Person:

Chowdhury, Dipanjan

Pancreatic ductal adenocarcinoma (PDAC) is amongst the deadliest of human cancers, due to its late diagnosis as well as its intense resistance to currently available therapeutics. To identify mechanisms as to why PDAC are refractory to DNA damaging cytoxic chemotherapy and radiation, we performed a global interrogation of the DNA damage response of PDAC. We find that PDAC cells generally harbor high levels of spontaneous DNA damage. Inhibition of Non-Homologous End Joining (NHEJ) repair either pharmacologically or by RNAi resulted in a further accumulation of DNA damage, inhibition of growth, and ultimately apoptosis even in the absence of exogenous DNA damaging agents. In response to radiation, PDAC cells rely on the NHEJ pathway to rapidly repair DNA double strand breaks. Mechanistically, when NHEJ is inhibited there is a compensatory increase in Homologous Recombination (HR). Despite this upregulation of HR, DNA damage persists and cells are significantly more sensitive to radiation. Together, these findings support the incorporation of NHEJ inhibition into PDAC therapeutic approaches, either alone, or in combination with DNA damaging therapies such as radiation.

Immune checkpoint inhibitors (e.g., anti-PD-1 and anti-PD-L1 antibodies) have demonstrated remarkable efficacy against hypermutated cancers such as melanomas and lung carcinomas. One explanation for this effect is that hypermutated lesions harbor more tumor-specific neoantigens that stimulate recruitment of an increased number of tumor-infiltrating lymphocytes (TILs), which is counterbalanced by overexpression of immune checkpoints such as PD-1 or PD-L1. Given that BRCA1/2-mutated high grade serous ovarian cancers (HGSOCs) exhibit a higher mutational load and a unique mutational signature with an elevated number of larger indels up to 50 bp, we hypothesized that they may also harbor more tumor-specific neoantigens, and, therefore, exhibit increased TILs and PD-1/PD-L1 expression. Here, we report significantly higher predicted neoantigens in BRCA1/2-mutated tumors compared to tumors without alterations in homologous recombination (HR) genes (HR-proficient tumors). Tumors with higher neoantigen load were associated with improved overall survival and higher expression of immune genes associated with tumor cytotoxicity such as genes of the TCR, the IFN-gamma and the TNFR pathways. Furthermore, immunohistochemistry studies demonstrated that BRCA1/2-mutated tumors exhibited significantly increased CD3+ and CD8+ TILs, as well as elevated expression of PD-1 and PD-L1 in tumor-associated immune cells compared to HR-proficient tumors. Survival analysis showed that both BRCA1/2-mutation status and number of TILs were independently associated with outcome. Of note, two distinct groups of HGSOCs, one with very poor prognosis (HR proficient with low number of TILs) and one with very good prognosis (BRCA1/2-mutated tumors with high number of TILs) were defined. These findings support a link between BRCA1/2-mutation status, immunogenicity and survival, and suggesting that BRCA1/2-mutated HGSOCs may be more sensitive to PD-1/PD-L1 inhibitors compared to HR-proficient HGSOCs.

Triple negative breast cancer (TNBC) has caught the attention of oncologists worldwide because of poor prognosis and paucity of targeted therapies. Gene pathways have been widely studied, but less is known about epigenetic factors such as microRNAs (miRNAs) and their role in tailoring an individual systemic and surgical approach for breast cancer patients. The aim of the study was to examine selected miRNAs in TNBC core biopsies sampled before preoperative chemotherapy and the subsequent pathologic response in mastectomy or breast conservation specimens. Prior to treatment, core needle biopsies were collected from 11 female patients with inoperable locally advanced TNBC or large resectable tumors suitable for down-staging. In all 11 TNBC core biopsies we analyzed 19 miRNAs per sample: 512, 190, 200, 346, 148, 449, 203, 577, 93, 126, 423, 129, 193, 182, 136, 135, 191, 122 and 222 (miRCURY LNA™ Universal RT microRNA polymerase chain reaction Custom Pick & Mixpanels). The Wilcoxon signed-rank test was used to compare related samples. Ingenuity pathway analysis was used to evaluate potential functional significance of differentially expressed miRNAs. Statistical analysis showed that 3 of 19 miRNAs differed in relation to pathologic response i.e. good versus poor. These differences failed to reach statistical significance, although a trend was observed (p = 0.06). Among these miRNAs, we identified—miR-200b-3p, miR-190a and miR-512-5p. In summary, our results indicate that higher miR-200b-3p, higher miR-190a and lower miR-512-5p expression levels in core biopsies sampled from TNBC patients may be associated with better pathologic response to chemotherapy and the increased feasibility of breast conserving surgery in these patients. Although these results were from a small cohort, they provide an important basis for larger, prospective, multicenter studies to investigate the potential role of miRNAs in neoadjuvant setting.

