis; Fig. 2 illustrates that BRCAl is required for paclitaxel-induced apoptosis; Fig. 3 illustrates that BRCAl functions as a differential modulator of sensitivity to chemotherapeutic agents; and Fig. 4 illustrates that BRCAl Functions as a differential modulator of chemotherapy induced apoptosis.

Detailed Description of the Invention

RESULTS

BRCAl enhances pacitaxel-induced apoptosis. To study the functional properties of BRCAl, inducible, tetracycline-regulated expression in a MDA435 breast cancer derived cell line, termed MBR62-bcl2, was generated (Harkin et al . , 1999; Mullan et al, 2001) . BRCAl expression levels were shown to increase approximately 4 -fold above endogenous levels in this cell line following tetracycline withdrawal (Figure 1A) . This is well within the physiological levels observed for this protein in mouse models where BRCAl expression has been shown to increase 10-fold during pregnancy a level that is maintained through postlactional involution (Marquis et al . , 1995). It has been previously demonstrated that induction of BRCAl in the presence of Taxol resulted in acute arrest at the G2/M phase of the cell cycle an effect which correlated with BRCAl mediated induction of GADD45 (Mullan et al, 2001) . In order to extend these studies further, the effect of inducible BRCAl expression on the sensitivity of MBR62-bcl2 cells to paclitaxel was evaluated. IC₅₀ curves following treatment of the MBR62-bcl2 cells with a range of different paclitaxel concentrations in the presence and absence of inducible BRCAl expression were generated. Inducible expression of BRCAl increased paclitaxel sensitivity 100-fold in the MBR62-bcl2 cells with an observed decrease in IC₅₀ from 7.7 x 10^"9 M to 9.6 x 10^"11 M (Figure IB). Inducible expression of BRCAl failed to alter the IC₅₀ concentrations of etoposide in these cells suggesting that overexpression of BRCAl did not enhance resistance over that conferred by endogenous expression of BRCAl in these cells (Figure IB) . To determine if BRCAl induced sensitivity to paclitaxel might reflect activation of an apoptotic pathway in these cells, PARP cleavage assays were carried out. The presence of the cleaved PARP 85 kDa product, which is an early hallmark of apoptosis was initially observed as early as 24 hours following inducible expression of BRCAl and was dramatically increased 48 hours following BRCAl induction in the presence of paclitaxel, suggesting that BRCAl can sensitise these cells to paclitaxel induced apoptosis (Figure 1C) . Paclitaxel treatment alone resulted in moderate PARP cleavage 48 hours following treatment as expected (Figures 1C) . Similarly, induction of BRCAl in the absence of paclitaxel treatment also resulted in moderate PARP cleavage consistent with our previous report that exogenous expression of BRCAl alone can induce apoptosis in these cells (Harkin et al . , 1999). BRCAl is required for Taxol mediated apoptosis .

The HCC1937 breast cancer cells that express a single copy of a C-terminal truncated BRCAl protein, which is transcriptionally inactive, was used as a model to investigate the requirement for BRCAl in paclitaxel mediated apoptosis. The HCC1937 derived cell lines, termed HCC-BR18 and HCC-BR116 with constitutive expression of exogenous wild type BRCAl and the control cell lines HCC-EV1 and HCCEV2 reconstituted with empty Rc/CMV vector, were generted. In order to confirm expression of exogenous BRCAl in these cells Northern blot analysis was carried out but was unable to detect the exogenous BRCAl transcript presumably due to extremely low expression levels (data not shown) . As an alternative vector-insert RT-PCR from cDNA derived from the various cell lines was carried out. RT-PCR analysis confirmed expression of exogenous BRCAl expression in both the HCC-BR18 and HCC-BR116 clones (Figure 2A) .

