ATM Kinase Inhibition Preferentially Sensitises PTEN-Deficient Prostate Tumour Cells to Ionising Radiation.

Radical radiotherapy, often in combination with hormone ablation, is a safe and effective treatment option for localised or locally-advanced prostate cancer. However, up to 30% of patients with locally advanced PCa will go on to develop biochemical failure, within 5 years, following initial radiotherapy. Improving radiotherapy response is clinically important since patients exhibiting biochemical failure develop castrate-resistant metastatic disease for which there is no curative therapy and median survival is 8-18 months. The aim of this research was to determine if loss of PTEN (highly prevalent in advanced prostate cancer) is a novel therapeutic target in the treatment of advanced prostate cancer. Previous work has demonstrated PTEN-deficient cells are sensitised to inhibitors of ATM, a key regulator in the response to DSBs. Here, we have shown the role of PTEN in cellular response to IR was both complex and context-dependent. Secondly, we have confirmed ATM inhibition in PTEN-depleted cell models, enhances ionising radiation-induced cell killing with minimal toxicity to normal prostate RWPE-1 cells. Furthermore, combined treatment significantly inhibited PTEN-deficient tumour growth compared to PTEN-expressing counterparts, with minimal toxicity observed. We have further shown PTEN loss is accompanied by increased endogenous levels of ROS and DNA damage. Taken together, these findings provide pre-clinical data for future clinical evaluation of ATM inhibitors as a neoadjuvant/adjuvant in combination with radiation therapy in prostate cancer patients harbouring PTEN mutations.

miR-191 promotes radiation resistance of prostate cancer through interaction with RXRA.

Radiation therapy is a common treatment for prostate cancer, however recurrence remains a problem. MicroRNA expression is altered in prostate cancer and may promote therapy resistance. Through bioinformatic analyses of TCGA and CPC-GENE patient cohorts, we identified higher miR-191 expression in tumor versus normal tissue, and increased expression in higher Gleason scores. In vitro and in vivo experiments demonstrated that miR-191 overexpression promotes radiation survival, and contributes to a more aggressive phenotype. Retinoid X receptor alpha, RXRA, was discovered to be a novel target of miR-191, and knockdown recapitulated radioresistance. Furthermore, treatment of prostate cancer cells with the RXRA agonist 9-cis-retinoic acid restored radiosensitivity. Supporting this relationship, patients with high miR-191 and low RXRA abundance experienced quicker biochemical recurrence. Reduced RXRA translated to a higher risk of distant failure after radiotherapy. Notably, this miR-191/RXRA interaction was conserved in a novel primary cell line derived from radiorecurrent prostate cancer. Together, our findings demonstrate that miR-191 promotes prostate cancer survival after radiotherapy, and highlights retinoids as a potential option to improve radiotherapy response.

Defining the molecular evolution of extrauterine high grade serous carcinoma.

OBJECTIVE: High grade serous carcinoma (HGSC) is the most common and most aggressive, subtype of epithelial ovarian cancer. It presents as advanced stage disease with poor prognosis. Recent pathological evidence strongly suggests HGSC arises from the fallopian tube via the precursor lesion; serous tubal intraepithelial carcinoma (STIC). However, further definition of the molecular evolution of HGSC has major implications for both clinical management and research. This study aims to more clearly define the molecular pathogenesis of HGSC. METHODS: Six cases of HGSC were identified at the Northern Ireland Gynaecological Cancer Centre (NIGCC) that each contained ovarian HGSC (HGSC), omental HGSC (OMT), STIC, normal fallopian tube epithelium (FTE) and normal ovarian surface epithelium (OSE). The relevant formalin-fixed paraffin embedded (FFPE) tissue samples were retrieved from the pathology archive via the Northern Ireland Biobank following attaining ethical approval (NIB11:005). Full microarray-based gene expression profiling was performed on the cohort. The resulting data was analysed bioinformatically and the results were validated in a HGSC-specific in-vitro model. RESULTS: The carcinogenesis of HGSC was investigated and showed the molecular profile of HGSC to be more closely related to normal FTE than OSE. STIC lesions also clustered closely with HGSC, indicating a common molecular origin. CONCLUSION: This study provides strong evidence suggesting that extrauterine HGSC arises from the fimbria of the distal fallopian tube. Furthermore, several potential pathways were identified which could be targeted by novel therapies for HGSC. These findings have significant translational relevance for both primary prevention and clinical management of the disease.

DNA Repair Deficiency in Breast Cancer: Opportunities for Immunotherapy.

Historically the development of anticancer treatments has been focused on their effect on tumor cells alone. However, newer treatments have shifted attention to targets on immune cells, resulting in dramatic responses. The effect of DNA repair deficiency on the microenvironment remains an area of key interest. Moreover, established therapies such as DNA damaging treatments such as chemotherapy and PARP inhibitors further modify the tumor microenvironment. Here we describe DNA repair pathways in breast cancer and activation of innate immune pathways in DNA repair deficiency, in particular, the STING (STimulator of INterferon Genes) pathway. Breast tumors with DNA repair deficiency are associated with upregulation of immune checkpoints including PD-L1 (Programmed Death Ligand-1) and may represent a target population for single agent or combination immunotherapy treatment.

