Nuala McCabe

Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy.

BRCA1 and BRCA2 are important for DNA double-strand break repair by homologous recombination, and mutations in these genes predispose to breast and other cancers. Poly(ADP-ribose) polymerase (PARP) is an enzyme involved in base excision repair, a key pathway in the repair of DNA single-strand breaks. We show here that BRCA1 or BRCA2 dysfunction unexpectedly and profoundly sensitizes cells to the inhibition of PARP enzymatic activity, resulting in chromosomal instability, cell cycle arrest and subsequent apoptosis. This seems to be because the inhibition of PARP leads to the persistence of DNA lesions normally repaired by homologous recombination. These results illustrate how different pathways cooperate to repair damage, and suggest that the targeted inhibition of particular DNA repair pathways may allow the design of specific and less toxic therapies for cancer.

BRCA1 and GADD45 mediated G2/M cell cycle arrest in response to antimicrotubule agents

BRCA1 is a tumour suppressor gene implicated in the predisposition to early onset breast and ovarian cancer. We have generated cell lines with inducible expression of BRCA1 to evaluate its role in mediating the cellular response to various chemotherapeutic drugs commonly used in the treatment of breast and ovarian cancer. Induction of BRCA1 in the presence of Taxol and Vincristine resulted in a dramatic increase in cell death; an effect that was preceded by an acute arrest at the G2/M phase of the cell cycle and which correlated with BRCA1 mediated induction of GADD45. A proportion of the arrested cells were blocked in mitosis suggesting activation of both a G2 and a mitotic spindle checkpoint. In contrast, no specific interaction was observed between BRCA1 induction and treatment of cells with a range of DNA damaging agents including Cisplatin and Adriamycin. Inducible expression of GADD45 in the presence of Taxol induced both G2 and mitotic arrest in these cells consistent with a role for GADD45 in contributing to these effects. Our results support a role for both BRCA1 and GADD45 in selectively regulating a G2/M checkpoint in response to antimicrotubule agents and raise the possibility that their expression levels in cells may contribute to the toxicity observed with these compounds.

DNA Polymerases as Potential Therapeutic Targets for Cancers Deficient in the DNA Mismatch Repair Proteins MSH2 or MLH1

Summary Synthetic sickness/lethality (SSL) can be exploited to develop therapeutic strategies for cancer. Deficiencies in the tumor suppressor proteins MLH1 and MSH2 have been implicated in cancer. Here we demonstrate that deficiency in MSH2 is SSL with inhibition of the DNA polymerase POLB, whereas deficiency in MLH1 is SSL with DNA polymerase POLG inhibition. Both SSLs led to the accumulation of 8-oxoG oxidative DNA lesions. MSH2/POLB SSL caused nuclear 8-oxoG accumulation, whereas MLH1/POLG SSL led to a rise in mitochondrial 8-oxoG levels. Both SSLs were rescued by silencing the adenine glycosylase MUTYH, suggesting that lethality could be caused by the formation of lethal DNA breaks upon 8-oxoG accumulation. These data suggest targeted, mechanism-based therapeutic approaches.

BRCA2-deficient CAPAN-1 cells are extremely sensitive to the inhibition of poly (ADP-ribose) polymerase: An issue of potency

We have previously demonstrated that deficiency of either the BRCA1 or BRCA2 breast cancer susceptibility proteins confers substantial cellular sensitivity to the inhibition of Poly(ADP-Ribose) polymerase (PARP). PARP is a key enzyme in the repair of single strand DNA damage via the Base Excision Repair pathway. We suggested that PARP inhibition produces persistent single-strand DNA breaks or gaps which degenerate into stalled replication forks and double-strand breaks, which may be repaired by homologous recombination, a process partially dependent on BRCA1 and BRCA2. It has recently been suggested that our results might be limited to certain BRCA2 mutations as the CAPAN-1 cell line, which carries a naturally occurring 6174delT mutation in one BRCA2 allele accompanied by loss of the wild-type allele, is apparently insensitive to two PARP inhibitors 3-aminobenzamide (IC50 33??M) and NU1025 (IC50 400nM). Here we show that CAPAN-1 cells are in fact very sensitive to the potent PARP inhibitors KU0058684 (IC50 3.2nM) and KU0058948 (IC50 3.4nM). In contrast, our results reveal much less sensitivity to a chemically related but much less active compound KU0051529 (IC50 730nM) and to NU1025. These results confirm that treatment with potent PARP inhibitors remains an exciting potential therapy for cancers involving BRCA1 or BRCA2 deficiency.

Targeting Tankyrase 1 as a therapeutic strategy for BRCA-associated cancer.

