Platinum Resistance In Epithelial Ovarian Cancer Research Articles

Clinical and bioinformatics analysis of the relationship between LAMA3 DNA methylation expression and platinum resistance and prognosis in epithelial ovarian cancer

Objective: To investigate the effect of DNA methylation of laminin α3 (LAMA3) on the prognosis of platinum-resistant epithelial ovarian cancer (EOC) and its possible mechanism. Methods: (1) The relationship between DNA methylation of LAMA3 and platinum resistance in EOC was evaluated by bioinformatics. (2) A total of 67 EOC patients treated at Guangxi Medical University Cancer Hospital from January 2000 to December 2012 were selected to detect the levels of LAMA3 DNA methylation in EOC tissues using pyrophosphate sequencing technology to explore its diagnostic efficacy for platinum resistance and prognosis in EOC patients. Furthermore, its impact on chemotherapy efficacy and prognosis of platinum resistant EOC patients were also analyzed. Results: (1) Ten proteins highly interacting with LAMA3 were screened from the Gene Interaction Retrieval Platform (STRING) database, including laminin β (LAMB) 3, laminin γ (LAMC) 3, integrin α (ITGA) 6, intestine protein β4 (ITGB4), ITGA3, LAMC1,LAMB2, dystrophin associated glycoprotein 1 (DAG1), LAMB1 and cytochrome P450c17α (COL17A1) protein; kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that LAMA3 and its related interacting proteins participate in the regulation of malignant tumor occurrence and development through signaling pathways such as apoptosis, cell cycle, DNA damage response, epithelial mesenchymal transition (EMT), androgen receptor (AR), estrogen receptor (ER), phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt), RAS/mitogen activated protein kinase (MAPK), receptor tyrosine kinase (RTK), tuberous sclerosis protein complex (TSC)/mammalian target of rapamycin (mTOR), and their expression levels were related to the sensitivity of chemotherapy drugs such as cisplatin in EOC. (2) Our clinical data analysis found that the LAMA3 DNA methylation level in EOC tissue of the platinum-sensitive group (35 cases) was 71% (25/35), which was higher than 69% (22/32) in the platinum-resistant group (32 cases), with statistically insignificant difference (χ2=0.057, P=0.811). The area under the curve (AUC) of LAMA3 DNA methylation level for assessing platinum resistance in EOC was 0.601, and the AUC for predicting EOC patient prognosis was 0.686. The chemotherapy efficacy of EOC patients with high methylation of LAMA3 DNA was worse than that of patients with low methylation, 50% (12/24) vs 15/15, with statistically significant difference (χ2=10.833, P=0.001). The level of LAMA3 DNA methylation had a significant impact on the progression free survival and overall survival of EOC patients (both P<0.05). Conclusion: The level of LAMA3 DNA methylation has certain diagnostic and predictive value for platinum resistance and prognosis in EOC patients, which may be closely related to the regulatory mechanism, platinum resistance and prognosis of EOC.

Platinum-resistance in epithelial ovarian cancer: an interplay of epithelial–mesenchymal transition interlinked with reprogrammed metabolism

