GLI2 and NANOG in epithelial ovarian cancer: molecular pathways driving carboplatin resistance

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.

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.

Programmed death-ligand 1 upregulation is associated with poor prognosis in patients with epithelial ovarian cancer

IntroductionPlatinum resistance seriously affects the survival of patients with epithelial ovarian cancer (EOC). Extracellular matrix play an important role in platinum resistance. Lumican (LUM) is an important proteoglycan in extracellular...

Phosphodiesterase 1A (PDE1A) regulates intracellular cyclic nucleotide signaling and has been implicated in tumor progression, but its clinical relevance and functional role in epithelial ovarian cancer (EOC), particularly in relation to the response to platinum remain unclear. This study aimed to evaluate the clinical significance of PDE1A in EOG and to clarify its functional role in tumor progression and response to platinum-based chemotherapy. PDE1A mRNA and protein levels were analyzed using public databases, RNA sequencing, and immunohistochemistry. Correlations between PDE1A expression, clinicopathological features, and prognosis were assessed. Functional roles were investigated in ovarian cancer cell lines. PDE1A was significantly overexpressed in EOC tissues compared with that in normal ovarian epithelial tissues. Overexpression correlated with advanced International Federation of Gynecology and Obstetrics (FIGO) stage, poor tumor grade, and reduced response to platinum-based chemotherapy. High PDE1A levels were linked to worse disease-free survival and overall survival, and multivariate analysis confirmed PDE1A as an independent prognostic factor. To elucidate its functional role, we performed in vitro experiments showing that PDE1A knockdown suppressed cell proliferation and colony formation, induced G1 arrest, and downregulated β-catenin signaling with reduced cyclin D1 and c-Myc expression. Notably, these inhibitory effects were partially rescued by lithium chloride (LiCl), a Wingless-related integration site (Wnt)/β-catenin activator. In conclusion, our findings identify PDE1A as a Wnt/β-catenin-linked biomarker of tumor progression and platinum resistance in EOC and provide a biological rationale for further investigation of PDE1A-targeted strategies in preclinical models.

Multi drug resistance protein 1 (MDR1) expression on tumor cells has been widely investigated in context of drug resistance. However, the role of MDR1 on the immune cell infiltrate of solid tumors remains unknown. The aim of this study was to analyze the prognostic significance of a MDR1+ immune cell infiltrate in epithelial ovarian cancer (EOC) and to identify the MDR1+ leucocyte subpopulation. MDR1 expression was analyzed by immunohistochemistry in 156 EOC samples. In addition to MDR1+ cancer cells, we detected a MDR1+ leucocyte infiltrate (high infiltrate >4 leucocytes per field of view). Correlations and survival analyses were calculated. To identify immune cell subpopulations immunofluorescence double staining was performed. The MDR1+ leucocyte infiltrate was associated with human epidermal growth factor receptor 2 (HER2) (cc = 0.258, p = 0.005) and tumor-associated mucin 1 (TA-MUC1) (cc = 0.202, p = 0.022) expression on cancer cells. A high MDR1+ leucocyte infiltrate was associated with impaired survival, especially in patients whose carcinoma showed either serous histology (median OS 28.80 vs. 50.64 months, p = 0.027, n = 91) or TA-MUC1 expression (median OS 30.60 vs. 63.36 months, p = 0.015, n = 110). Similar findings for PFS suggest an influence of MDR1+ immune cells on the development of chemoresistance. A Cox regression analysis confirmed the independency of a high MDR1+ leucocyte infiltrate as prognostic factor. M2 macrophages were identified as main part of the MDR1+ leucocyte infiltrate expressing MDR1 as well as the M2 marker CD163 and the pan-macrophage marker CD68. Infiltration of MDR1+ leucocytes, mostly M2 macrophages, is associated with poor prognosis of EOC patients. Further understanding of the interaction of M2 macrophages, MDR1 and TA-MUC1 appears to be a key aspect to overcome chemoresistance in 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.

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.

The copper transporters <italic>CTR1</italic>, <italic>CTR2</italic>, <italic>ATP7A</italic>, and <italic>ATP7B</italic> regulate intracellular concentration of platinum by mediating its uptake and efflux in cells. We sought to explore the effect of genetic polymorphisms in <italic>CTR1</italic>, <italic>CTR2</italic>, <italic>ATP7A</italic>, and <italic>ATP7B</italic> on platinum resistance in patients suffering from epithelial ovarian cancer (EOC). A total of 152 Chinese EOC patients were enrolled in this study, all of whom underwent adjuvant chemotherapy using platinum and taxane after maximal debulking surgery. In total, 11 single-nucleotide polymorphisms (SNPs) in <italic>CTR1, CTR2, ATP7A</italic>, and <italic>ATP7B</italic> were genotyped in these patients. The <italic>CTR1</italic> rs10981694 polymorphism was observed to be associated with carboplatin resistance, while patients with the rs10981694 G allele showed a significantly higher rate of carboplatin resistance (OR=4.00, 95% CI 1.309-12.23, p<0.01). In addition, we found that <italic>ATP7A</italic> rs2227291 was associated with cisplatin resistance and that carriers of the C allele were more sensitive to cisplatin (OR=0.40, 95% CI: 0.17-0.94, p=0.03). Our findings suggest that the <italic>CTR1</italic> and <italic>ATP7A</italic> genetic polymorphisms could affect platinum resistance. The <italic>CTR1</italic> and <italic>ATP7A</italic> genes might be considered a predictive marker for carboplatin and cisplatin resistance, respectively. .

