Telomeric Allelic Imbalance Indicates Defective DNA Repair and Sensitivity to DNA-Damaging Agents
ABSTRACT: DNA repair competency is one determinant of sensitivity to certain chemotherapy drugs, such as cisplatin. Cancer cells with intact DNA repair can avoid the accumulation of genome damage during growth and also can repair platinum-induced DNA damage. We sought genomic signatures indicative of defective DNA repair in cell lines and tumors and correlated these signatures to platinum sensitivity. The number of subchromosomal regions with allelic imbalance extending to the telomere (NtAI) predicted cisplatin sensitivity in vitro and pathologic response to preoperative cisplatin treatment in patients with triple-negative breast cancer (TNBC). In serous ovarian cancer treated with platinum-based chemotherapy, higher levels of NtAI forecast a better initial response. We found an inverse relationship between BRCA1 expression and NtAI in sporadic TNBC and serous ovarian cancers without BRCA1 or BRCA2 mutation. Thus, accumulation of telomeric allelic imbalance is a marker of platinum sensitivity and suggests impaired DNA repair. Overall design: SNP data from 27 and 40 primary triple negative breast cancer tumor samples from two clinical trials treated with cisplatin and cisplatin + bevacizumab. Labeling, hybridization and data processing was performed by Affymetrix using 70k MIP arrays and 330k MIP arrays. In the cisplatin trial, matched normal samples based on blood from all patients and an additional three samples based on FFPE negative lymph nodes were used as references (30 normal references in total). In the cisplatin+bevacizumab trial, mathed normal samples based on blood from 10 patients were used as references.
Project description:DNA repair competency is one determinant of sensitivity to certain chemotherapy drugs, such as cisplatin. Cancer cells with intact DNA repair can avoid the accumulation of genome damage during growth and also can repair platinum-induced DNA damage. We sought genomic signatures indicative of defective DNA repair in cell lines and tumors and correlated these signatures to platinum sensitivity. The number of subchromosomal regions with allelic imbalance extending to the telomere (NtAI) predicted cisplatin sensitivity in vitro and pathologic response to preoperative cisplatin treatment in patients with triple-negative breast cancer (TNBC). In serous ovarian cancer treated with platinum-based chemotherapy, higher levels of NtAI forecast a better initial response. We found an inverse relationship between BRCA1 expression and NtAI in sporadic TNBC and serous ovarian cancers without BRCA1 or BRCA2 mutation. Thus, accumulation of telomeric allelic imbalance is a marker of platinum sensitivity and suggests impaired DNA repair. SNP data from 27 and 40 primary triple negative breast cancer tumor samples from two clinical trials treated with cisplatin and cisplatin + bevacizumab. Labeling, hybridization and data processing was performed by Affymetrix using 70k MIP arrays and 330k MIP arrays. In the cisplatin trial, matched normal samples based on blood from all patients and an additional three samples based on FFPE negative lymph nodes were used as references (30 normal references in total). In the cisplatin+bevacizumab trial, mathed normal samples based on blood from 10 patients were used as references.
Project description:Gene expression data from 21 triple negative breast cancer samples treated with cisplatin & bevacizumab in the neoadjuvant setting as part of a clinical trial. Gene expression data RMA normalized was used to find predictors of platinum sensitivity Overall design: Patients were treated in the neoadjuvant setting, RNA was purified from core biopsy pre-treatment and pre-surgery, profiled for RNA expression on Affymetrix U133 Plus 2.0 gene expression array following manufacturers protocol.
Project description:Triple negative breast cancers (TNBCs) are characterised by a wide spectrum of genomic aberrations representing underlying repair defects that may be targeted therapeutically. However, means to measure these defects in tumours and an understanding of their effect on sensitivity to DNA damaging agents is limited. We sought to address this by establishing methods to trace underlying deficiencies in DNA repair processes using patterns of genomic instability. Here, we demonstrate that a pattern related to Homologous Recombination defects, allelic-imbalanced Copy Number Aberration, predicts response to platinum containing chemotherapeutics in TNBC patients. These patterns also enabled us to identify a meiotic gene HORMAD1, as a functional driver of allelic-imbalanced Copy Number Aberration and genomic instability in TNBC. Additionally, HORMAD1 expression is also a predictive marker of carboplatin response in TNBC. Mechanistically, expression of HORMAD1 in cell lines inhibited Homologous Recombination representing outÐof-context activation of its meiotic function.