A Novel Role for Cathepsin S as a Potential Biomarker in Triple Negative Breast Cancer.

Cathepsin S (CTSS) has previously been implicated in a number of cancer types, where it is associated with poor clinical features and outcome. To date, patient outcome in breast cancer has not been examined with respect to this protease. Here, we carried out immunohistochemical (IHC) staining of CTSS using a breast cancer tissue microarray in patients who received adjuvant therapy. We scored CTSS expression in the epithelial and stromal compartments and evaluated the association of CTSS expression with matched clinical outcome data. We observed differences in outcome based on CTSS expression, with stromal-derived CTSS expression correlating with a poor outcome and epithelial CTSS expression associated with an improved outcome. Further subtype characterisation revealed high epithelial CTSS expression in TNBC patients with improved outcome, which remained consistent across two independent TMA cohorts. Further in silico gene expression analysis, using both in-house and publicly available datasets, confirmed these observations and suggested high CTSS expression may also be beneficial to outcome in ER-/HER2+ cancer. Furthermore, high CTSS expression was associated with the BL1 Lehmann subgroup, which is characterised by defects in DNA damage repair pathways and correlates with improved outcome. Finally, analysis of matching IHC analysis reveals an increased M1 (tumour destructive) polarisation in macrophage in patients exhibiting high epithelial CTSS expression. In conclusion, our observations suggest epithelial CTSS expression may be prognostic of improved outcome in TNBC. Improved outcome observed with HER2+ at the gene expression level furthermore suggests CTSS may be prognostic of improved outcome in ER- cancers as a whole. Lastly, from the context of these patients receiving adjuvant therapy and as a result of its association with BL1 subgroup CTSS may be elevated in patients with defects in DNA damage repair pathways, indicating it may be predictive of tumour sensitivity to DNA damaging agents.

FES-related tyrosine kinase activates the insulin-like growth factor-1 receptor at sites of cell adhesion.

IGF-1 receptor (IGF-1R) and integrin cooperative signaling promotes cancer cell survival, proliferation, and motility, but whether this influences cancer progression and therapy responses is largely unknown. Here we investigated the non-receptor tyrosine adhesion kinase FES-related (FER), following its identification as a potential mediator of sensitivity to IGF-1R kinase inhibition in a functional siRNA screen. We found that FER and the IGF-1R co-locate in cells and can be co-immunoprecipitated. Ectopic FER expression strongly enhanced IGF-1R expression and phosphorylation on tyrosines 950 and 1131. FER phosphorylated these sites in an IGF-1R kinase-independent manner and also enhanced IGF-1-mediated phosphorylation of SHC, and activation of either AKT or MAPK-signaling pathways in different cells. The IGF-1R, ß1 Integrin, FER, and its substrate cortactin were all observed to co-locate in cell adhesion complexes, the disruption of which reduced IGF-1R expression and activity. High FER expression correlates with phosphorylation of SHC in breast cancer cell lines and with a poor prognosis in patient cohorts. FER and SHC phosphorylation and IGF-1R expression could be suppressed with a known anaplastic lymphoma kinase inhibitor (AP26113) that shows high specificity for FER kinase. Overall, we conclude that FER enhances IGF-1R expression, phosphorylation, and signaling to promote cooperative growth and adhesion signaling that may facilitate cancer progression.

Activation of MAPK signalling results in resistance to saracatinib (AZD0530) in ovarian cancer.

SRC tyrosine kinase is frequently overexpressed and activated in late-stage, poor prognosis ovarian tumours, and preclinical studies have supported the use of targeted SRC inhibitors in the treatment of this disease. The SAPPROC trial investigated the addition of the SRC inhibitor saracatinib (AZD0530) to weekly paclitaxel for the treatment of platinum resistant ovarian cancer; however, this drug combination did not provide any benefit to progression free survival (PFS) of women with platinum resistant disease. In this study we aimed to identify mechanisms of resistance to SRC inhibitors in ovarian cancer cells. Using two complementary strategies; a targeted tumour suppressor gene siRNA screen, and a phospho-receptor tyrosine kinase array, we demonstrate that activation of MAPK signalling, via a reduction in NF1 (neurofibromin) expression or overexpression of HER2 and the insulin receptor, can drive resistance to AZD0530. Knockdown of NF1 in two ovarian cancer cell lines resulted in resistance to AZD0530, and was accompanied with activated MEK and ERK signalling. We also show that silencing of HER2 and the insulin receptor can partially resensitize AZD0530 resistant cells, which was associated with decreased phosphorylation of MEK and ERK. Furthermore, we demonstrate a synergistic effect of combining SRC and MEK inhibitors in both AZD0530 sensitive and resistant cells, and that MEK inhibition is sufficient to completely resensitize AZD0530 resistant cells. This work provides a preclinical rationale for the combination of SRC and MEK inhibitors in the treatment of ovarian cancer, and also highlights the need for biomarker driven patient selection for clinical trials.

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.

IGF-1R inhibition sensitizes breast cancer cells to ATM-related kinase (ATR) inhibitor and cisplatin.