BackgroundEpithelial ovarian cancer is the most lethal gynaecological cancer worldwide. Chemotherapy resistance represents a significant clinical challenge and is the main reason for poor ovarian cancer prognosis. We identified novel expression of markers related to epithelial mesenchymal transitions (EMT) in a carboplatin resistant ovarian cancer cell line by proteomics. This was validated in the platinum resistant versus sensitive parental cell lines, as well as platinum resistant versus sensitive human ovarian cancer patient samples. The prognostic significance of the different proteomics-identified marker proteins in prognosis prediction on survival as well as their correlative association and influence on immune cell infiltration was determined by public domain data bases.MethodsWe explored the proteomic differences between carboplatin-sensitive OVCAR5 cells (parental) and their carboplatin-resistant counterpart, OVCAR5 CBPR cells. qPCR and western blots were performed to validate differentially expressed proteins at the mRNA and protein levels, respectively. Association of the identified proteins with epithelial–mesenchymal transition (EMT) prompted the investigation of cell motility. Cellular bioenergetics and proliferation were studied to delineate any biological adaptations that facilitate cancer progression. Expression of differentially expressed proteins was assessed in ovarian tumors obtained from platinum-sensitive (n = 15) versus platinum-resistant patients (n = 10), as well as matching tumors from patients at initial diagnosis and following relapse (n = 4). Kaplan–Meier plotter and Tumor Immune Estimation Resource (TIMER) databases were used to determine the prognostic significance and influence of the different proteomics-identified proteins on immune cell infiltration in the tumor microenvironment (TME).ResultsOur proteomics study identified 2422 proteins in both cell lines. Of these, 18 proteins were upregulated and 14 were downregulated by ≥ twofold (p < 0.05) in OVCAR5 CBPR cells. Gene ontology enrichment analysis amongst upregulated proteins revealed an overrepresentation of biological processes consistent with EMT in the resistant cell line. Enhanced mRNA and/or protein expression of the identified EMT modulators including ITGA2, TGFBI, AKR1B1, ITGAV, ITGA1, GFPT2, FLNA and G6PD were confirmed in OVCAR5 CBPR cells compared to parental OVCAR5 cell line. Consistent with the altered EMT profile, the OVCAR5 CBPR cells demonstrated enhanced migration and reduced proliferation, glycolysis, and oxidative phosphorylation. The upregulation of G6PD, AKR1B1, ITGAV, and TGFβ1 in OVCAR5 CBPR cells was also identified in the tumors of platinum-resistant compared to platinum-sensitive high grade serous ovarian cancer (HGSOC) patients. Matching tumors of relapsed versus newly diagnosed HGSOC patients also showed enhanced expression of AKR1B1, ITGAV, TGFβ1 and G6PD protein in relapsed tumors. Among the identified proteins, significant enhanced expression of GFPT2, FLNA, TGFBI (CDGG1), ITGA2 predicted unfavorable prognosis in ovarian cancer patients. Further analysis suggested that the expression of TGFBI to correlate positively with the expression of identified and validated proteins such as GFPT2, FLNA, G6PD, ITGAV, ITGA1 and ITGA2; and with the infiltration of CD8+ T cells, macrophages, neutrophils, and dendritic cells in the TME.ConclusionsOur research demonstrates proteomic-based discovery of novel EMT-related markers with an altered metabolic profile in platinum-resistant versus sensitive ovarian cancer cell lines. The study also confirms the expression of selected identified markers in the tumors of platinum-resistant versus sensitive, and in matching relapsed versus newly diagnosed HGSOC patients. The study provides insights into the metabolic adaptation of EMT-induced carboplatin resistant cells that confers on them reduced proliferation to provide effective migratory advantage; and the role of some of these identified proteins in ovarian cancer prognosis. These observations warrant further investigation of these novel target proteins in platinum-resistant patients.

RNA sequencing and bioinformatics analysis revealed PACSIN3 as a potential novel biomarker for platinum resistance in epithelial ovarian cancer

Failure to respond to treatment in epithelial ovarian cancer can often be attributed to platinum-based chemotherapy resistance. However, the possible mechanisms or candidate biomarkers associated with platinum resistance are yet to be elucidated, even though many researchers have performed related studies. We performed RNA sequencing of clinical specimens obtained from patients with platinum-sensitive or resistant epithelial ovarian cancer (EOC). Furthermore, various bioinformatics approaches, including spatial analysis of functional enrichment, were used to identify key regulators and associated underlying mechanisms of platinum resistance in EOC. Through RNA-sequencing, we identified 263 differentially expressed genes (98 upregulated and 165 downregulated) and subjected them to Gene Oncology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses, which were characterized to the traditional platinum-resistant characteristics. Subsequently, the gene interaction network and module analysis by spatial analysis of functional enrichment software demonstrated protein kinase C and casein kinase substrate in neurons 3 (PACSIN3) as the only upregulated hub gene, and neurotensin (NTS) and KIAA0319 as downregulated hub genes in platinum-resistant EOC. We selected PACSIN3 for further analysis because it has not been studied in relation to response to platinum-based chemotherapy. PACSIN3 was significantly upregulated in ovarian cancer cells compared to immortalized human ovarian surface epithelial cells. In addition, cisplatin-induced apoptosis was measured in PACSIN3 knockout OVCA433 and BRCA-mutated EOC cell line, SNU251, by a fluorescence-activated cell sorting-based Annexin-V/propium iodide double staining assay, which revealed a significant increase in apoptosis. Taken together, the present study presents PACSIN3 as a promising predictive biomarker associated with platinum resistance, especially in BRCA-mutated epithelial ovarian cancers.