Project description:Veliparib showed limited single agent cytotoxicity but selectively potentiated (≥50% reduction in IC50) cisplatin, carboplatin and etoposide in vitro in 5 of 9 SCLC cell lines. Veliparib with cisplatin, etoposide or with both cisplatin and etoposide showed greater delay in tumor growth than chemotherapy alone in H146 but not H128. The potentiating effect of veliparib was associated with in vitro cell line sensitivity to cisplatin (CC=0.672; p=0.048) and DNA-PKcs protein modulation. Gene expression profiling identified differential expression of a 5-gene panel (GLS, UBEC2, HACL1, MSI2 and LOC100129585) in cell lines with relatively greater sensitivity to platinum and veliparib combination. Veliparib potentiates standard cytotoxic agents against SCLC in a cell specific manner. This potentiation correlates with platinum sensitivity, DNA-PKcs expression and a 5-gene expression profile. Overall design: In vitro cytotoxicity of veliparib, cisplatin, carboplatin, and etoposide singly and combined was determined by MTS in 9 SCLC cell lines (H69, H128, H146, H526, H187, H209, DMS53, DMS153, and DMS114). Subcutaneous xenografts in athymic nu/nu mice of H146 and H128 with relatively high and low platinum sensitivity, respectively, were employed for in vivo testing. Mechanisms of differential sensitivity of SCLC cell lines to PARP inhibition were investigated by comparing protein and gene expression profiles of the platinum sensitive and the less sensitive cell lines.
Project description:Introduction: Although High Grade Serous Ovarian Cancer (HGSOC) is considered a chemo-responsive disease, a proportion of patients do not respond to platinum-based chemotherapy at presentation or have progression-free survival of <6 months. Validated biomarkers of lack of response would enable alternative treatment stratification for these patients and identify novel mechanisms of resistance. Methods: Differential DNA methylation was investigated in independent tumour sets using Illumina 27K HumanMethylation arrays and validated by bisulphite pyrosequencing. Gene expression was by Affymetrix arrays and qRT-PCR. The role of Msh homeobox 1 (MSX1) in drug sensitivity was investigated by gene reintroduction into ovarian cancer cell lines. Results: CpG sites at contiguous genomic locations within the MSX1 gene have significantly lower levels of methylation in HGSOC which recur by 6 months compared to after 12 months and/or with RECIST response (p<0.05, q<0.05). A decrease in methylation at these intragenic CpG sites was significantly correlated with decreased MSX1 gene expression. Low expression of MSX1 was associated with poor progression-free survival independent of known clinical prognostic features (p=0.014). Three mutant or wild-type TP53 expressing ovarian cancer cell lines, resistant to cisplatin, have reduced MSX1 expression compared to matched parental, platinum sensitive, lines. Re-expression of MSX1 in resistant lines led to cisplatin sensitisation, increased apoptosis, increased p21 and BAX expression. However, in two TP53-null cell lines, MSX1 failed to change cisplatin sensitivity. Conclusion: Hypomethylation of MSX1 is a biomarker of resistant HGSOC disease at presentation and identifies a novel mechanism of platinum drug resistance. Bisulphite converted DNA from the 86 samples were hybridised to the Illumina Infinium 27k Human Methylation Beadchip v1.2
Project description:Platinum compounds display clinical activity against a wide variety of solid tumors. However, resistance to these agents is a major limitation in cancer therapy. Reduced platinum uptake and increased platinum export are examples of resistance mechanisms that limit the extent of DNA damage. Here, we report the discovery and characterization of the role of ATP11B, a P-type ATPase membrane protein, in cisplatin resistance. ATP11B gene silencing restored the sensitivity of ovarian cancer cell lines to cisplatin in vitro. Combined therapy of cisplatin and ATP11B-siRNA significantly decreased cancer growth in mice bearing ovarian tumors derived from cisplatin-sensitive and -resistant cells. In vitro mechanistic studies on cellular platinum content and cisplatin efflux-kinetics indicated that ATP11B enhances the export of cisplatin from cells. The co-localization of ATP11B with fluorescent cisplatin and with vesicular trafficking proteins such as syntaxin-6 (STX6) and vesicular associated membrane protein 4 (VAMP4) strongly suggests that ATP11B contributes to secretory vesicular transport of cisplatin from Golgi to plasma membrane. In conclusion, silencing ATP11B expression might be a therapeutic strategy to overcome cisplatin resistance. We performed the transfection of control-siRNA and ATP11B-siRNA to both cisplatin-sensitive A2780-PAR and cisplatin-resistant A2780-CP20 cells respectively.