To investigate the ability of BRCAl to sensitise these cells to paclitaxel mediated apoptosis PARP and caspase-3 cleavage assays were carried out. PARP assays demonstrated that the empty vector transfected HCC-EV1 and EV2 cells were resistant to paclitaxel-mediated apoptosis following treatment with 10^"8 M paclitaxel (Figure 2B) . In contrast the BRCAl reconstituted HCC-BR18 and HCC-BR116 cells displayed the presence of the 85kDa cleaved PARP product, indicative of apoptosis following treatment with identical concentrations of paclitaxel (Figure 2B) . Western blots were re-probed with an anti-β- tubulin antibody to confirm equal loading of protein lysates (Figure 2B) . Interestingly, exogenous BRCAl alone failed to induce apoptosis, probably reflecting the extremely low BRCAl expression levels observed in these cells (Figure 2A, 2B, 2C) . Identical data was obtained following caspase-3 cleavage assays . Reconstitution of BRCAl in the HCC- BR18 and HCC-BR116 cells resulted in a dramatic increase in the 17 and 19 kDa cleaved caspase-3 products following treatment with 10^"8 M paclitaxel (Figure 2C) . In contrast the HCC-EV1 and HCC-EV2 cells failed to display the presence of cleaved caspase-3 suggesting that these cells are resistant to paclitaxel mediated apoptosis (Figure 2C) . These studies were extended to look at paclitaxel sensitivity in the T47D breast cancer cell line following inhibition of endogenous BRCAl using an siRNA approach. T47D cells transfected with a BRCAl specific siRNA oligonucleotide displayed a greater than 100-fold increase in resistance to paclitaxel compared to cells transfected with a scrambled control siRNA oligonucleotide with the IC₅₀ values calculated as >1 X 10^'4 M and 2.2 X 10^"6 M respectively (Figure 2D) . Inhibition of endogenous BRCAl following transfection with the BRCAl specific siRNA oligonucleotide was confirmed by RT-PCR and Western blot analysis relative to controls transfected with the scrambled siRNA oligonucleotide (Figure 2E) . BRCAl acts as a differential modulator of sensitivity to chemotherapeutic agents.

The ability of BRCAl to mediate sensitivity to paclitaxel-induced apoptosis prompted the examination of the role played by BRCAl in mediating sensitivity or resistance to a range of chemotherapeutic agents commonly used in cancer treatment. In order to do this, dose response curves were generated for the HCC-BR116 and HCC-EV1 cells following treatment with the antimicrotubule agents paclitaxel and vinorelbine which disrupt the mitotic spindle, the radiomimetic bleomycin which gives rise to double strand breaks, the topoisomerase II poison etoposide, the inter and intra-strand DNA crosslinking agent cisplatin and the antimetabolite, 5-fluorouracil. The HCC-EV1 and HCC-BR116 cells were plated at equal densities and exposed to a range of concentrations of each drug for 72 hours after which time cell counts were carried out. Reconstitution of BRCAl in the HCC1937 cells resulted in a 1000-fold increase in sensitivity of the HCC-BR116 cells to paclitaxel, with the IC₅₀ value decreasing from 6.2 x 10^_δ M in the HCC-EV1 cells compared to 7.7 x 10^"9 M in the HCC-BR116 cells (Figure 3A) . A similar effect was observed following exposure of these cells to vinorelbine. The HCC-EV1 cells were acutely resistant to vinorelbine with an IC₅₀ value of 4.0 x 10^"5 M. In contrast the BRCAl expressing HCC-BR116 cells displayed a greater than 100-fold increase in sensitivity to vinorelbine with an IC₅₀ value calculated at 2.2 x 10^"7 M (Figure 3B) . The opposite effects was observed when these cell lines are exposed to DNA damaging agents. The IC₅₀ value for the HCC-EV1 cells following bleomycin treatment was calculated at 5.0 x 10^~4 M. However, we failed to obtain an IC₅₀ value for the HCC-BR116 cells obtaining a value of >1 x 10^"3 M, suggesting that BRCAl induces extreme resistance to bleomycin in these cells (Figure 3C) . Similarly, treatment with the topoisomerase II poison etoposide, resulted in an IC₅₀ value of 9.1 x 10^"7 M in the HCC-EV1 cells compared to an IC₅₀ value of > 1 x 10^~4 M in the HCC- BR116 cells representing at least a 1000-fold increase in resistance (Figure 3D) . BRCAl also induced a 10-fold resistance to cisplatin with an IC₅₀ value of 2.3 x 10^~7 M observed in the HCC-EV1 cells compared to an IC₅₀ value of 4 x 10^"6 M in the HCC-BR116 cells (Figure 3E) . Interestingly, BRCAl failed to modulate resistance or sensitivity to the antimetabolite 5- fluorouracil with equivalent IC₅₀ values obtained for both cell lines (Figure 3F) , probably reflecting the distinct mode of action of this agent.

