Cisplatin Response Research Articles

HPV induction of APOBEC3 enzymes mediate overall survival and response to cisplatin in head and neck cancer

Human papillomavirus (HPV) is associated with the development of head and neck squamous cell carcinomas (HNSC). Cisplatin is used to treat HNSC and induces DNA adducts including interstrand crosslinks (ICLs). Previous reports have shown that HPV positive HNSC patients respond better to cisplatin therapy. Our previous reports highlight that loss of base excision repair (BER) and mismatch repair (MMR) results in cisplatin resistance. Of importance, uracil DNA glycosylase (UNG) is required to initiate the BER response to cisplatin treatment and maintain drug sensitivity. These previous results highlight that specific cytidine deaminases could play an important role in the cisplatin response by activating the BER pathway to mediate drug sensitivity. The APOBEC3 (A3) family of cytidine deaminases are enzymes that restrict HPV as part of the immune defense to viral infection. In this study, the Cancer Genome Atlas (TCGA) HNSC data were used to assess the association between the expression of the seven proteins in the A3 cytidine deaminase family, HPV-status and survival outcomes. Higher A3 G expression in HPV-positive tumors corresponds with better overall survival (OS) (HR 0.33, 95 % CI 0.11-0.93, p = 0.04). FaDu and Scc-25 HNSC cell lines were used to assess alterations in A3, BER and MMR expression in response to cisplatin. We demonstrate that A3, Polβ, and MSH6 knockdown in HNSC cells results in resistance to cisplatin and carboplatin as well as an increase in the rate of ICL removal in FaDu and Scc-25 HNSC cells. Our results suggest that A3s activate BER in HNSC, mediate repair of cisplatin ICLs and thereby, sensitize cells to cisplatin which likely contributes to the improved patient responses observed in HPV infected patients.

MicroRNA-103a-3p potentiates chemoresistance to cisplatin in non-small cell lung carcinoma by targeting neurofibromatosis 1.

Lung cancer remains the leading cause of cancer-associated mortality worldwide, and non-small-cell lung cancer (NSCLC) contributes to ~80% of these deaths. However, both primary and acquired cisplatin resistance frequently occurs within the disease and represents a huge clinical treatment problem. The underlying molecular mechanisms are not yet completely understood, but in recent years, microRNAs (miR) have been reported to play vital roles in the development of lung cancer and chemoresistance. In the present study, it was revealed that there were increased expression levels of miR-103a-3p in both NSCLC cell lines and human NSCLC samples that exhibited resistance to cisplatin. The results also revealed that the inhibition of miR-103a-3p in A549/cisplatin cells significantly sensitized these cells to cisplatin, while inhibition of miR-103a-3p expression inhibited tumor growth and enhanced the function of cisplatin in a xenograft animal model. Furthermore, the present study demonstrated that miR-103a-3p regulates cisplatin resistance by targeting neurofibromatosis 1 (NF1) via activating ERK signaling in vitro and in vivo. In conclusion, NF1 was identified as a special miR-103a-3p target in the present study, and it was revealed that targeting NF1 via miR-103a-3p may help reverse chemoresistance and provide a biomarker to cisplatin responsiveness in NSCLC.

Links between cancer metabolism and cisplatin resistance.

Cisplatin is one of the most potent and widely used chemotherapeutic agent in the treatment of several solid tumors, despite the high toxicity and the frequent relapse of patients due to the onset of drug resistance. Resistance to chemotherapeutic agents, either intrinsic or acquired, is currently one of the major problems in oncology. Thus, understanding the biology of chemoresistance is fundamental in order to overcome this challenge and to improve the survival rate of patients. Studies over the last 30 decades have underlined how resistance is a multifactorial phenomenon not yet completely understood. Recently, tumor metabolism has gained a lot of interest in the context of chemoresistance; accumulating evidence suggests that the rearrangements of the principal metabolic pathways within cells, contributes to the sensitivity of tumor to the drug treatment. In this review, the principal metabolic alterations associated with cisplatin resistance are highlighted. Improving the knowledge of the influence of metabolism on cisplatin response is fundamental to identify new possible metabolic targets useful for combinatory treatments, in order to overcome cisplatin resistance.