Relationship between ascites volume and clinical outcomes in epithelial ovarian cancer

Ascites is a tumor microenvironment, ascites and massive ascites-induce compression could promote the progression of epithelial ovarian cancer (EOC); however, the impact of ascites volume on clinical outcomes has not been studied extensively. We aimed to investigate the association between ascites volume and clinical outcomes especially platinum resistance in EOC. We retrospectively evaluated a total of 546 EOC patients with respect to the amount of ascites, clinicopathologic factors, and survival. Using the threshold of 1500 ml to classify patients into small- and large-volume ascites groups, we analyzed the correlation between ascites volume and clinicopathological factors, including platinum-free interval (PFI), and prognosis. Patients with large volume ascites were more likely to present with later stage disease, primary platinum-resistant (PPR) cancer, and suboptimal cytoreduction. Prolonged PFI was associated with decreased ascites volume. The large-volume ascites group showed worse progression-free survival (PFS) and overall survival (OS). An increase in ascites volume was associated with an increased risk of disease recurrence (hazard ratio [HR] = 1.115, 95% confidence interval [CI]: 1.035-1.200) and death (HR = 1.213, 95% CI: 1.090-1.350). Ascites was an independent predictor of PFS and OS in EOC patients. A large volume of ascites predicated a shortened PFI, an increased incidence of PPR and suboptimal cytoreduction. Thus, the volume of ascites is a simply available clinical parameter, which could be used to evaluate the prognosis and platinum resistance of EOC patients early, it contributes to formulate individualized treatment plan and improve the outcome of EOC patients.

Notch1 Affects Chemo-resistance Through Regulating Epithelial-Mesenchymal Transition (EMT) in Epithelial Ovarian cancer cells.

Background: Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, chemo-resistance is the main cause for treatment failure. Our previous studies have found that SKOV3 could promote immune escape and tumor progression via Notch1 pathway. Therefore, Notch1 is suspected to be involved in chemo-resistance. The current study is to investigate the possible mechanisms of platinum-resistance in epithelial ovarian cancer mediated by Notch1.Methods: The expressions of Notch1, Snail, MMP-2, N-cadherin, Vimentin and E-cadherin were detected by Western-blot. A stable high expression or low expression of Notch1 in ovarian cancer cells was established by using lentiviral gene engineering. The cell migration and invasion ability were observed by scratch test and transwell test. Cell apoptosis rate and cell cycle were analyzed by flow cytometry.Results: The expression levels of Notch1, Snail, MMP-2, N-cadherin and Vimentin in ovarian cancer were high, while the expression levels of E-cadherin were low.Notch1 promoted the expression of Snail, vimentin, N-cadherin and MMP2 protein, but inhibiting the expression of E-cadherin, promoting cell migration and invasion. Notch1 affected apoptosis of cells through Epithelial-Mesenchymal Transition (EMT), increasing the proportion of cells in S phase and G2 phase, thus affecting drug resistance.Conclusion: Notch1 affects EOC cells chemo-resistance by regulating EMT. This may provide a new target for the treatment of ovarian cancer.