To investigate regulatory effects and associated mechanisms of myeloid differentiation factor 88 (MyD88) on cisplatin chemoresistance in human ovarian cancer (OC) cells. The expression of MyD88 in SKOV3/DDP cells was restrained by short interfering RNA (siRNA) of MyD88. Cell viability and proliferation in the presence or absence of cisplatin were detected by Cell Counting Kit-8 (CCK-8). The expression of p-Akt protein, X-linked inhibitor of apoptosis protein (XIAP), and multidrug resistance protein 1 (MRP1) was detected by Western blot analysis. After Protein Kinase B (PKB/Akt) signal pathway was inhibited by the p-Akt inhibitor (LY294002) and the expression of MRP1 was restrained by siRNA of MRP1, CCK-8 was used to examine the cell proliferation after treatment with cisplatin. After the expression of MyD88 in SKOV3/DDP cells was restrained, the cell proliferation was inhibited, the cisplatin resistance decreased and the half maximal inhibitory concentration (IC50) reduced to 38 % of the control group (P < 0.01). The increased expression of p-Akt, XIAP, and MRP1 in SKOV3/DDP cells after cisplatin treatment was also repressed by the downregulation of MyD88. Furthermore, the inhibition of PKB/Akt signal pathway or expression of MRP1 both could decrease the cisplatin resistance of SKOV3/DDP cells and the IC50 decreased to 75 and 53 % of the control group (P < 0.01, P < 0.05), respectively. MyD88 promoted cisplatin chemoresistance in human OC cells through activating PKB/Akt signal pathway, and enhancing the expression of XIAP and MRP1. MyD88 might be a new target of the novel pharmacological treatments for cisplatin-resistant OC.

Evaluation of Chemoresistance Markers in Women with Epithelial Ovarian Carcinoma

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

Overexpression of plasma membrane MRP-1 can lead to multidrug resistance. In this study we describe for the first time the expression of mitochondrial MRP-1 in untreated human normal and cancer cells and tissues and investigate the functional significance of mitochondrial MRP-1 with a view to identifying a new therapeutic starategy. MRP-1 expression and subcellular localisation in normal and cancer cells and tissues was examined by differential centrifugation and western blotting, and immunofluorescence microscopy. Protein extracts were de-glycosylated using the Protein Deglycosylation Mix (NEB). Viable mitochondria were isolated and MRP-1 efflux activity measured using the calcein-AM functional assay. MRP-1 expression was increased by retroviral infection and specific overexpression confirmed by RNA array. Cell viability was determined by trypan blue exclusion and annexin V-PI labelling of cells. Mitochondrial membranes were solubilised by incubation with increasing concentrations of the detergent digitonin and MRP-1 localisation within the mitochondria evaluated by Western blot; protein markers of the mitochondrial outer and inner membranes, the matrix and intermembrane space were used to confirm localisation within the mitochondria. For the first time we have detected MRP-1 in the mitochondria of untreated cancer and normal cells and tissues by Western blot and confirmed these findings by immunofluorescence and confocal microscopy. The size of the MRP-1 protein in the mitochondria of cancer and normal cells was between 150–250 kDa, reflecting differences in glycosylation. The efflux activity of mitochondrial MRP-1 was more efficient (55–64%) than plasma membrane MRP-1 (11–22%; p&amp;lt;0.001). Specific overexpression of MRP-1 in TC-32 cells by retroviral infection induced a preferential increase in mitochondrial MRP-1 (150%), resulting in an 85% increase in MRP-1 efflux activity (p&amp;lt;0.0001), suggesting selective targeting to this organelle. This was associated with an increase in the IC50 of the MRP-1 substrates vincristine (4.5nM to 14.7nM; p&amp;lt;0.001) and etoposide (101.7nM to 121.6nM (p&amp;lt;0.001) but not the non MRP-1 substrate actinomycin D (0.5nM). Treatment with a non-lethal concentration of doxorubicin (0.85nM, 8h) increased mitochondrial and plasma membrane MRP-1, increasing resistance to MRP-1 substrates. The glycosylation of MRP-1 and the efflux of calcein-F from whole mitochondria are consistent with localisation of MRP-1 to the outer mitochondrial membrane. This was confirmed by the increased expression of MRP-1 protein in the mitochondrial supernatant after treatment with digitonin and centrifugation. The expression pattern of MRP-1 was similar to that of porin, an outer mitochondrial membrane marker. Analysis of the MRP-1 sequence (using Predator and MitoProtII) failed to reveal published mitochondrial targeting sequences, and so we are currently investigating the mechanism of MRP-1 transport to the mitochondria to identify a druggable therapeutic target. MRP-1 expressed in the outer mitochondrial membrane has increased efflux activity compared to plasma membrane MRP-1, and so represents an exciting new therapeutic target where historically conventional MRP-1 inhibitor strategies targeting plasma membrane MRP-1 have had limited clinical success. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B79.

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