Association of LncRNA MEG3 polymorphisms with efficacy of neoadjuvant chemotherapy in breast cancer

BackgroundBreast cancer is the most common malignancy in women, and neoadjuvant chemotherapy has been recommended to the patients with locally advanced breast cancer as the initial treatments. Long non-coding RNA (lncRNA) MEG3, an identified tumor suppressor, has been implicated in the development of various cancers. However, there is no data to evaluate the effect of MEG3 polymorphisms on neoadjuvant treatment in the breast cancer.MethodsGenotyping was performed using Nanodispenser Spectro CHIP chip spotting and Mass ARRAY Compact System. Univariate and multivariate logistic regression analyses were used to analyze the associations between the MEG3 polymorphisms and the pathological complete response (pCR). The disease-free survival (DFS) was estimated by the Kaplan-Meier method, and multivariate Cox proportional hazards models were used to calculate the hazard ratios (HRs) with a 95% confidential interval (CI).ResultsA total of 144 patients with available pretreatment blood species were enrolled in the SHPD002 clinic trial of neoadjuvant chemotherapy for breast cancer. MEG3 rs10132552 were significantly associated with good response (Adjusted OR = 2.79, 95% CI 1.096–7.103, p = 0.031) in dominant model. Median follow-up time was 20 months. In multiple regression analysis, rs10132552 TC + CC (adjusted HR = 0.127, 95% CI 0.22–0.728, p = 0.02) and rs941576 AG + GG (adjusted HR = 0.183, 95% CI 0.041–0.807, p = 0.025) were significantly associated with good DFS. MEG3 rs7158663 (OR = 0.377, 95% CI 0.155–0.917, p = 0.032) were associated with a low risk of hemoglobin decrease in dominant models.ConclusionsLncRNA MEG3 polymorphisms were associated with the chemotherapy response and toxicity of paclitaxel and cisplatin. The result indicates that MEG3 polymorphisms can be considered as the predictive and prognostic markers for the breast cancer patients.Trial registrationRetrospectively registered (ClinicalTrials. Gov identifier: NCT02221999); date of registration: Aug 20th, 2014.

Hsp90B enhances MAST1-mediated cisplatin resistance by protecting MAST1 from proteosomal degradation

Microtubule-associated serine/threonine kinase 1 (MAST1) is a central driver of cisplatin resistance in human cancers. However, the molecular mechanism regulating MAST1 levels in cisplatin-resistant tumors is unknown. Through a proteomics screen, we identified the heat shock protein 90 B (hsp90B) chaperone as a direct MAST1 binding partner essential for its stabilization. Targeting hsp90B sensitized cancer cells to cisplatin predominantly through MAST1 destabilization. Mechanistically, interaction of hsp90B with MAST1 blocked ubiquitination of MAST1 at lysines 317 and 545 by the E3 ubiquitin ligase CHIP and prevented proteasomal degradation. The hsp90B-MAST1-CHIP signaling axis and its relationship with cisplatin response were clinically validated in cancer patients. Furthermore, combined treatment with a hsp90 inhibitor and the MAST1 inhibitor lestaurtinib further abrogated MAST1 activity and consequently enhanced cisplatin-induced tumor growth arrest in a patient-derived xenograft model. Our study not only uncovers the regulatory mechanism of MAST1 in tumors but also suggests a promising combinatorial therapy to overcome cisplatin resistance in human cancers.

TAp73 expression and P1 promoter methylation, a potential marker for chemoresponsiveness to cisplatin therapy and survival in muscle-invasive bladder cancer (MIBC)

ABSTRACTIntrinsic and/or acquired resistance to cisplatin is a significant obstacle in the treatment of muscle-invasive bladder cancer. p73, a p53 homolog and determinant of chemosensitivity, is rarely mutated in bladder cancer (BC). However p73 expression and therefore function can be repressed through epigenetic changes. In this study, we sought to identify DNA methylation status of p73, expression of TAp73 isoform, and their role in cisplatin sensitivity in BC. Primary tumor samples from 338 bladder cancer patients showed decreased TAp73 expression in MIBC compared to superficial BC. Low TAp73 protein expression was associated with shorter overall survival. To investigate if the loss of expression was methylation dependent, we utilized Illumina 450K methylation arrays to interrogate over 150 BC patient samples. We found 12 distinct CpGs in the p73 gene locus that were hypermethylated in tumors compared to adjacent normal tissues. Patients with high p73 promoter methylation specifically at CpG site cg07382920 had worse survival. In vitro, treatment with a DNA demethylating agent, decitabine (DAC), decreased TAp73 methylation and upregulated expression in both CR-T24 (cisplatin resistant T24 cells) and wild type T24 cells. Furthermore, treatment with DAC increased cisplatin response in wild type T24 and CR-T24. Our studies indicate that TAp73 expression and P1 promoter methylation, specifically at the cg073892920 site, may have prognostic and diagnostic value in MIBC. In the setting of P1 promoter hypermethylation, DAC could be used as a potentiating agent of cisplatin-based chemotherapy.