Project description:Breast cancers lacking receptors for estrogen, progesterone or HER2 on their cell surface are called triple-negative breast cancers (TNBCs). TNBCs account for ~15-20% of all invasive breast cancers and do not benefit from anti-hormonal or anti-HER2 treatments. Although patients with TNBC can initially respond to chemotherapy, they do have worse overall prognosis compared to other breast cancer subtypes. Unfortunately, TNBCs lack clear targetable ‘driver’ oncogenes. Thus, there is an unmet need for strategies to improve the therapeutic options for these patients. We used microarrays to assess differences in gene expression in triple-negative breast cancer cells in response to the platinum-based chemotherapeutic agent cisplatin. The purpose was to find drug induced changes in gene expression level that could differentiate cisplatin sensitive from cisplatin resistant TNBC cell lines. Overall design: Four triple-negative breast cancer cell lines, MDA-MB-231, MDA-MB-157, BT549 and HCC38, were treated with 2 µM cisplatin for 0, 24 or 72 hours. Two independent replicates were collected for each cell line.
Project description:Platinum chemotherapies induce damages in DNA that distort the helical structure. In human cells, these adducts are removed primarily by the Nucleotide Excision Repair pathway. In this study, we mapped both cisplatin and oxaliplatin induced damages and their repair at single nucleotide resolution across the human genome. Overall design: We performed Damage-seq to map 2 types of damages in two different human cell lines. The damages were cisplatin and oxaliplatin induced DNA adducts. The two cell lines were normal human skin fibroblasts NHF1 and the GM12878 human lymphoblastoid cells. As control, cisplatin damages were induced in genomic DNA from GM12878. For comparison to repair, XR-seq was performed for cisplatin and oxaliplatin treated GM12878 cells.
Project description:Evidence suggests that BRCA1 mutation associated tumors have increased sensitivity to DNA damaging agents like cisplatin. Sporadic triple negative breast cancers (TNBC) have many phenotypic similarities to BRCA1 tumors and may have a similar sensitivity to cisplatin. We tested the efficacy of cisplatin monotherapy in 28 TNBC patients in a single arm neoadjuvant trial with outcome measured by pathologic treatment response quantified using the Miller-Payne scale. We used microarrays gene expression profiles to determine tumor subtype of each trial tumor sample and to test various expression signatures for association with pathologic response to cisplatin. Pretreatment tumor samples from the clinical trial (N=24 with adequate tissue) were used for RNA extraction, linear amplification, biotin labeling and hybridization to Affymetrix U133 plus 2.0 arrays. A reference set of 51 primary breast tumors representing all subtypes of breast cancer were processed in a similar manner to include linear amplification, and hybridized to Affymetrix arrays.
Project description:Purpose:Triple negative breast cancer (TNBC) commonly metastasizes to the brain and predicts poor prognosis with limited therapeutic options. TNBC frequently harbors BRCA mutations translating to platinum sensitivity; platinum response may be augmented by additional suppression of DNA repair mechanisms through poly(ADP-ribose)polymerase (PARP) inhibition. We evaluated brain penetrance and efficacy of Carboplatin +/- the PARP inhibitor ABT888, and investigated gene expression changes in murine intracranial (IC) TNBC models stratified by BRCA and molecular subtype status. Experimental design:Athymic mice were inoculated intra-cerebrally with BRCA-mutant: SUM149 (basal), MDA-MB-436 (claudin-low), or BRCA-wild-type: MDA-MB-468 (basal), MDA-MB-231BR (claudin-low) TNBC cells and treated with PBS control (IP, weekly), Carboplatin (50mg/kg/week, IP), ABT888 (25mg/kg/day, OG), or their combination. DNA-damage (?-H2AX) and apoptosis (cleaved-Caspase-3(cC3)) were assessed via IHC of IC tumors. Gene expression of BRCA-mutant IC tumors was measured. Results: Carboplatin+/-ABT888 significantly improved survival in BRCA-mutant IC models compared to control, but did not improve survival in BRCA-wild-type IC models. Carboplatin+ABT888 revealed a modest survival advantage versus Carboplatin in BRCA-mutant models. ABT888 yielded a marginal survival benefit in the MDA-MB-436 but not in the SUM149 model. BRCA-mutant SUM149 expression of ?-H2AX and cC3 proteins was elevated in all treatment groups compared to Control, while BRCA-wild-type MDA-MB-468 cC3 expression did not increase with treatment. Carboplatin treatment induced common gene expression changes in BRCA-mutant models.Conclusions: Carboplatin+/-ABT888 improves survival in BRCA-mutant IC TNBC models with corresponding DNA damage and gene expression changes. Combination therapy represents a promising treatment strategy for patients with TNBC brain metastases warranting further clinical investigation. reference x sample