BRCAl is a differential modulator of apoptosis.

To investigate if the observed chemosensitivity or resistance induced by BRCAl was due to activation or inhibition of apoptosis PARP assays were carried out 24 hours following treatment with paclitaxel (10^~8 M) , vinorelbine (10^~7M) , cisplatin (10^~6M) , etoposide (10^~6M) , bleomycin (10^~4 M) and 5-fluorouracil (10^~ ⁵M) . PARP assays demonstrated cleavage of the 116kDa PARP product into its 85kDa cleaved fragment when HCC-BR116 cells were treated with either pacitaxel or vinorelbine (Figure 4A, 4B) . In agreement with the drug sensitivity assays the HCC-EV1 cells were completely resistant to paclitaxel and vinorelbine induced apoptosis at these concentrations (Figure 4A, 4B) . The exact opposite was observed following treatment of the HCC-BR116 cells with the DNA damaging agents, bleomycin, etoposide and cisplatin. HCC-BR116 cells were resistant to apoptosis induced by these agents while the HCC-EV1 cells displayed the characteristic 85kDa PARP cleavage product indicative of apoptosis (Figure 4A) .

It is possible that the differences in sensitivity or resistance observed in the HCC-BR116 and HCC-EV1 cells may be due to clonal variation. Therefore, to rule this out these experiments were repeated with the HCC-BR18 and HCC-EV2 cells using paclitaxel and etoposide as representative agents. In keeping with our previous observations reconstitution of BRCAl in the HCC-BR18 cells resulted in a greater than 3000- fold increase in sensitivity to paclitaxel compared to the HCC-EV2 cells (Figure 4C) . Similarly, the HCC-BR18 cells were observed to be 100-fold more resistant to etoposide compared to the HCC-EV2 cells (Figure 4C) . Finally, to illustrate that this difference reflected a corresponding increase/decrease in apoptosis we carried out PARP cleavage assays in these cells. The HCC-BR18 cells displayed the characteristic 85 kDa PARP cleavage product following treatment with paclitaxel but not with etoposide while the reverse was true for the HCC-EV2 cells (Figure 4C) .