Pleiotropic Effects of Epithelial Mesenchymal Crosstalk on Head and Neck Cancer: EMT and beyond

Epithelial mesenchymal crosstalk (EMC) describes the interaction of the tumor stroma and associated fibroblasts with epithelial cancer cells. In this study we analysed the effects of EMC on head and neck cancer cells. In tumor cell lines EMC was induced using media conditioned from a mix-culture of cancer cells and fibroblasts. Cell proliferation and chemotherapy response were assessed using direct cell counting. Flow cytometry, immunohistochemistry of markers of epithelial-mesenchymal transition (EMT) and subsequent TissueFaxs™ acquisition and quantification and western blot analysis were performed. Holotomographic microscopy imaging was used to visualize the effects of EMC on Cisplatin response of SCC-25 cells. EMC induced a hybrid epithelial-mesenchymal phenotype in SCC-25 cells with co-expression of vimentin and cytokeratin. This hybrid phenotype was associated with chemotherapy resistance and increased proliferation of the cells. The EMC conditioned medium led to an activation of the IL-6/STAT3 pathway with subsequent phosphorylation of STAT3. EMC induced a hybrid epithelial-mesenchymal phenotype in HNSCC cells accompanied by increased therapy resistance and cell proliferation. The IL-6/STAT3 pathway might be one of the major pathways involved in these EMC-related effects.

AKR1C1 controls cisplatin-resistance in head and neck squamous cell carcinoma through cross-talk with the STAT1/3 signaling pathway

BackgroundCisplatin is the first-line chemotherapy used against most upper aerodigestive tract carcinomas. In head and neck cancer, sensitivity to cisplatin remains the key issue in treatment response and outcome. Genetic heterogeneity and aberrant gene expression may be the intrinsic factors that cause primary cisplatin-resistance.MethodsCombination of the HNSCC gene expression data and the cisplatin sensitivity results from public database. We found that aldo-keto reductase family 1 member C1 (AKR1C1) may be associated with cisplatin sensitivity in HNSCC treatment of naïve cells. We examined the AKR1C1 expression and its correlation with cisplatin IC50 and prognosis in patients. The in vitro and in vivo AKR1C1 functions in cisplatin-resistance through overexpression or knockdown assays, respectively. cDNA microarrays were used to identify the upstream regulators that modulate AKR1C1-induced signaling in HNSCC. Finally, we used the cigarette metabolites to promote AKR1C1 expression and ruxolitinib to overcome AKR1C1-induced cisplatin-resistance.ResultsAKR1C1 positively correlates to cisplatin-resistance in HNSCC cells. AKR1C1 is a poor prognostic factor for recurrence and death of HNSCC patients. Silencing of AKR1C1 not only reduced in vitro IC50 but also increased in vivo cisplatin responses and vise versa in overexpression cells. Cigarette metabolites also promote AKR1C1 expression. Transcriptome analyses revealed that STAT1 and STAT3 activation enable AKR1C1-induced cisplatin-resistance and can be overcome by ruxolitinib treatment.ConclusionsAKR1C1 is a crucial regulator for cisplatin-resistance in HNSCC and also poor prognostic marker for patients. Targeting the AKR1C1-STAT axis may provide a new therapeutic strategy to treat patients who are refractory to cisplatin treatment.

ATR mediates cisplatin resistance in 3D-cultured breast cancer cells via translesion DNA synthesis modulation

Tissue architecture and cell–extracellular matrix (cell–ECM) interaction determine the organ specificity; however, the influences of these factors on anticancer drugs preclinical studies are highly neglected. For considering such aspects, three-dimensional (3D) cell culture models are relevant tools for accurate analysis of cellular responses to chemotherapy. Here we compared the MCF-7 breast cancer cells responses to cisplatin in traditional two-dimensional (2D) and in 3D-reconstituted basement membrane (3D-rBM) cell culture models. The results showed a substantial increase of cisplatin resistance mediated by 3D microenvironment. This phenotype was independent of p53 status and autophagy activity and was also observed for other cellular models, including lung cancer cells. Such strong decrease on cellular sensitivity was not due to differences on drug-induced DNA damage, since similar levels of γ-H2AX and cisplatin–DNA adducts were detected under both conditions. However, the processing of these cisplatin-induced DNA lesions was very different in 2D and 3D cultures. Unlike cells in monolayer, cisplatin-induced DNA damage is persistent in 3D-cultured cells, which, consequently, led to high senescence induction. Moreover, only 3D-cultured cells were able to progress through S cell cycle phase, with unaffected replication fork progression, due to the upregulation of translesion (TLS) DNA polymerase expression and activation of the ATR-Chk1 pathway. Co-treatment with VE-821, a pharmacological inhibitor of ATR, blocked the 3D-mediated changes on cisplatin response, including low sensitivity and high TLS capacity. In addition, ATR inhibition also reverted induction of REV3L by cisplatin treatment. By using REV3L-deficient cells, we showed that this TLS DNA polymerase is essential for the cisplatin sensitization effect mediated by VE-821. Altogether, our results demonstrate that 3D-cell architecture-associated resistance to cisplatin is due to an efficient induction of REV3L and TLS, dependent of ATR. Thus co-treatment with ATR inhibitors might be a promising strategy for enhancement of cisplatin treatment efficiency in breast cancer patients.