The promise of PARP-inhibitors(PARPis) in the management of epithelial ovarian cancer(EOC) is tempered by the fact that approximately 50% of patients with homologous recombination (HR)-proficient tumors do not respond well to these agents. Combination of PARPis with agents that inhibit HR may represent an effective strategy to enhance their activity in HR-proficient tumors. Using a bioinformatics approach, we identified that heat shock protein 90 inhibitors(HSP90i) may suppress HR and thus revert HR-proficient to HR-deficient tumors. Analysis of publicly available gene expression data showed that exposure of HR-proficient breast cancer cell lines to HSP90i 17-AAG(17-allylamino-17-demethoxygeldanamycin) downregulated HR, ATM and Fanconi Anemia pathways. In HR-proficient EOC cells, 17-AAG suppressed HR as assessed using the RAD51 foci formation assay and this was further confirmed using the Direct Repeat-GFP reporter assay. Furthermore, 17-AAG downregulated BRCA1 and/or RAD51 protein levels, and induced significantly more γH2AX activation in combination with olaparib compared to olaparib alone. Finally, sublethal concentrations of 17-AAG sensitized HR-proficient EOC lines to olaparib and carboplatin but did not affect sensitivity of the HR-deficient OVCAR8 line arguing that the 17-AAG mediated sensitization is dependent on suppression of HR. These results provide a preclinical rationale for using a combination of olaparib/17-AAG in HR-proficient EOC.

Homologous recombination (HR)-mediated repair of DNA double-strand break (DSB)s is restricted to the post-replicative phases of the cell cycle. Initiation of HR in the G1 phase blocks non-homologous end joining (NHEJ) impairing DSB repair. Completion of HR in G1 cells can lead to the loss-of-heterozygosity (LOH), which is potentially carcinogenic. We conducted a gain-of-function screen to identify miRNAs that regulate HR-mediated DSB repair, and of these miRNAs, miR-1255b, miR-148b*, and miR-193b* specifically suppress the HR-pathway in the G1 phase. These miRNAs target the transcripts of HR factors, BRCA1, BRCA2, and RAD51, and inhibiting miR-1255b, miR-148b*, and miR-193b* increases expression of BRCA1/BRCA2/RAD51 specifically in the G1-phase leading to impaired DSB repair. Depletion of CtIP, a BRCA1-associated DNA end resection protein, rescues this phenotype. Furthermore, deletion of miR-1255b, miR-148b*, and miR-193b* in independent cohorts of ovarian tumors correlates with significant increase in LOH events/chromosomal aberrations and BRCA1 expression. DOI: http://dx.doi.org/10.7554/eLife.02445.001

Phosphorylation of replication protein A (RPA) by Cdk2 and the checkpoint kinase ATR (ATM and Rad3 related) during replication fork stalling stabilizes the replisome, but how these modifications safeguard the fork is not understood. To address this question, we used single-molecule fiber analysis in cells expressing a phosphorylation-defective RPA2 subunit or lacking phosphatase activity toward RPA2. Deregulation of RPA phosphorylation reduced synthesis at forks both during replication stress and recovery from stress. The ability of phosphorylated RPA to stimulate fork recovery is mediated through the PALB2 tumor suppressor protein. RPA phosphorylation increased localization of PALB2 and BRCA2 to RPA-bound nuclear foci in cells experiencing replication stress. Phosphorylated RPA also stimulated recruitment of PALB2 to single-strand deoxyribonucleic acid (DNA) in a cell-free system. Expression of mutant RPA2 or loss of PALB2 expression led to significant DNA damage after replication stress, a defect accentuated by poly-ADP (adenosine diphosphate) ribose polymerase inhibitors. These data demonstrate that phosphorylated RPA recruits repair factors to stalled forks, thereby enhancing fork integrity during replication stress.

