Arvind Arora

Targeting BRCA1-BER deficient breast cancer by ATM or DNA-PKcs blockade either alone or in combination with cisplatin for personalized therapy.

BRCA1, a key factor in homologous recombination (HR) repair may also regulate base excision repair (BER). Targeting BRCA1‐BER deficient cells by blockade of ATM and DNA‐PKcs could be a promising strategy in breast cancer. We investigated BRCA1, XRCC1 and pol β protein expression in two cohorts (n = 1602 sporadic and n = 50 germ‐line BRCA1 mutated) and mRNA expression in two cohorts (n = 1952 and n = 249). Artificial neural network analysis for BRCA1‐DNA repair interacting genes was conducted in 249 tumours. Pre‐clinically, BRCA1 proficient and deficient cells were DNA repair expression profiled and evaluated for synthetic lethality using ATM and DNA‐PKcs inhibitors either alone or in combination with cisplatin. In human tumours, BRCA1 negativity was strongly associated with low XRCC1, and low pol β at mRNA and protein levels (p < 0.0001). In patients with BRCA1 negative tumours, low XRCC1 or low pol β expression was significantly associated with poor survival in univariate and multivariate analysis compared to high XRCC1 or high pol β expressing BRCA1 negative tumours (ps < 0.05). Pre‐clinically, BRCA1 negative cancer cells exhibit low mRNA and low protein expression of XRCC1 and pol β. BRCA1‐BER deficient cells were sensitive to ATM and DNA‐PKcs inhibitor treatment either alone or in combination with cisplatin and synthetic lethality was evidenced by DNA double strand breaks accumulation, cell cycle arrest and apoptosis. We conclude that XRCC1 and pol β expression status in BRCA1 negative tumours may have prognostic significance. BRCA1‐BER deficient cells could be targeted by ATM or DNA‐PKcs inhibitors for personalized therapy.

Untangling the ATR-CHEK1 network for prognostication, prediction and therapeutic target validation in breast cancer

ATR‐CHEK1 signalling is critical for genomic stability. ATR‐CHEK1 signalling may be deregulated in breast cancer and have prognostic, predictive and therapeutic significance. We investigated ATR, CHEK1 and phosphorylated CHEK1 Ser345 protein (pCHEK1) levels in 1712 breast cancers. ATR and CHEK1 mRNA expression was evaluated in 1950 breast cancers. Pre‐clinically, biological consequences of ATR gene knock down or ATR inhibition by the small molecule inhibitor (VE‐821) were investigated in MCF7 and MDA‐MB‐231 breast cancer cell lines and in non‐tumorigenic breast epithelial cells (MCF10A). High ATR and high cytoplasmic pCHEK1 levels were significantly associated with higher tumour stage, higher mitotic index, pleomorphism and lymphovascular invasion. In univariate analyses, high ATR and high cytoplasmic pCHEK1 levels were associated with poor breast cancer specific survival (BCSS). In multivariate analysis, high ATR level remains an independent predictor of adverse outcome. At the mRNA level, high CHEK1 remains associated with aggressive phenotypes including lymph node positivity, high grade, Her‐2 overexpression, triple negative, aggressive molecular phenotypes and adverse BCSS. Pre‐clinically, CHEK1 phosphorylation at serine345 following replication stress was impaired in ATR knock down and in VE‐821 treated breast cancer cells. Doxycycline inducible knockdown of ATR suppressed growth, which was restored when ATR was re‐expressed. Similarly, VE‐821 treatment resulted in a dose dependent suppression of cancer cell growth and survival (MCF7 and MDA‐MB‐231) but was less toxic in non‐tumorigenic breast epithelial cells (MCF10A). We provide evidence that ATR and CHEK1 are promising biomarkers and rational drug targets for personalized therapy in breast cancer.

Camptothecin targets WRN protein: mechanism and relevance in clinical breast cancer