Global Transcriptomic Profile of Dorsal Root Ganglion and Physiological Correlates of Cisplatin-Induced Peripheral Neuropathy.

Multiple cell signaling pathways are implicated in the development, progression, and persistence of cisplatin-induced peripheral neuropathy. Although advances have been made in terms of understanding specific neurotoxic mechanisms, there are few predictive factors identified that can help inform the clinician approach to symptom prevention or management. We investigate the differential sensitivity to cisplatin-induced peripheral neuropathy and examine the contribution of dorsal root ganglion (DRG) transcriptional profiles across two inbred strains of mice. Cisplatin (4 mg/kg intraperitoneal or vehicle control) was administered twice a week for 4 weeks to adult female C57BL/6J and A/J mice-the C57BL/6J strain of mice characterized by a robust mechanical allodynia and the A/J with a mild largely resistant allodynia phenotype. Peripheral nerve conduction velocities (NCVs), electrophysiological evaluation of wide dynamic range (WDR) neurons, morphological examination of DRG neurons, and microarray analysis of spinal cord tissues were compared across the 4 weeks. The A/J strain presents with an early, mild nocifensive response to cisplatin with reduced neuronal activity in WDR neurons and small changes in cross-sectional nucleus size in DRG neurons at 4 weeks. The more nocifensive-sensitive C57BL/6J strain presents with no early changes in WDR neuron responsiveness; however, there were significant changes in DRG size. Both strains demonstrate a drop in NCV after 4 weeks of treatment, with the greatest reduction present in the A/J strain. Transcriptome data implicate neuroimmune modulation in the differential response to cisplatin in the DRGs of A/J and C57BL/6J mice. Nocifensive responses in both strains implicate involvement of small myelinated and unmyelinated fibers in neurotoxic cisplatin response, whereas reductions in NCV reflect involvement of the largest myelinated fibers in the peripheral nerves. Microarray data analysis identifies neuropathy-relevant gene sets with differential activation of pathways, suggesting a role for antigen presentation in the differential neurotoxic response to cisplatin across strains. Further research is indicated to determine the relative contributions of each of these potential pathological mechanisms to both the neurotoxic response to cisplatin and to the potential for targeted therapy.

The ratio of Bcl-2/Bim as a predictor of cisplatin response provides a rational combination of ABT-263 with cisplatin or radiation in small cell lung cancer.

Cisplatin-based chemotherapy and radiotherapy are the most commonly used treatments for small cell lung cancer (SCLC). However, despise initially dramatic response, the response duration of SCLC patients is variable and resistance to chemo- and radio-therapy inevitably develops. The aim of the study is to investigate the role of Bcl-2 family proteins in predicting SCLC sensitivity to cisplatin treatment, and to identify the potential sensitizer of cisplatin or ratiation treatment in SCLC. We collected cisplatin sensitivity data from public available database, and evaluated its possible association with mRNA or protein expression of Bcl-2 family members in SCLC cell lines. The IC50 value of cisplatin was significantly correlated with the ratio of Bcl-2/Bim mRNA expression in 33 SCLC cell lines (P= 0.041) as well as the ratio of Bcl-2/Bim protein expression in 7 SCLC cell lines (P= 0.0252). Furthermore, a BH3-mimetic ABT-263 was found to be able to sensitize SCLC cells to cisplatin or radiation. The synergistic and additive antitumor activity of ABT-263 combined with cisplatin or radiation was associated with the enhanced apoptosis, which may be caused by the disruption of Bcl-2 binding to Bim by ABT-263. Our study indicates that the ratio of Bcl-2/Bim could be a SCLC response predictor to cisplatin, and ABT-263 addition could be an effective strategy to improve the activity of chemo- or radio-therapy in SCLC.

ERCC2 Helicase Domain Mutations Confer Nucleotide Excision Repair Deficiency and Drive Cisplatin Sensitivity in Muscle-Invasive Bladder Cancer.