Hypermethylation of mismatch repair gene hMSH2 associates with platinum-resistant disease in epithelial ovarian cancer

PurposeOne major reason of the high mortality of epithelial ovarian cancer (EOC) is due to platinum-based chemotherapy resistance. Aberrant DNA methylation may be a potential mechanism underlying the development of platinum resistance in EOC. The purpose of this study is to discover potential aberrant DNA methylation that contributes to drug resistance.MethodsBy initially screening of 16 platinum-sensitive/resistant samples from EOC patients with reduced representation bisulfite sequencing (RRBS), the upstream region of the hMSH2 gene was discovered hypermethylated in the platinum-resistant group. The effect of hMSH2 methylation on the cellular response to cisplatin was explored by demethylation and knockdown assays in ovarian cancer cell line A2780. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was employed to examine the methylation levels of hMSH2 upstream region in additional 40 EOC patient samples. RT-qPCR and IHC assay was used to detect the hMSH2 mRNA and protein expression in extended 150 patients.ResultsRRBS assay discovered an upstream region from − 1193 to − 1125 of hMSH2 was significant hypermethylated in resistant EOC patients (P = 1.06 × 10−14). In vitro analysis demonstrated that global demethylation increased cisplatin sensitivity along with a higher expression of the hMSH2 mRNA and protein. Knockdown hMSH2 reduced the cell sensitivity to cisplatin. MALDI-TOF mass spectrometry assay validated the strong association of hypermethylation of hMSH2 upstream region with platinum resistance. Spearman’s correlation analysis revealed a significantly negative connection between methylation level of hMSH2 upstream region and its expression. The Kaplan-Meier analyses showed the high methylation of hMSH2 promoter region, and its low expressions are associated with worse survival. In multivariable models, hMSH2 low expression was an independent factor predicting poor outcome (P = 0.03, HR = 1.91, 95%CI = 1.85–2.31).ConclusionThe hypermethylation of hMSH2 upstream region is associated with platinum resistant in EOC, and low expression of hMSH2 may be an index for the poor prognosis.

HPIP expression predicts chemoresistance and poor clinical outcomes in patients with epithelial ovarian cancer

This study aims to investigate the expression of the hematopoietic pre-B-cell leukemia transcription factor-interacting protein (HPIP) and its association with platinum resistance in epithelial ovarian cancer (EOC). The protein expression of HPIP was analyzed by immunohistochemistry in 248 patients with EOC. Furthermore, we analyzed the association between HPIP expression and clinicopathological features including prognosis in EOC samples. High HPIP expression was correlated with platinum resistance in EOCs. HPIP in platinum-resistant cases was overexpressed compared with that in platinum-sensitive cases (P<.001). Platinum resistance was independently correlated with the International Federation of Gynecology and Obstetrics stage and HPIP overexpression. High HPIP expression was positively correlated with International Federation of Gynecology and Obstetrics stage, histologic grade, residual tumor size, lymph node metastasis, serum levels of CA125, and poor prognosis. Our findings indicate that HPIP overexpression is an independent predictor of platinum-based chemotherapy resistance and that it may also be a potential biomarker for targeted therapy.

MicroRNA 206 is associated with longer overall survival and platinum sensitivity in serous epithelial ovarian cancer

Objectives: Most women with high-grade serous epithelial ovarian cancer (EOC) eventually develop platinum-resistant disease. MicroRNAs (miRNAs) are pleiotropic and regulate many biologic processes. Our objective was to identify and validate miRNAs associated with platinum resistance in EOC.

TNFAIP8 overexpression is associated with platinum resistance in epithelial ovarian cancers with optimal cytoreduction.

Here, we correlated tumor necrosis factor α-induced protein 8 (TNFAIP8) messenger RNA (mRNA) expression with clinicopathological parameters and investigated the involvement of TNFAIP8 overexpression in platinum resistance of epithelial ovarian cancer (EOC). The status of TNFAIP8 protein was evaluated by Western blot analysis (n = 25) and immunohistochemistry (n = 134). TNFAIP8 mRNA expression was assessed with real-time polymerase chain reaction in fresh frozen EOC tissues (n = 40). TNFAIP8 overexpression at both mRNA and protein levels in platinum-resistant disease was clearly higher than that in platinum-sensitive disease (P < .05). Platinum resistance was independently correlated with residual tumor size (P = .025), ascites (P = .027), and TNFAIP8 overexpression (P = .003). In particular, TNFAIP8 overexpression was correlated with platinum resistance in EOCs with optimal cytoreduction (P = .001). TNFAIP8 mRNA expression was strongly associated with residual tumor size (P = .019). In conclusion, our findings indicate that TNFAIP8 overexpression is an independent predictor of platinum resistance and may be a potential biomarker for targeted therapy.