Werner syndrome protein (WRN) is a RecQ helicase that participates in DNA repair, genome stability and cellular senescence. The five human RecQ helicases, RECQL1, Bloom, WRN, RECQL4 and RECQL5 play critical roles in DNA repair and cell survival after treatment with the anticancer drug camptothecin (CPT). CPT derivatives are widely used in cancer chemotherapy to inhibit topoisomerase I and generate DNA double-strand breaks during replication. Here we studied the effects of CPT on the stability and expression dynamics of human RecQ helicases. In the cells treated with CPT, we observed distinct effects on WRN compared to other human RecQ helicases. CPT altered the cellular localization of WRN and induced its degradation by a ubiquitin-mediated proteasome pathway. WRN knockdown cells as well as CPT treated cells became senescent and stained positive for senescence-associated β-galactosidase at a higher frequency compared to control cells. However, the senescent phenotype was attenuated by ectopic expression of WRN suggesting functional implication of WRN degradation in CPT treated cells. Approximately 5-23% of breast cancer tumors are known to respond to CPT-based chemotherapy. Interestingly, we found that the extent of CPT-induced WRN degradation correlates with increasing sensitivity of breast cancer cells to CPT. The abundance of WRN decreased in CPT-treated sensitive cells; however, WRN remained relatively stable in CPT-resistant breast cancer cells. In a large clinical cohort of breast cancer patients, we find that WRN and topoisomerase I expression correlate with an aggressive tumor phenotype and poor prognosis. Our novel observations suggest that WRN abundance along with CPT-induced degradation could be a promising strategy for personalizing CPT-based cancer chemotherapeutic regimens.

Are DNA repair factors promising biomarkers for personalized therapy in gastric cancer

Chronic inflammation is a driving force for gastric carcinogenesis. Reactive oxygen species (ROS) generated during the inflammatory process generates DNA damage that is processed through the DNA repair pathways. In this study, we profiled key DNA repair proteins (single-strand-selective monofunctional uracil-DNA glycosylase 1 [SMUG1], Flap endonuclease 1 [FEN1], X-ray repair cross-complementing gene 1 [XRCC1], and Ataxia telangiectasia mutated [ATM]) involved in ROS-induced oxidative DNA damage repair in gastric cancer and correlated to clinicopathological outcomes. High expression of SMUG1, FEN1, and XRCC1 correlated to high T-stage (T3/T4) (p-values: 0.001, 0.005, and 0.02, respectively). High expression of XRCC1 and FEN1 also correlated to lymph node-positive disease (p-values: 0.009 and 0.02, respectively). High expression of XRCC1, FEN1, and SMUG1 correlated with poor disease-specific survival (DSS) (p-values: 0.001, 0.006, and 0.05, respectively) and poor disease-free survival (DFS) (p-values: 0.001, 0.001, and 0.02, respectively). Low expression of ATM correlated to lymph node positivity (p=0.03), vascular invasion (p=0.05), and perineural invasion (p=0.005) and poor DFS (p=0.001) and poor DSS (p=0.003). In the multivariate Cox model, high XRCC1 and low ATM were independently associated with poor survival (p=0.008 and 0.011, respectively). Our observation supports the hypothesis that DNA repair factors are promising biomarkers for personalized therapy in gastric cancer.

Human Helicase RECQL4 Drives Cisplatin Resistance in Gastric Cancer by Activating an AKT–YB1–MDR1 Signaling Pathway

Elevation of the DNA-unwinding helicase RECQL4, which participates in various DNA repair pathways, has been suggested to contribute to the pathogenicity of various human cancers, including gastric cancer. In this study, we addressed the prognostic and chemotherapeutic significance of RECQL4 in human gastric cancer, which has yet to be determined. We observed significant increases in RECQL4 mRNA or protein in >70% of three independent sets of human gastric cancer specimens examined, relative to normal gastric tissues. Strikingly, high RECQL4 expression in primary tumors correlated well with poor survival and gastric cancer lines with high RECQL4 expression displayed increased resistance to cisplatin treatment. Mechanistic investigations revealed a novel role for RECQL4 in transcriptional regulation of the multidrug resistance gene MDR1, through a physical interaction with the transcription factor YB1. Notably, ectopic expression of RECQL4 in cisplatin-sensitive gastric cancer cells with low endogenous RECQL4 was sufficient to render them resistant to cisplatin, in a manner associated with YB1 elevation and MDR1 activation. Conversely, RECQL4 silencing in cisplatin-resistant gastric cancer cells with high endogenous RECQL4 suppressed YB1 phosphorylation, reduced MDR1 expression, and resensitized cells to cisplatin. In establishing RECQL4 as a critical mediator of cisplatin resistance in gastric cancer cells, our findings provide a therapeutic rationale to target RECQL4 or the downstream AKT-YB1-MDR1 axis to improve gastric cancer treatment. Cancer Res; 76(10); 3057-66. ©2016 AACR.