DNA-damaging agents comprise the backbone of systemic treatment for many tumor types; however, few reliable predictive biomarkers are available to guide use of these agents. In muscle-invasive bladder cancer (MIBC), cisplatin-based chemotherapy improves survival, yet response varies widely among patients. Here, we sought to define the role of the nucleotide excision repair (NER) gene ERCC2 as a biomarker predictive of response to cisplatin in MIBC. Somatic missense mutations in ERCC2 are associated with improved response to cisplatin-based chemotherapy; however, clinically identified ERCC2 mutations are distributed throughout the gene, and the impact of individual ERCC2 variants on NER capacity and cisplatin sensitivity is unknown. We developed a microscopy-based NER assay to profile ERCC2 mutations observed retrospectively in prior studies and prospectively within the context of an institution-wide tumor profiling initiative. In addition, we created the first ERCC2-deficient bladder cancer preclinical model for studying the impact of ERCC2 loss of function. We used our functional assay to test the NER capacity of clinically observed ERCC2 mutations and found that most ERCC2 helicase domain mutations cannot support NER. Furthermore, we show that introducing an ERCC2 mutation into a bladder cancer cell line abrogates NER activity and is sufficient to drive cisplatin sensitivity in an orthotopic xenograft model. Our data support a direct role for ERCC2 mutations in driving cisplatin response, define the functional landscape of ERCC2 mutations in bladder cancer, and provide an opportunity to apply combined genomic and functional approaches to prospectively guide therapy decisions in bladder cancer.See related commentary by Grivas, p. 907.

Proteome analysis of non-small cell lung cancer cell line secretomes and patient sputum reveals biofluid biomarker candidates for cisplatin response prediction

Molecular markers are urgently needed to select non-small cell lung cancer (NSCLC) patients most likely to benefit from platinum-based chemotherapies. Of particular interest are proteins that can be found in biofluids like sputum for non-invasive detection. Therefore, we profiled the secretomes of 6 NSCLC cell lines with varying IC50-values for cisplatin, using label-free GeLC-MS/MS-based proteomics. Out of a total dataset of 2610 proteins, 304 proteins showed significant differences in expression levels between cisplatin sensitive and insensitive cell lines. Functional data mining revealed that the secretion of typically extracellular factors was associated with a higher sensitivity towards cisplatin, while cisplatin insensitivity correlated with increased secretion of theoretically intra-cellular proteins. Stringent statistical analysis and quantitative filtering yielded 58 biomarker candidates, 34 of which could be detected in clinical biofluids of lung cancer patients such as sputum using label-free LC-MS/MS-based proteomics. To assess performance of these biofluid biomarker candidates, we correlated protein expression with patient survival using a publically available clinical gene expression data set (GSE14814). We thus identified 3 top candidates with potential predictive value in determining cisplatin response (UGGT1, COL6A1 and MAP4) for future development as non-invasive biomarkers to guide treatment decisions. SignificancePlatinum-based chemotherapies are still the standard of care for NSCLC and other lung cancer types in the clinic today. However, due to chemoresistance, many patients suffer from the toxic side effects of these treatments without gaining any benefit in terms of survival. To date, no molecular biomarkers are available to predict clinical outcome of platinum-based chemotherapy. Because proteins present the functional read-out of genetic, epigenetic and translational events in the cell, a protein test is likely to be particularly suitable for response prediction. Of high relevance are proteins that are shed or secreted from cells, for example at primary tumor sites, and can be found in easily accessible biofluids like sputum for non-invasive detection. Here, we report the proteome profiling of the conditioned media (secretomes) of a panel of NSCLC cell lines in relation to cisplatin IC50 values, as a pre-clinical model, and of patient sputum as a clinical, lung cancer relevant biofluid. Using this approach in conjunction with exploration of the predictive potential in a transcriptome lung cancer patient dataset, we reveal biofluid biomarker candidates that, with further validation, may be used for non-invasive cisplatin response prediction in the future.

Predicting responses to platin chemotherapy agents with biochemically-inspired machine learning