It is demonstrated herein that BRCAl acts as a molecular determinant of response to a range of chemotherapeutic agents commonly used in the treatment of a variety of cancers. In particular, BRCAl dramatically sensitises HCC1937 cells to apoptosis induced by the antimicrotubule agents paclitaxel and vinorelbine while inducing resistance to a range of DNA damaging agents most notably those that induce double strand breaks in DNA including bleomycin and etoposide. The observation that BRCAl is required for apoptosis induced by mitotic spindle poisons supports a number of previous studies, which have implicated BRCAl in the regulation of the mitotic checkpoint. A dominant negative BRCAl construct encoding the COOH terminus was shown to abrogate G2/M arrest in response to the spindle poison colchicine (Larson et al . , 1997). Moreover, BRCAl has been shown to localise to centromeres via a physical interaction with γ-tubulin, suggesting that BRCAl plays a direct role in the accurate segregation of duplicated chromosomes during mitosis (Hsu and White, 1998) . It has also been reported that BRCAl is required for the decatenation and therefore accurate separation of sister chromatids at anaphase lending further support for a role in mitotic checkpoint regulation (Deming et al, 2001) . Genetic studies support these observations revealing aneuploidy characteristic of a defect in the mitotic checkpoint in BRCAl exon 11 isoform-de icient cells, (Xu, et al., 1999). Our observation that BRCAl can specifically sensitise breast cancer cell lines to apoptosis induced by antimicrotubule agents is intriguing in the light of these other studies. Antimicrotubule agents function by promoting the stabilization or destabilization of tubulin which in turn prevents sister chromatid separation and arrest at the metaphase to anaphase transition (Wang et al . , 2000) . The mechanism through which spindle poisons such as paclitaxel induce apoptosis are still a matter of speculation however it has been suggested that different pathways are activated depending on the concentrations used. Higher concentrations of paclitaxel in the region of 200 nM (2 x 10^"4 M) to 30 μM (3 x 10^"3 M) give rise to microtubule damage and activation of JNK dependent apoptosis (Wang et al . , 2000). In contrast, paclitaxel concentrations of 10-100 nM (1 x 10^"5 - 1 x 10^"4 M) result in suppression of microtubule dynamics and mitotic arrest in the absence of microtubule damage (Wang et al . , 2000) . In this study we demonstrate that BRCAl sensitises HCC1937 cells to apoptosis at concentrations as low as 10 nM (lO^-5 M) suggesting that BRCAl may specifically play a role in mediated apoptosis at lower concentrations. Indeed, cellular proliferation assays clearly display a biphasic decrease in cell viability with BRCAl mediating sensitivity at paclitaxel concentrations lower than 10^"5 M followed by a BRCAl independent increase in sensitivity at concentrations higher than 10^"5 M paclitaxel (Figure 3A) . Furthermore we demonstrate that inhibition of exogenous BRCAl results in a dramatic increase in resistance of breast cancer cells to paclitaxel induced apoptosis suggesting that BRCAl expression levels are critical for mediating apoptosis in response to this class of chemotherapeutic agent.

The observation that BRCAl sensitises HCC1937 cells to antimicrotubule induced apoptosis is in direct contrast to that observed following exposure to DNA damaging agents where BRCAl clearly mediates resistance. We observed a dramatic increase in resistance to apoptosis in HCC1937 cells reconstituted with wild type BRCAl following treatment with the radiomemetic bleomycin and the topoisomerase II inhibitor etoposide both of which introduce double strand breaks in DNA although through different mechanisms. It is also demonstrated that BRCAl can induce resistance to cisplatin-mediated apoptosis suggesting that BRCAl may also be involved in the repair of inter and intra strand crosslinks. These data are in agreement with previous reports which demonstrate that reconstitution of BRCAl in HCC1937 cells can rescue the radioresistant phenotype although this had not been linked to inhibition of apoptosis (Lee et al . , 2000; Moynahan et al . , 2001; Bhattacharyya et al . , 2000) . Inhibition of endogenous BRCAl has however been reported to decrease the efficiency of repair and enhance apoptosis in response to cisplatin (Husain et al . , 1998) . The mechanism through which BRCAl rescues the resistant phenotype in response to these agents has yet to be defined however it is likely to be linked to activation of BRCAl dependent DNA repair pathways (Moynahan et al . , 1999) . It has been reported that BRCAl induces activation of genes involved in nucleotide excision repair in a p53 independent manner following exposure to UV- irradiation (Hartman and Ford, 2002) . In addition it has been demonstrated that exogenous expression of BRCAl mediates the induction of growth arrest and DNA repair genes in a p53 dependent manner in the absence of DNA damage (MacLachlan et al 2002) . Since the HCC1937 cell line also harbours mutant p53, our data would suggest that BRCAl mediated inhibition of apoptosis in response to DNA damage and conversely induction of apoptosis in response to spindle poisons occurs independent of p53. The mechanistic basis of BRCAl induced apoptosis in response to paclitaxel and vinorelbine remains to be defined, however it is reasonable to speculate that BRCAl may also regulate key pro-apoptotic target genes following disruption of the mitotic spindle. Further elucidation of this pathway may therefore provide critical new information on the mechansim of paclitaxel sensitivity and resistance in breast tumours. BRCAl failed to modulate the cellular response to 5-fluorouracil, which functions by inhibiting thymidylate synthase (TS) , resulting in thymidine starvation leading to apoptosis. This observation is consistent with the fact that 5- fluorouracil does not directly damage DNA, although it metabolite xx does get incorporated into DNA and RNA and can indirectly lead to DNA damage. In summary therefore the present invention is based on the surprising finding that BRCAl functions as a molecular determinant of response to a range of different chemotherapeutic agents.