The BRCA1 and BRCA2 proteins are involved in the maintenance of genome stability and germ-line loss-of-function mutations in either BRCA1 or BRCA2 strongly predispose carriers to cancers of the breast and other organs. It has been demonstrated previously that inhibiting elements of the cellular DNA maintenance pathways represents a novel therapeutic approach to treating tumors in these individuals. Here, we show that inhibition of the telomere-associated protein, Tankyrase 1, is also selectively lethal with BRCA deficiency. We also demonstrate that the selectivity caused by inhibition of Tankyrase 1 is associated with an exacerbation of the centrosome amplification phenotype associated with BRCA deficiency. We propose that inhibition of Tankyrase 1 could be therapeutically exploited in BRCA-associated cancers.

BRCA1 Interacts with and Is Required for Paclitaxel-Induced Activation of Mitogen-Activated Protein Kinase Kinase Kinase 3

BRCA1 has been implicated in a number of cellular processes, including transcriptional regulation, DNA damage repair, cell cycle arrest, and apoptosis. We identified mitogen-activated protein kinase (MAPK) kinase kinase 3 (MEKK3), an upstream regulator of the c-Jun NH2-terminal kinase/stress-activated protein kinase and p38/MAPK pathways, as a novel BRCA1-interacting protein in a yeast two-hybrid screen and confirmed the interaction by coimmunoprecipitation in mammalian cells. Deletion mapping demonstrated that amino acids 1611–1863 are required to mediate the interaction with MEKK3 in yeast. BRCA1 disease-associated mutations abrogated the interaction in yeast, and BRCA1 failed to interact with MEKK3 in BRCA1 mutant HCC1937 breast cancer cells. We demonstrate that small interfering RNA-based inhibition of endogenous BRCA1 reduces MEKK3 kinase activity and conversely that inducible expression of BRCA1 activates MEKK3 and p38/MAPK. Finally, we demonstrate using complementary approaches that BRCA1 is required for paclitaxel-induced activation of MEKK3. These data indicate that BRCA1 is a key regulator of the paclitaxel-induced stress response pathway and suggest that the ability of BRCA1 to associate with, and mediate the activation of, MEKK3 represents a potential mechanism through which this pathway is regulated.

Potentiation of Inflammatory CXCL8 Signalling Sustains Cell Survival in PTEN-deficient Prostate Carcinoma

BACKGROUND Inflammation and genetic instability are enabling characteristics of prostate carcinoma (PCa). Inactivation of the tumour suppressor gene phosphatase and tensin homolog (PTEN) is prevalent in early PCa. The relationship of PTEN deficiency to inflammatory signalling remains to be characterised. OBJECTIVE To determine how loss of PTEN functionality modulates expression and efficacy of clinically relevant, proinflammatory chemokines in PCa. DESIGN, SETTING, AND PARTICIPANTS Experiments were performed in established cell-based PCa models, supported by pathologic analysis of chemokine expression in prostate tissue harvested from PTEN heterozygous (Pten(+/-)) mice harbouring inactivation of one PTEN allele. INTERVENTIONS Small interfering RNA (siRNA)- or small hairpin RNA (shRNA)-directed strategies were used to repress PTEN expression and resultant interleukin-8 (CXCL8) signalling, determined under normal and hypoxic culture conditions. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Changes in chemokine expression in PCa cells and tissue were analysed by real-time polymerase chain reaction (PCR), immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry; effects of chemokine signalling on cell function were assessed by cell cycle analysis, apoptosis, and survival assays. RESULTS AND LIMITATIONS Transient (siRNA) or prolonged (shRNA) PTEN repression increased expression of CXCL8 and its receptors, chemokine (C-X-C motif) receptor (CXCR) 1 and CXCR2, in PCa cells. Hypoxia-induced increases in CXCL8, CXCR1, and CXCR2 expression were greater in magnitude and duration in PTEN-depleted cells. Autocrine CXCL8 signalling was more efficacious in PTEN-depleted cells, inducing hypoxia-inducible factor-1 (HIF-1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription and regulating genes involved in survival and angiogenesis. Increased expression of the orthologous chemokine KC was observed in regions displaying atypical cytologic features in Pten(+/-) murine prostate tissue relative to normal epithelium in wild-type PTEN (Pten(WT)) glands. Attenuation of CXCL8 signalling decreased viability of PCa cells harbouring partial or complete PTEN loss through promotion of G1 cell cycle arrest and apoptosis. The current absence of clinical validation is a limitation of the study. CONCLUSIONS PTEN loss induces a selective upregulation of CXCL8 signalling that sustains the growth and survival of PTEN-deficient prostate epithelium.