The selection of effective genes that accurately predict chemotherapy responses might improve cancer outcomes. We compare optimized gene signatures for cisplatin, carboplatin, and oxaliplatin responses in the same cell lines and validate each signature using data from patients with cancer. Supervised support vector machine learning is used to derive gene sets whose expression is related to the cell line GI50 values by backwards feature selection with cross-validation. Specific genes and functional pathways distinguishing sensitive from resistant cell lines are identified by contrasting signatures obtained at extreme and median GI50 thresholds. Ensembles of gene signatures at different thresholds are combined to reduce the dependence on specific GI50 values for predicting drug responses. The most accurate gene signatures for each platin are: cisplatin: BARD1, BCL2, BCL2L1, CDKN2C, FAAP24, FEN1, MAP3K1, MAPK13, MAPK3, NFKB1, NFKB2, SLC22A5, SLC31A2, TLR4, and TWIST1; carboplatin: AKT1, EIF3K, ERCC1, GNGT1, GSR, MTHFR, NEDD4L, NLRP1, NRAS, RAF1, SGK1, TIGD1, TP53, VEGFB, and VEGFC; and oxaliplatin: BRAF, FCGR2A, IGF1, MSH2, NAGK, NFE2L2, NQO1, PANK3, SLC47A1, SLCO1B1, and UGT1A1. Data from The Cancer Genome Atlas (TCGA) patients with bladder, ovarian, and colorectal cancer were used to test the cisplatin, carboplatin, and oxaliplatin signatures, resulting in 71.0%, 60.2%, and 54.5% accuracies in predicting disease recurrence and 59%, 61%, and 72% accuracies in predicting remission, respectively. One cisplatin signature predicted 100% of recurrence in non-smoking patients with bladder cancer (57% disease-free; N = 19), and 79% recurrence in smokers (62% disease-free; N = 35). This approach should be adaptable to other studies of chemotherapy responses, regardless of the drug or cancer types.

A comparative study of nab-paclitaxel versus cisplatin concurrent chemoradiotherapy in locally advanced cervical cancer

Background: Approximately, 80%–90% patients presented with locally advanced stage with bulky central disease in our center, thus induction followed by concurrent chemoradiotherapy (CCRT) plays a predominant role in the treatment of cervical cancer. Aim: The purpose of this study was to compare the effects, toxicities, treatment response, and progression-free survival (PFS) of nab-paclitaxel and cisplatin in the management of cervical cancer as CCRT. Materials and Methods: This was a prospective, observational study performed at a tertiary care hospital. A total of 120 patients of squamous cell carcinoma of cervical cancer had received three cycles of induction chemotherapy (CT), paclitaxel 175 mg/m2, and cisplatin 75 mg/m2, three weekly regimen. All patients were divided into two CCRT arm, A and B. In arm A, patients received external beam radiation therapy (EBRT) with weekly cisplatin 40 mg/m2 plus intracavitary brachytherapy (ICBT). In arm B, patients received EBRT with weekly nab-paclitaxel 70 mg/m2 plus ICBT. Results: In this study, International Federation of Gynecology and Obstetrics Stage III B, 53.33% in arm A and 46.66% in arm B. After EBRT, complete response was 48.33% in arm A and 73.33% patients in arm B, and 51.66% in arm A and 26.66% patients in arm B had partial response. Median duration of follow-up was 33 months (range 24–48). The PFS, P = 0.0093 was significant. Conclusion: With this study, we can consider the justification for future approach for locally advanced cervical cancer which incorporates induction CT followed by concurrent nab-paclitaxel with EBRT followed by ICBT.

ARF-derived peptide sensitizes ovarian cancer cells to Cisplatin

Background: Forkhead box M1 (FOXM1) is over-expressed in multiple cancers, and its over-expression induces chemoresistance and contributes to unfavorable prognosis in patients with ovarian cancer. P19 ARF is a tumor suppressor that contains a motif, which suppresses the transcriptional activity of FOXM1. In this study we investigated the potential role of alternative reading frame (ARF)-derived peptide as a cisplatin sensitizer for ovarian cancer. Methods: FOXM1 expression in ovarian cancer tissues were analyzed using the online databases Oncomine and protein atlas. The association between FOXM1 expression and cisplatin response was analyzed and plotted in a panel of ovarian cancer cell lines. The expression of FOXM1 downstream target genes were accessed in SKOV3 cells treated with ARF peptide using quantitative polymerase chain reaction (QPCR) and western blot. Clonogenicity and apoptosis in SKOV3 cells treated with ARF peptide in the presence or absence of cisplatin was used to evaluate the effect of ARF peptides. In addition, tumor size in nude mice bearing SKOV3 xenografts were measured in the presence or absence of wild-type or mutant ARF peptide and with or without cisplatin. Results: FOXM1 was over-expressed in ovarian cancer tissues and cell lines. FOXM1 expression was associated with cisplatin response in the ovarian cancer cell lines. ARF peptide decreased both mRNA and protein expression of FOXM1 downstream target genes, polo-like kinase 1 (PLK1) and exonuclease 1 (EXO1). In vitro , SKOV3 cells pretreated with ARF peptide showed increased apoptotic cells, decreased clonogenicity, and increased cleavage of caspase-3 in response to cisplatin treatment. In vivo , combination of wild-type ARF peptide and cisplatin had increased inhibition of tumor growth compared to mutant peptide. Conclusions: FOXM1 expression levels are associated with cisplatin response. Treatment with ARF peptide enhanced cisplatin sensitivity in ovarian cancer cells. These results suggest a new therapeutic strategy to overcome chemoresistance in FOXM1 overexpressing ovarian cancers.