EXPERIMENTAL PROCEDURES

Assaying for BRCAl mutation or expression levels. BRCAl mutations are detected by a range of methodologies including two-dimensional gene scanning, denaturing high performance liquid chromatography, enzymatic mutation detection, single strand conformation polymorphism analysis, RNA/DNA- based protein truncation test, protein truncation assays (Andrulis et al, 2002) . More recent methods include allele-specific gene expression analysis (Montagna et al, 2002) . BRCAl expression levels are typically estimated by immunohistochemistry or by quantitative real time PCR (Rio et al 1999; Russell et al 2000) .

Generation of cell lines MBR62-bcl2 cells were generated as previously described (Mullan et al . , 2001). HCCEV1 and HCCBR1 cells were generated by the stable transfection of the HCC1937 breast cancer cell line with the Rc/CMV (Invitrogen) or Rc/CMV-BRCAl constructs under selection with geneticin (G418) (Sigma) . The Rc/CMV construct that expresses the neomycin resistance gene was obtained from Invitrogen and the Rc/CMV- BRCAl construct was generated as previously described (Andrews et al . , 2002). These constructs were transfected into the HCC1937 cell line using Genejuice methodology (Invitrogen) according to manufacturer's instructions. Transfectant cells were selected in 0.2mg/ml G418, assessed by RT-PCR for expression of BRCAl and the resultant HCC-EV1, HCC- EV2 and HCC-BR116 and HCC-BR18 clones were selected.

Cell Culture The MBR62-bcl2 cell line was maintained as described previously (Mullan et al . , 2002). The HCC1937 and T47D breast cancer cell lines were maintained in RPMI supplemented with 20% Foetal Calf Serum, lmM sodium pyruvate, (lOOμg/ml) penicillin-streptomycin (all from Life Technologies, Inc) . The HCC-EV1, EV2 , BR116 and HCC-BR18 cell lines were grown in HCC1937 medium supplemented with 0.2mg/ml G418.

Drug sensitivity and growth assays For growth assays MBR62-bcl2 cells were seeded in 24 well plates and induced to express BRCAl by withdrawal of tetracycline from medium and at the same time were treated or untreated with 10^~8M Taxol (Bristol Myers Squibb) . For drug sensitivity assays HCC-EV1 and HCCBR1 cells were seeded onto 24-well tissue culture plates at a density of 100,000 cells per well. After 24 hours, cells were incubated in HCC1937 medium supplemented with the described concentration ranges of pacitaxel (Bristol Myers Squibb) , Vinorelbine (Novartis) , Cisplatin (Faulding Pharmaceuticals) , Etopo-phos (Bristol Myers Squibb) and 5-Fluorouracil (David Bull Laboratories) . After 72 hours of continuous drug exposure, cells were detached and counted using a Z2 particle and size analyser (Coulter, Miami, FL) . IC₅₀ values were calculated for each drug from the respective sigmoidal dose response curves using Prism software.

Antibodies and Western blot analysis MBR62-bcl2, HCC-EV and HCC-BR116 cells were treated in the presence of 10^"8M Taxol, 10^"7M Vinorelbine, 10^" ⁶M Cisplatin, 10^"6M Etoposide and 10^"5M 5- Fluorouracil . Protein lysates were extracted in RIPA buffer (50μM Hepes, pH 7.0, 150mM NaCl, 0.1% Triton X-100, 0.1% Sodium Deoxycholate, 0.1% SDS) , separated on a 12% SDS polyacrylamide gel and transferred to PVDF membrane followed by immunoblotting. BRCAl IP Westerns were carried out using the rabbit polyclonal C-20 (Santa Cruz) and the mouse monoclonal AB1 (Calbiochem) . For PARP assays, the monoclonal antibody #556494 (BD Bioscience) was used which specifically recognises the full length 116kD PARP protein and its 85kD and 25kD cleaved products. Cleaved caspase-3 was detected using the rabbit polyclonal Cleaved Caspase-3 (Asp 175) antibody #9661 (Cell Signaling) that specifically recognises the cleaved 17 and 19 kDa Caspase-3 products. β-Tubulin was detected using the monoclonal antibody T-4026 (Sigma) .