ATM, ATR and DNA-PKcs expressions correlate to adverse clinical outcomes in epithelial ovarian cancers

Background Ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia mutated and rad3 related (ATR) and DNA-dependent protein kinase catalytic sub-unit (DNA-PKcs) play critical roles in DNA damage response (DDR) by linking DNA damage sensing to DDR effectors that regulate cell cycle progression and DNA repair. Our objective was to evaluate if ATM, ATR and DNA-PKcs expressions could predict response to therapy and clinical outcome in epithelial ovarian cancers. Methods We investigated ATM, ATR, and DNA-PKcs expressions in ovarian epithelial cancers [protein expression (n = 194 patients), mRNA expression (n = 156 patients)] and correlated to clinicopathological outcomes as well as expression of X-ray repair cross-complementing protein 1 (XRCC1), cell division cycle-45 (CDC45), cyclin-dependent kinase 1(CDK1) and Ki-67 in tumours. Results High ATM protein expression was associated with serous cystadenocarcinomas (p = 0.021) and platinum resistance (p = 0.017). High DNA-PKcs protein expression was associated with serous cystadenocarcinomas (p = 0.006) and advanced stage tumours (p = 0.018). High ATM protein (p = 0.001), high ATM mRNA (p = 0.018), high DNA-PKcs protein (p = 0.002), high DNA-PKcs mRNA (p = 0.044) and high ATR protein (p = 0.001) expressions are correlated with poor ovarian cancer specific survival (OCSS). In multivariate Cox model, high DNA-PKcs (p = 0.006) and high ATR (p = 0.043) protein expressions remain independently associated with poor OCSS. Conclusions ATM, ATR and DNA-PKcs expressions may have prognostic and predictive significances in epithelial ovarian cancer. General significance The data presented here provides evidence that ATM, ATR and DNA-PKcs involved in DDR are not only promising biomarkers but are also rational targets for personalized therapy in ovarian cancer.

Calpain system protein expression in carcinomas of the pancreas, bile duct and ampulla

BackgroundPancreatic cancer, including cancer of the ampulla of Vater and bile duct, is very aggressive and has a poor five year survival rate; improved methods of patient stratification are required.MethodsWe assessed the expression of calpain-1, calpain-2 and calpastatin in two patient cohorts using immunohistochemistry on tissue microarrays. The first cohort was composed of 68 pancreatic adenocarcinomas and the second cohort was composed of 120 cancers of the bile duct and ampulla.ResultsIn bile duct and ampullary carcinomas an association was observed between cytoplasmic calpastatin expression and patient age (P = 0.036), and between nuclear calpastatin expression and increased tumour stage (P = 0.026) and the presence of vascular invasion (P = 0.043). In pancreatic cancer, high calpain-2 expression was significantly associated with improved overall survival (P = 0.036), which remained significant in multivariate Cox-regression analysis (hazard ratio = 0.342; 95% confidence interva l = 0.157-0.741; P = 0.007). In cancers of the bile duct and ampulla, low cytoplasmic expression of calpastatin was significantly associated with poor overall survival (P = 0.012), which remained significant in multivariate Cox-regression analysis (hazard ratio = 0.595; 95% confidence interval = 0.365-0.968; P = 0.037).ConclusionThe results suggest that calpain-2 and calpastatin expression is important in pancreatic cancers, influencing disease progression. The findings of this study warrant a larger follow-up study.

Clinicopathological Significance of ATM-Chk2 Expression in Sporadic Breast Cancers: a Comprehensive Analysis in Large Cohorts

ATM-Chk2 network is critical for genomic stability, and its deregulation may influence breast cancer pathogenesis. We investigated ATM and Chk2 protein levels in two cohorts [cohort 1 (n = 1650) and cohort 2 (n = 252)]. ATM and Chk2 mRNA expression was evaluated in the Molecular Taxonomy of Breast Cancer International Consortium cohort (n = 1950). Low nuclear ATM protein level was significantly associated with aggressive breast cancer including larger tumors, higher tumor grade, higher mitotic index, pleomorphism, tumor type, lymphovascular invasion, estrogen receptor (ER)−, PR −, AR −, triple-negative, and basal-like phenotypes (Ps < .05). Breast cancer 1, early onset negative, low XRCC1, low SMUG1, high FEN1, high MIB1, p53 mutants, low MDM2, low Bcl-2, low p21, low Bax, high CDK1, and low Chk2 were also more frequent in tumors with low nuclear ATM level (Ps < .05). Low ATM protein level was significantly associated with poor survival including in patients with ER-negative tumors who received adjuvant anthracycline or cyclophosphamide, methotrexate, and 5-fluorouracil–based adjuvant chemotherapy (Ps < .05). Low nuclear Chk2 protein was likely in ER −/PR −/AR −; HER-2 positive; breast cancer 1, early onset negative; low XRCC1; low SMUG1; low APE1; low polβ; low DNA-PKcs; low ATM; low Bcl-2; and low TOPO2A tumors (P < .05). In patients with ER + tumors who received endocrine therapy or ER-negative tumors who received chemotherapy, nuclear Chk2 levels did not significantly influence survival. In p53 mutant tumors, low ATM (P < .000001) or high Chk2 (P < .01) was associated with poor survival. When investigated together, low-ATM/high-Chk2 tumors have the worst survival (P = .0033). Our data suggest that ATM-Chk2 levels in sporadic breast cancer may have prognostic and predictive significance.