Targeting the DNA Repair Endonuclease ERCC1-XPF with Green Tea Polyphenol Epigallocatechin-3-Gallate (EGCG) and Its Prodrug to Enhance Cisplatin Efficacy in Human Cancer Cells.

The 5′-3′ structure-specific endonuclease ERCC1/XPF (Excision Repair Cross-Complementation Group 1/Xeroderma Pigmentosum group F) plays critical roles in the repair of cisplatin-induced DNA damage. As such, it has been identified as a potential pharmacological target for enhancing clinical response to platinum-based chemotherapy. The goal of this study was to follow up on our previous identification of the compound NSC143099 as a potent inhibitor of ERCC1/XPF activity by performing an in silico screen to identify structural analogues that could inhibit ERCC1/XPF activity in vitro and in vivo. Using a fluorescence-based DNA-endonuclease incision assay, we identified the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) as a potent inhibitor of ERCC1/XPF activity with an IC50 (half maximal inhibitory concentration) in the nanomolar range in biochemical assays. Using DNA repair assays and clonogenic survival assays, we show that EGCG can inhibit DNA repair and enhance cisplatin sensitivity in human cancer cells. Finally, we show that a prodrug of EGCG, Pro-EGCG (EGCG octaacetate), can enhance response to platinum-based chemotherapy in vivo. Together these data support a novel target of EGCG in cancer cells, namely ERCC1/XPF. Our studies also corroborate previous observations that EGCG enhances sensitivity to cisplatin in multiple cancer types. Thus, EGCG or its prodrug makes an ideal candidate for further pharmacological development with the goal of enhancing cisplatin response in human tumors.

Small Molecules Identified from a Quantitative Drug Combinational Screen Resensitize Cisplatin's Response in Drug-Resistant Ovarian Cancer Cells

Drug resistance to chemotherapy occurs in many ovarian cancer patients resulting in failure of treatment. Exploration of drug resistance mechanisms and identification of new therapeutics that overcome the drug resistance can improve patient prognosis. Following a quantitative combination screen of 6060 approved drugs and bioactive compounds in a cisplatin-resistant A2780-cis ovarian cancer cell line, 38 active compounds with IC50s under 1 μM suppressed the growth of cisplatin-resistant ovarian cancer cells. Among these confirmed compounds, CUDC-101, OSU-03012, oligomycin A, VE-821, or Torin2 in a combination with cisplatin restored cisplatin's apoptotic response in the A2780-cis cells, while SR-3306, GSK-923295, SNX-5422, AT-13387, and PF-05212384 directly suppressed the growth of A2780-cis cells. One of the mechanisms for overcoming cisplatin resistance in these cells is mediated by the inhibition of epidermal growth factor receptor (EGFR), though not all the EGFR inhibitors are equally active. The increased levels of total EGFR and phosphorylated-EGFR (p-EGFR) in the A2780-cis cells were reduced after the combined treatment of cisplatin with EGFR inhibitors. In addition, a knockdown of EGFR mRNA reduced cisplatin resistance in the A2780-cis cells. Therefore, the top active compounds identified in this work can be studied further as potential treatments for cisplatin-resistant ovarian cancer. The quantitative combinational screening approach is a useful method for identifying effective compounds and drug combinations against drug-resistant cancer cells.

Abstract 2844: Cisplatin response of NSCLC cells irresponsive to knocking down VRAC subunits LRRC8A or LRRC8D