Abstract B38: The role of TWIST1 in chemoresistance and metastasis in ovarian cancer – potential therapeutic applications

Abstract Introduction: Epithelial Ovarian Cancer (EOC) is a leading cause of death among women with gynecologic cancers. Most patients respond to primary treatment with platinum-based therapies, but eventually develop platinum-resistant metastatic disease. Novel treatment strategies to address both metastasis and platinum-resistance are therefore urgently needed. A promising target for such therapy is TWIST1, a basic helix-loop-helix transcription factor which is aberrantly reactivated in many cancers, and which acts as a master regulator of epithelial to mesenchymal transition (EMT), a process by which epithelial cancer cells gain the ability to migrate and metastasize. A growing body of literature suggests EMT as a chemoresistance mechanism, though little research has focused on the role of TWIST1 in chemoresistance, or as potential therapeutic target in EOC. Purpose: To a. characterize the relationship between TWIST1 and platinum-resistance in EOC, and b. to develop dendrimer delivery of RNA-based TWIST1 inhibitors to prevent metastatic spread and platinum- resistance in EOC. Methods: TWIST1 expression was evaluated via Western Blot in A2780 and A2780R cells, which are platinum-sensitive and platinum-resistant EOC cell lines, respectively. TWIST1 was knocked down via siRNA against TWIST1 complexed with poly(amidoamine) dendrimers, or using shRNA (without dendrimer). Cellular uptake of siRNA-dendrimer complexes was assayed via fluorescent microscopy of AlexaFluor tagged siRNA. Expression of TWIST1 was quantified by Western blotting and quantitative real-time RT-PCR. To assay chemoresistance, the platinum-resistant EOC cells, A2780R, were treated with logarithmically increasing doses of cisplatin (CDDP), and cytotoxicity assayed by sulforhodamine B staining, and compared with a platinum-sensitive A2870 EOC cells, as well as A2780R knockdown of TWIST1. Lastly, TWIST1 expression was correlated with EOC progression, in twenty-two EOC patient samples (eighteen Stage III/IV, four Stage I/II, obtained at City of Hope) via immunohistochemistry (IHC) using anti-Twist polyclonal antibody (Glackin 14-2). Results: TWIST1 was significantly expressed in A2780R cells, with little to no protein expression in A2780 cells. Development of siRNA-dendrimer complexes has resulted in effective, long-term TWIST1 silencing in EOC cells, with peak efficacy up to one week following transfection A2780 cells. TWIST1 knockdown in platinum-resistant EOC cells results in reversal of resistance, with sensitivity approaching that of parental, platinum-sensitive cells. A CDDP dose of 10 μM produced 40% cell death in resistant cells with TWIST1 knockdown, compared to 0% cell death in untreated resistant cells. By comparison, treatment of sensitive cells with the same dose yielded approximately 60% cell death. TWIST1 expression in human EOC tissues by IHC shows strong nuclear expression in advanced stage III/IV cancers, compared to low nuclear expression in early stage I/II disease. Interestingly, stage I/II cancers displayed TWIST1 expression in stromal cells surrounding the tumor as opposed to strong nuclear expression in advanced stage samples, possibly as a result of an inflammatory response. Conclusions: TWIST1 is a promising novel therapeutic target in advanced EOC given its role in metastasis and chemoresistance. It is predominantly expressed in advanced stage disease and platinum-resistant EOC cell lines. Long-term silencing of this transcription factor is feasible with dendrimer complexing, and results in significant reversal of platinum resistance in vitro. Preclinical mouse models to confirm these in vitro results will pave the way for clinical applications to silence EMT transcriptional suppressors and potentially reverse chemoresistance and prevent metastasis. Citation Format: Cai Roberts, Ernest S. Han, Gina Lowe, James Finlay, Sharon Wilczynski, Lucy Ghoda, Carlotta Glackin, Thanh H. Dellinger. The role of TWIST1 in chemoresistance and metastasis in ovarian cancer – potential therapeutic applications. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B38.