Background: Risk of normal tissue toxicity limits the amount of thoracic radiation therapy (RT) that can be routinely prescribed to treat non-small cell lung cancer (NSCLC). An early biomarker of response to thoracic RT may provide a way to predict eventual toxicities—such as radiation pneumonitis—during treatment, thereby enabling dose adjustment before the symptomatic onset of late effects. MicroRNAs (miRNAs) were studied as potential serological biomarkers for thoracic RT. As a first step, we sought to identify miRNAs that correlate with delivered dose and standard dosimetric factors. Methods: We performed miRNA profiling of plasma samples obtained from five patients with Stage IIIA NSCLC at five dose-points each during radical thoracic RT. Candidate miRNAs were then assessed in samples from a separate cohort of 21 NSCLC patients receiving radical thoracic RT. To identify a cellular source of circulating miRNAs, we quantified in vitro miRNA expression intracellularly and within secreted exosomes in five NSCLC and stromal cell lines. Results: miRNA profiling of the discovery cohort identified ten circulating miRNAs that correlated with delivered RT dose as well as other dosimetric parameters such as lung V20. In the validation cohort, miR-29a-3p and miR-150-5p were reproducibly shown to decrease with increasing radiation dose. Expression of miR-29a-3p and miR-150-5p in secreted exosomes decreased with radiation. This was concomitant with an increase in intracellular levels, suggesting that exosomal export of these miRNAs may be downregulated in both NSCLC and stromal cells in response to radiation. Conclusions: miR-29a-3p and miR-150-5p were identified as circulating biomarkers that correlated with delivered RT dose. miR-150 has been reported to decrease in the circulation of mammals exposed to radiation while miR-29a has been associated with fibrosis in the human heart, lungs, and kidneys. One may therefore hypothesize that outlier levels of circulating miR-29a-3p and miR-150-5p may eventually help predict unexpected responses to radiation therapy, such as toxicity. Electronic supplementary material The online version of this article (doi:10.1186/s13014-016-0636-4) contains supplementary material, which is available to authorized users.

Background: Genomic instability promotes evolution and heterogeneity of tumors. Unraveling its mechanistic basis is essential for the design of appropriate therapeutic strategies. In a previous study, we reported an unexpected oncogenic property of p21WAF1/Cip1, showing that its chronic expression in a p53-deficient environment causes genomic instability by deregulation of the replication licensing machinery. Results: We now demonstrate that p21WAF1/Cip1 can further fuel genomic instability by suppressing the repair capacity of low- and high-fidelity pathways that deal with nucleotide abnormalities. Consequently, fewer single nucleotide substitutions (SNSs) occur, while formation of highly deleterious DNA double-strand breaks (DSBs) is enhanced, crafting a characteristic mutational signature landscape. Guided by the mutational signatures formed, we find that the DSBs are repaired by Rad52-dependent break-induced replication (BIR) and single-strand annealing (SSA) repair pathways. Conversely, the error-free synthesis-dependent strand annealing (SDSA) repair route is deficient. Surprisingly, Rad52 is activated transcriptionally in an E2F1-dependent manner, rather than post-translationally as is common for DNA repair factor activation. Conclusions: Our results signify the importance of mutational signatures as guides to disclose the repair history leading to genomic instability. We unveil how chronic p21WAF1/Cip1 expression rewires the repair process and identifies Rad52 as a source of genomic instability and a candidate therapeutic target. Electronic supplementary material The online version of this article (10.1186/s13059-018-1401-9) contains supplementary material, which is available to authorized users.

Background: Senescence is a fundamental biological process implicated in various pathologies, including cancer. Regarding carcinogenesis, senescence signifies, at least in its initial phases, an anti-tumor response that needs to be circumvented for cancer to progress. Micro-RNAs, a subclass of regulatory, non-coding RNAs, participate in senescence regulation. At the subcellular level micro-RNAs, similar to proteins, have been shown to traffic between organelles influencing cellular behavior. The differential function of micro-RNAs relative to their subcellular localization and their role in senescence biology raises concurrent in situ analysis of coding and non-coding gene products in senescent cells as a necessity. However, technical challenges have rendered in situ co-detection unfeasible until now. Methods: In the present report we describe a methodology that bypasses these technical limitations achieving for the first time simultaneous detection of both a micro-RNA and a protein in the biological context of cellular senescence, utilizing the new commercially available SenTraGorTM compound. The method was applied in a prototypical human non-malignant epithelial model of oncogene-induced senescence that we generated for the purposes of the study. For the characterization of this novel system, we applied a wide range of cellular and molecular techniques, as well as high-throughput analysis of the transcriptome and micro-RNAs. Results: This experimental setting has three advantages that are presented and discussed: i) it covers a “gap” in the molecular carcinogenesis field, as almost all corresponding in vitro models are fibroblast-based, even though the majority of neoplasms have epithelial origin, ii) it recapitulates the precancerous and cancerous phases of epithelial tumorigenesis within a short time frame under the light of natural selection and iii) it uses as an oncogenic signal, the replication licensing factor CDC6, implicated in both DNA replication and transcription when over-expressed, a characteristic that can be exploited to monitor RNA dynamics. Conclusions: Consequently, we demonstrate that our model is optimal for studying the molecular basis of epithelial carcinogenesis shedding light on the tumor-initiating events. The latter may reveal novel molecular targets with clinical benefit. Besides, since this method can be incorporated in a wide range of low, medium or high-throughput image-based approaches, we expect it to be broadly applicable. Electronic supplementary material The online version of this article (10.1186/s12864-017-4375-1) contains supplementary material, which is available to authorized users.