Abstract Platinum compounds represent the backbone of combination chemotherapy protocols for advanced lung cancer. Their efficacies are limited chiefly by either primal or chemotherapeutically acquired cellular platinum resistance whose complex mechanisms became more accessible through recent discoveries (Voets et al., 2015, EMBO J 34(24): 2985-2987). By stepwise increasing the cisplatin concentration in the culture medium, we generated cisplatin-resistant sublines of the wild type A240286S cell strain which derived from the suprarenal metastasis of a lung adenocarcinoma (Heuser et al., 2005, Cancer Lett 223(1): 57-66). All IC50 values were determined under flavin-protecting conditions (Granzow et al., 1995, Cancer Res 55(21): 4837-4843). For cisplatin, IC50 values were determined of 0.46±0.05 µM in the wild type A240286S cells, 6.03±1.39 µM in the Pt2.0 A240286S subline (growing in the presence of 2 µM cisplatin), 9.09±0.56 µM in the Pt4.0 A240286S subline (growing in the presence of 4 µM cisplatin), and 18.49±1.96 µM in the Pt8.0 A240286S subline (growing in the presence of 8 µM cisplatin). Testing for cross resistance to oxaliplatin of these tumor cell lines yielded respective IC50 values of 0.19±0.01 µM, 0.50±0.06 µM, 0.58±0.18 µM, and 0.78±0.11 µM oxaliplatin. Cultivation in the absence of cisplatin for seven months reduced the respective IC50 values for cisplatin to 0.99±0.07 µM in the Pt2.0 A240286S subline and to 4.02±0.23 µM in the Pt4.0 A240286S subline. As described by Planells-Cases et al., 2015, EMBO J 34(24): 2993-3008, the subunit composition of VRAC may determine the cellular resistance to platinum-based anti-cancer drugs in haploid KBM7 leukemia cells. In our study, the LRRC8D expression decreased with increasing cisplatin levels. In the Pt8.0 A240286S subline, LRRC8D expression was nearly abolished. However, knockdown of the LRRC8A or LRRC8D subunits of VRAC showed no effect on the cisplatin response in A240286S wild type cells. The respective IC50 values determined were 0.47±0.03 µM in the LRRC8A knockdown cells and 0.61±0.01 µM in the LRRC8D knockdown cells. In conclusion, in A240286S NSCLC cells, knockdown of the subunit composition of VRAC had no effect on cisplatin response. In addition, and in agreement with clinical experience (Stordal et al., 2007, Cancer Treat Rev 33(4): 347-357), the induction of cisplatin resistance in these cells was accompanied by cross resistance to oxaliplatin. Acknowledgments: We thank Rolf Jaggi and Irene Keller for contributing excellent technical support. Citation Format: Nico O. Ruprecht, Martin Hungerbühler, Christof Granzow, Christoph M. Kempf, Johannes T. Heverhagen. Cisplatin response of NSCLC cells irresponsive to knocking down VRAC subunits LRRC8A or LRRC8D [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2844.

Abstract 2876: Targeting VCP to enhance pancreatic cancer response to DNA damaging agents

Abstract Dysregulation of DNA damage response and repair (DDR) is common in pancreatic and other cancers, and impairs their sensitivity to DNA damaging agents. Targeting DDR mediators is therefore an attractive strategy to improve response to DNA damaging agents. We have identified the AAA ATPase VCP as a lead candidate for this via siRNA screening. VCP-chemotherapy synergistic combinations have not as yet been explored in solid tumours. To investigate the mechanism by which VCP modulates response to cisplatin-induced DNA damage, the effect of VCP functionality loss in decreasing cell viability was validated using two pharmacological VCP inhibitors, NMS-873 and CB-5083, in a panel of pancreatic cancer cell lines. Effects on ovarian cancer cell lines were also assessed for comparison, and IC50 values (72 hours) were found to range from 0.03 - 1.4μM. Greater sensitivity to both inhibitors corresponded with accumulation of K48-ubiquitinated protein levels, and increased expression of the unfolded protein response-related genes BiP, DDIT3, and the spliced form of XBP1. NMS-873 (10nM) or CB-5083 (50nM) were also found to increase cisplatin sensitivity (decreased IC50 values, 48 hours) in MIA-PaCa-2, SKOV-3, PEO1, and PEO4 cell lines, but not in AsPC-1 or PANC-1 cells. To investigate if cisplatin sensitization relates to changes in DDR activity, DNA double strand break repair (DSB) assays, using GFP-reporter constructs, were carried out but lower efficiency of homologous recombination (HR) and non-homologous end-joining pathways were found in both PANC-1 and SKOV-3 cells following VCP inhibition. Consistent with decreased HR activity, VCP inhibition reduced numbers of cisplatin-induced Rad51 foci in both cell lines. Unexpectedly, this was associated with increased proteasomal degradation of Rad51, as indicated by western blotting. Phosphorylation of VCP at serine 784 was detected following cisplatin treatment, and interestingly was found to be reduced following inhibition of DNA-PKcs, one of the main PI3K-related kinases (PIKKs) which directs DSB repair. To explore the role of this modification, a phospho-deficient VCP, with mutations in 4 predicted PIKK-targeted sites (including serine 784), was generated. A significant increase in apoptosis, as measured by Caspase-Glo®3/7 assay, was observed in cisplatin-treated SKOV-3 cells, as an initial model system, expressing this phospho-deficient VCP. Studies are underway to further elucidate the importance of each individual phosphorylation site on cisplatin response and DSB repair. Our findings suggest VCP could be a potential therapeutic target for pancreatic and other cancers. Confirming how VCP regulates DSB repair will allow development of novel strategies to enhance the efficacy of DNA damaging agents in cancer. Citation Format: Yuliana Astuti, Charlene Lam, Harpreet S. Wasan, Elaina N. Maginn. Targeting VCP to enhance pancreatic cancer response to DNA damaging agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2876.