SiRNA T47D cells were transfected with a BRCAl specific siRNA ologinucleotide or a scrambled control oligonucleotide and treated with a range of paclitaxel concentrations. After 72 hours of continuous drug exposure, cells were detached and counted using a Z2 particle and size analyser (Coulter, Miami, FL) . IC₅₀ values were calulated from the respective sigmoidal dose response curves. BRCAl siRNA oligonucleotide 5'- aac ctg tct cca caa agt gtg - 3 ' Control siRNA oligonucleotide 5'- aaa ace cgu cua ggc ugu uac - 3 '

The invention is not limited to the embodiments hereinbefore described which may be varied in detail without departing from the invention.

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Claims

1. A method of determining a suitable chemotherapeutic agent for an individual, the method comprising the steps of : -

- assaying a biological sample from the individual for BRCAl activity; and

- when the BRCAl activity is greater than or equel to a normal level, recommending an antimicrotubule agent as a chemotherapeutic agent; or

- when the BRCAl activity is below a normal level, recommending a DNA damaging agent as a chemotherapeutic agent.

2. A method as claimed in Claim 1 for use with individuals having established cancers.

3. A method as claimed in Claim 2 for use with individuals having established breast cancer.

4. A method as claimed in any preceding Claim in which the biological sample to be assayed is a tumour biopsy.

5. A method as claimed in any preceding Claim in which the step of assaying for BRCAl activity comprises the step of assaying for BRCAl mutations.

6. A method as claimed in any of Claims 1 to 4 in which the step of assaying for BRCAl activity comprises assaying for BRCAl expression levels.

7. A method as claimed in any preceding Claim in which the step of assaying for BRCAl activity comprises assaying for BRCAl mutations and assaying for BRCAl expression levels.

8. A method as claimed in any preceding Claim in which BRCAl expression levels are assayable by quantitative real time RT-PCR.

9. A method as claimed in any preceding Claim in which the antimicrobule agent is selected from the group comprising: taxol, paclitaxel, taxatere, docetaxel and vinorelbine.

10. A method as claimed in any preceding in which the DNA damaging chemotherapeutic agent is selected from the group comprising: cisplatin, etoposide, doxorubicin, bleomycin and cyclophosphamide.

11. A method of treating cancer in an individual, the method comprising the steps of : -

- assaying a biological sample from the individual for BRCAl activity; and

- when the BRCAl activity is below a threshold level, treating the individual with a DNA damaging chemotherapeutic agent.

12. A method as claimed in Claim 11 for use with an individual having an established cancer.

13. A method as claimed in Claim 12 in which the established cancer is breast cancer.

14. A method as claimed in any of Claims 11 to 13 in which the biological sample is a tumour biopsy.

15. A method as claimed in any of Claims 11 to 14 in which the antimicrobule agent is selected from the group comprising: taxol, paclitaxel, taxatere, docetaxel and vinorelbine.

16. A method as claimed in any of Claims 11 to 14 in which the DNA damaging chemotherapeutic agent is selected from the group comprising: cisplatin, etoposide, doxorubicin and bleomycin.

17. Use of a BRCAl activity assay to determine a suitable chemotherapeutic agent for an individual.

18. Use as claimed in Claim 17 in which the BRCAl activity assay comprises an assay for one or more mutations in BRCAl.

19. Use as claimed in Claim 17 in which the BRCAl activity assay comprises an assay for BRCAl expression levels.