Activation of STING-Dependent Innate Immune Signaling By S-Phase-Specific DNA Damage in Breast Cancer.

Background: Previously we identified a DNA damage response–deficient (DDRD) molecular subtype within breast cancer. A 44-gene assay identifying this subtype was validated as predicting benefit from DNA-damaging chemotherapy. This subtype was defined by interferon signaling. In this study, we address the mechanism of this immune response and its possible clinical significance. Methods: We used immunohistochemistry (IHC) to characterize immune infiltration in 184 breast cancer samples, of which 65 were within the DDRD subtype. Isogenic cell lines, which represent DDRD-positive and -negative, were used to study the effects of chemokine release on peripheral blood mononuclear cell (PBMC) migration and the mechanism of immune signaling activation. Finally, we studied the association between the DDRD subtype and expression of the immune-checkpoint protein PD-L1 as detected by IHC. All statistical tests were two-sided. Results: We found that DDRD breast tumors were associated with CD4+ and CD8+ lymphocytic infiltration (Fisher’s exact test P < .001) and that DDRD cells expressed the chemokines CXCL10 and CCL5 3.5- to 11.9-fold more than DNA damage response–proficient cells (P < .01). Conditioned medium from DDRD cells statistically significantly attracted PBMCs when compared with medium from DNA damage response–proficient cells (P < .05), and this was dependent on CXCL10 and CCL5. DDRD cells demonstrated increased cytosolic DNA and constitutive activation of the viral response cGAS/STING/TBK1/IRF3 pathway. Importantly, this pathway was activated in a cell cycle–specific manner. Finally, we demonstrated that S-phase DNA damage activated expression of PD-L1 in a STING-dependent manner. Conclusions: We propose a novel mechanism of immune infiltration in DDRD tumors, independent of neoantigen production. Activation of this pathway and associated PD-L1 expression may explain the paradoxical lack of T-cell-mediated cytotoxicity observed in DDRD tumors. We provide a rationale for exploration of DDRD in the stratification of patients for immune checkpoint–based therapies.

Mechanistic Rationale to Target PTEN-Deficient Tumor Cells with Inhibitors of the DNA Damage Response Kinase ATM

Ataxia telangiectasia mutated (ATM) is an important signaling molecule in the DNA damage response (DDR). ATM loss of function can produce a synthetic lethal phenotype in combination with tumor-associated mutations in FA/BRCA pathway components. In this study, we took an siRNA screening strategy to identify other tumor suppressors that, when inhibited, similarly sensitized cells to ATM inhibition. In this manner, we determined that PTEN and ATM were synthetically lethal when jointly inhibited. PTEN-deficient cells exhibited elevated levels of reactive oxygen species, increased endogenous DNA damage, and constitutive ATM activation. ATM inhibition caused catastrophic DNA damage, mitotic cell cycle arrest, and apoptosis specifically in PTEN-deficient cells in comparison with wild-type cells. Antioxidants abrogated the increase in DNA damage and ATM activation in PTEN-deficient cells, suggesting a requirement for oxidative DNA damage in the mechanism of cell death. Lastly, the ATM inhibitor KU-60019 was specifically toxic to PTEN mutant cancer cells in tumor xenografts and reversible by reintroduction of wild-type PTEN. Together, our results offer a mechanistic rationale for clinical evaluation of ATM inhibitors in PTEN-deficient tumors.

Uncovering BRCA1-regulated signalling pathways by microarray-based expression profiling

The introduction of microarray technology to the scientific and medical communities has dramatically changed the way in which we now address basic biomedical questions. Expression profiling using microarrays facilitates an experimental approach where alterations in the transcript level of entire transcriptomes can be simultaneously assayed in response to defined stimuli. We have used microarray analysis to identify downstream transcriptional targets of the BRCA1 (Breast Cancer 1) tumour-suppressor gene as a means of defining its function. BRCA1 has been implicated in the predisposition to early onset breast and ovarian cancer and while its exact function remains to be defined, roles in DNA repair, cell-cycle control and transcriptional regulation have been implied. In the current study we have generated cell lines with tetracycline-regulated, inducible expression of BRCA1 as a tool to identify genes, which might represent important effectors of BRCA1 function. Oligonucleotide array-based expression profiling identified a number of genes that were upregulated at various times following inducible expression of BRCA1 including the DNA damage-responsive gene GADD45 (Growth Arrest after DNA Damage). Identified targets were confirmed by Northern blot analysis and their functional significance as BRCA1 targets examined.