Chk1 phosphorylated at serine345 is a predictor of early local recurrence and radio-resistance in breast cancer

Radiation‐induced DNA damage activates the DNA damage response (DDR). DDR up‐regulation may predict radio‐resistance and increase the risk of early local recurrence despite radiotherapy in early stage breast cancers. In 1755 early stage breast cancers, DDR signalling [ATM, ATR, total Ckh1, Chk1 phosphorylated at serine345 (pChk1), Chk2, p53], base excision repair [PARP1, POLβ, XRCC1, FEN1, SMUG1], non‐homologous end joining (Ku70/Ku80, DNA‐PKcs) and homologous recombination [RAD51, BRCA1, γH2AX, BLM, WRN, RECQL5, PTEN] protein expression was correlated to time to early local recurrence. Pre‐clinically, radio‐sensitization by inhibition of Chk1 activation by ATR inhibitor (VE‐821) and inhibition of Chk1 (V158411) were investigated in MDA‐MB‐231 (p53 mutant) and MCF‐7 (p53 wild‐type) breast cancer cells. In the whole cohort, 208/1755 patients (11.9%) developed local recurrence of which 126 (61%) developed local recurrence within 5 years of initiation of primary therapy. Of the 20 markers tested, only pChk1 and p53 significantly associated with early local recurrence (p value = 0.015 and 0.010, respectively). When analysed together, high cytoplasmic pChk1‐nuclear pChk1 (p = 0.039), high cytoplasmic pChk1‐p53 (p = 0.004) and high nuclear pChk1‐p53 (p = 0.029) co‐expression remain significantly linked to early local recurrence. In multivariate analysis, cytoplasmic pChk1 level independently predicted early local recurrence (p = 0.025). In patients who received adjuvant local radiotherapy (n = 949), p53 (p = 0.014) and high cytoplasmic pChk1‐p53 (p = 0.017) remain associated with early local recurrence. Pre‐clinically, radio‐sensitisation by VE‐821 or V158411 was observed in both MCF‐7 and MDA‐MB‐231 cells and was more pronounced in MCF‐7 cells. We conclude that pChk1 is a predictive biomarker of radiotherapy resistance and early local recurrence.

Transcriptomic and Protein Expression Analysis Reveals Clinicopathological Significance of Bloom Syndrome Helicase (BLM) in Breast Cancer

Bloom syndrome helicase (BLM) has key roles in homologous recombination repair, telomere maintenance, and DNA replication. Germ-line mutations in the BLM gene causes Bloom syndrome, a rare disorder characterized by premature aging and predisposition to multiple cancers, including breast cancer. The clinicopathologic significance of BLM in sporadic breast cancers is unknown. We investigated BLM mRNA expression in the Molecular Taxonomy of Breast Cancer International Consortium cohort (n = 1,950) and validated in an external dataset of 2,413 tumors. BLM protein level was evaluated in the Nottingham Tenovus series comprising 1,650 breast tumors. BLM mRNA overexpression was significantly associated with high histologic grade, larger tumor size, estrogen receptor–negative (ER−), progesterone receptor–negative (PR−), and triple-negative phenotypes (ps < 0.0001). BLM mRNA overexpression was also linked to aggressive molecular phenotypes, including PAM50.Her2 (P < 0.0001), PAM50.Basal (P < 0.0001), and PAM50.LumB (P < 0.0001) and Genufu subtype (ER+/Her2−/high proliferation; P < 0.0001). PAM50.LumA tumors and Genufu subtype (ER+/Her2−/low proliferation) were more likely to express low levels of BLM mRNA (ps < 0.0001). Integrative molecular clusters (intClust) intClust.1 (P < 0.0001), intClust.5 (P < 0.0001), intClust.9 (P < 0.0001), and intClust.10 (P < 0.0001) were also more likely in tumors with high BLM mRNA expression. BLM mRNA overexpression was associated with poor breast cancer–specific survival (BCSS; ps < 0.000001). At the protein level, altered subcellular localization with high cytoplasmic BLM and low nuclear BLM was linked to aggressive phenotypes. In multivariate analysis, BLM mRNA and BLM protein levels independently influenced BCSS. This is the first and the largest study to provide evidence that BLM is a promising biomarker in breast cancer. Mol Cancer Ther; 14(4); 1057–65. ©2015 AACR.