Cisplatin-sensitive Ovarian Cancer Cells Research Articles

Abstract 5860: Understanding the biological role of MMP16 in cisplatin resistance in ovarian cancer

Abstract High-Grade Serous Ovarian Cancer (HGSOC) is the most common and lethal of Ovarian Cancer (OC) types, which accounts for eighty percent of deaths. Treatment for OC includes surgery and platinum-based therapy. However, around 70% of women with HGSOC become resistant to chemotherapy. Therefore, new therapeutic agents with higher specificity and efficacy are needed for women with advanced and drug-resistant OC. Matrix metalloproteases (MMPs) are a group of enzymes involved in the degradation of the extracellular matrix (ECM). Deregulation of MMPs contributes to tumor growth and metastasis in several cancer types. Preliminary studies of our research team found that MMP16 is higher in cisplatin-resistant as compared with cisplatin-sensitive OC cells. The aim of this project is to understand the role of MMP16 in the cisplatin resistance of OC cells. We hypothesize that MMP16 increased expression may contribute to cancer progression and drug-resistance. We performed experiments in A2780/A2780CP20 and OVCAR3/OVCAR3CIS human OC cells. To assess the MMP16 protein levels we used western blot analysis. To measure the mRNA levels, we used qRT-PCR. Kaplan-Meier survival curves were generated by interrogation of the KM Plotter patient database. The MMP16 protein and qRT-PCR experiments confirmed that protein and mRNA levels are more abundant in cisplatin-resistant as compared with cisplatin-sensitive cells. Interrogation of the KM plotter database showed that OC patients with higher MMP16 mRNA levels live less as compared with OC patients with lower MMP16 levels. Ongoing experiments are testing the biological effect of small interference RNA (siRNA) targeting MMP16 in cisplatin-resistant OC cells. Citation Format: Jesennia Bonilla-Liriano, Mariela Rivera-Serrano, Victor Reyes-Burgos, Jose Rolon-Perez, Fatima Valiyeva, Pablo Vivas-Mejia. Understanding the biological role of MMP16 in cisplatin resistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5860.

CD39+MDSC facilitates cisplatin resistance of ovarian cancer cells by affecting adenosine synthesis.

Chemoresistance is a major cause of relapse or death in ovarian cancer (OC) patients. New evidence suggests the crucial role of myeloid-derived suppressor cells (MDSCs) in mediating chemoresistance of cancer cells. We aimed to dissect the way MDSCs affect the cisplatin resistance phenotype of OC and the related mechanisms. MDSCs were isolated from the spleen of OC mice isograft. CCK-8 and colony formation assays revealed the effects of an MDSC-conditioned medium with dysregulated CD39 on the proliferation and cisplatin sensitivity of OC cells. Fluorescence assay was used to reveal the effects of MDSCs with dysregulated CD39 on adenosine triphosphate (ATP) hydrolysis and adenosine (ADO) synthesis. MDSCs with highly expressed CD39 could facilitate the proliferation and cisplatin resistance of OC cells, while MDSCs with downregulated CD39 caused the opposite results. In addition, MDSCs with upregulated CD39 could facilitate the hydrolysis of immunogenic ATP to immunosuppressive ADO, while the introduction of CD39 inhibitor could repress such hydrolysis of ATP and generation of ADO, thereby abating the proliferation and cisplatin resistance of OC cells. CD39+MDSC could promote the proliferation and cisplatin resistance of OC cells by generating high concentrations of ADO, which indicates that targeting CD39+MDSC might be a feasible way to improve cisplatin resistance in OC.

Cu-Catalyzed Asymmetric Kinetic Boron Conjugate Addition of γ-Substituted α,β-Unsaturated γ-Lactams.

Chiral α,β-unsaturated γ-lactams bearing simple γ- substituents are found in biologically active molecules and natural products, however, their synthesis still remains difficult. Herein, we report an efficient kinetic resolution (KR) of γ-substituted α,β-unsaturated γ-lactams via a Cu-catalyzed asymmetric boron conjugate addition, which also leads to the efficient synthesis of chiral β-hydroxy-γ-lactams with β,γ-stereogenic carbon centers. The KR proceeded smoothly with a wide range of γ-alkyl or aryl substituted substrates including those bearing aromatic heterocycles and different N-protected substrates in up to 347 of s value. Their highly versatile transformations, synthetic utility in biologically active molecules, and inhibitory activities against cisplatin-sensitive ovarian cancer cell A2780 have also been demonstrated. Differing from the well-known mechanism involving Cu-B species in Cu-catalyzed boron conjugate additions, our mechanistic studies using density functional theory (DFT) calculations and experiments indicate that a Lewis acid CuI -catalyzed mechanism is the likely pathway in the catalytic reaction.