The complexity of the IGF-1 signalling axis is clearly a roadblock in targeting this receptor in cancer therapy. Here, we sought to identify mediators of resistance, and potential co-targets for IGF-1R inhibition. By using an siRNA functional screen with the IGF-1R tyrosine kinase inhibitor (TKI) BMS-754807 in MCF-7 cells we identified several genes encoding components of the DNA damage response (DDR) pathways as mediators of resistance to IGF-1R kinase inhibition. These included ATM and Ataxia Telangiectasia and RAD3-related kinase (ATR). We also observed a clear induction of DDR in cells that were exposed to IGF-1R TKIs (BMS-754807 and OSI-906) as indicated by accumulation of γ-H2AX, and phosphorylated Chk1. Combination of the IGF-1R/IR TKIs with an ATR kinase inhibitor VE-821 resulted in additive to synergistic cytotoxicity compared to either drug alone. In MCF-7 cells with stably acquired resistance to the IGF-1R TKI (MCF-7-R), DNA damage was also observed, and again, dual inhibition of the ATR kinase and IGF-1R/IR kinase resulted in synergistic cytotoxicity. Interestingly, dual inhibition of ATR and IGF-1R was more effective in MCF-7-R cells than parental cells. IGF-1R TKIs also potentiated the effects of cisplatin in a panel of breast cancer cell lines. Overall, our findings identify induction of DDR by IGF-1R kinase inhibition as a rationale for co-targeting the IGF-1R with ATR kinase inhibitors or cisplatin, particularly in cells with acquired resistance to TKIs.

Prior knowledge transfer across transcriptional data sets and technologies using compositional statistics yields new mislabelled ovarian cell line.

Here, we describe gene expression compositional assignment (GECA), a powerful, yet simple method based on compositional statistics that can validate the transfer of prior knowledge, such as gene lists, into independent data sets, platforms and technologies. Transcriptional profiling has been used to derive gene lists that stratify patients into prognostic molecular subgroups and assess biomarker performance in the pre-clinical setting. Archived public data sets are an invaluable resource for subsequent in silico validation, though their use can lead to data integration issues. We show that GECA can be used without the need for normalising expression levels between data sets and can outperform rank-based correlation methods. To validate GECA, we demonstrate its success in the cross-platform transfer of gene lists in different domains including: bladder cancer staging, tumour site of origin and mislabelled cell lines. We also show its effectiveness in transferring an epithelial ovarian cancer prognostic gene signature across technologies, from a microarray to a next-generation sequencing setting. In a final case study, we predict the tumour site of origin and histopathology of epithelial ovarian cancer cell lines. In particular, we identify and validate the commonly-used cell line OVCAR-5 as non-ovarian, being gastrointestinal in origin. GECA is available as an open-source R package.

When the guardian becomes the enemy: Targeting ATM in PTEN-deficient cancers.

Ataxia telangiectasia mutated (ATM) is an important signaling molecule in the DNA damage response and inhibitors of ATM are under clinical development. We identified a synthetic lethal interaction between ATM inhibition and phosphatase and tensin homolog (PTEN) loss that was the result of increased oxidative stress. Inhibition of ATM therefore represents a novel strategy to target PTEN-associated cancers.

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.

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.

DNA polymerases as potential therapeutic targets for cancers deficient in the DNA mismatch repair proteins MSH2 or MLH1.

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.

Deficiency in the repair of DNA damage by homologous recombination and sensitivity to poly(ADP-ribose) polymerase inhibition.

Deficiency in either of the breast cancer susceptibility proteins BRCA1 or BRCA2 induces profound cellular sensitivity to the inhibition of poly(ADP-ribose) polymerase (PARP) activity. We hypothesized that the critical role of BRCA1 and BRCA2 in the repair of double-strand breaks by homologous recombination (HR) was the underlying reason for this sensitivity. Here, we examine the effects of deficiency of several proteins involved in HR on sensitivity to PARP inhibition. We show that deficiency of RAD51, RAD54, DSS1, RPA1, NBS1, ATR, ATM, CHK1, CHK2, FANCD2, FANCA, or FANCC induces such sensitivity. This suggests that BRCA-deficient cells are, at least in part, sensitive to PARP inhibition because of HR deficiency. These results indicate that PARP inhibition might be a useful therapeutic strategy not only for the treatment of BRCA mutation-associated tumors but also for the treatment of a wider range of tumors bearing a variety of deficiencies in the HR pathway or displaying properties of 'BRCAness.'

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 microM) and NU1025 (IC50 400 nM). Here we show that CAPAN-1 cells are in fact very sensitive to the potent PARP inhibitors KU0058684 (IC50 3.2 nM) and KU0058948 (IC50 3.4 nM). In contrast, our results reveal much less sensitivity to a chemically related but much less active compound KU0051529 (IC50 730 nM) 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 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 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 NH(2)-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.

X-chromosome inactivation: X marks the spot for BRCA1.

X-chromosome inactivation equalizes the dosage of X-linked genes in XX females with that in XY males. Recent findings reveal that the BRCA1 breast cancer susceptibility gene has an important function in this epigenetic phenomenon.