Novel Homoleptic and Heteroleptic Pt(II) β‐oxodithiocinnamic ester Complexes: Synthesis, Characterization, Interactions with 9‐methylguanine and Antiproliferative Activity

AbstractThree new series of homoleptic and heteroleptic platinum(II) β‐oxodithiocinnamic ester complexes, [Pt(L1–L9)2], [Pt(L1–L9)(DMS)Cl] and [Pt(L1–L9)(DMSO)Cl], were synthesized and characterized using elemental analysis, mass spectrometry, and different NMR spectroscopy (1H, 13C{1H} and 195Pt). The β‐oxodithiocinnamic esters coordinate towards the platinum(II) centre as O,S‐bidentate chelating ligands. The structures of HL3, [Pt(L2)2], [Pt(L6)(DMS)Cl] as well as [Pt(L2)(DMSO)Cl] have been confirmed through the X‐ray crystallography, where the platinum(II) complexes exhibit a slightly distorted square planar geometry. In this article, we also investigated the solvolysis of three representative Pt(II) complexes, as well as the interaction with 9‐methylguanine as a DNA model system, by utilizing the LC‐ESI‐MS technique. A selection of the complexes was assessed for their use as anticancer agents, and cytotoxicity assays with these complexes showed modest toxicity on both Cisplatin sensitive and resistant ovarian cancer cell lines. However, the compounds cytotoxicity was not affected by the Cisplatin resistance mechanisms and a specific selection of the ligands may modify the cell line specificity.

Increased Expression of the RBPMS Splice Variants Inhibits Cell Proliferation in Ovarian Cancer Cells.

RNA-Binding Protein with Multiple Splicing (RBPMS) is a member of family proteins that bind to nascent RNA transcripts and regulate their splicing, localization, and stability. Evidence indicates that RBPMS controls the activity of transcription factors associated with cell growth and proliferation, including AP-1 and Smads. Three major RBPMS protein splice variants (RBPMSA, RBPMSB, and RBPMSC) have been described in the literature. We previously reported that reduced RBPMS levels decreased the sensitivity of ovarian cancer cells to cisplatin treatment. However, little is known about the biological role of the RBPMS splice variants in ovarian cancer cells. We performed RT-PCR and Western blots and observed that both RBPMSA and RBPMSC are reduced at the mRNA and protein levels in cisplatin resistant as compared with cisplatin sensitive ovarian cancer cells. The mRNA and protein levels of RBPMSB were not detectable in any of the ovarian cancer cells tested. To better understand the biological role of each RBPMSA and RBPMSC, we transfected these two splice variants in the A2780CP20 and OVCAR3CIS cisplatin resistant ovarian cancer cells and performed cell proliferation, cell migration, and invasion assays. Compared with control clones, a significant reduction in the number of colonies, colony size, cell migration, and invasion was observed with RBPMSA and RBPMSC overexpressed cells. Moreover, A2780CP20-RBPMSA and A2780CP20-RBPMSC clones showed reduced senescence-associated β-galactosidase (β-Gal)-levels when compared with control clones. A2780CP20-RBPMSA clones were more sensitive to cisplatin treatment as compared with A2780CP20-RBPMSC clones. The A2780CP20-RBPMSA and A2780CP20-RBPMSC clones subcutaneously injected into athymic nude mice formed smaller tumors as compared with A2780CP20-EV control group. Additionally, immunohistochemical analysis showed lower proliferation (Ki67) and angiogenesis (CD31) staining in tissue sections of A2780CP20-RBPMSA and A2780CP20-RBPMSC tumors compared with controls. RNAseq studies revealed many common RNA transcripts altered in A2780CP20-RBPMSA and A2780CP20-RBPMSC clones. Unique RNA transcripts deregulated by each RBPMS variant were also observed. Kaplan-Meier (KM) plotter database information identified clinically relevant RBPMSA and RBPMSC downstream effectors. These studies suggest that increased levels of RBPMSA and RBPMSC reduce cell proliferation in ovarian cancer cells. However, only RBPMSA expression levels were associated with the sensitivity of ovarian cancer cells to cisplatin treatment.

Role of RNA Binding Protein with Multiple Splicing (RBPMS) Variants in Cell Proliferation, Invasion and Angiogenesis in Ovarian Cancer

Worldwide, the number of cancer‐related deaths continues to increase due to the ability of cancer cells to become chemotherapy‐resistant and metastasize. For women with ovarian cancer, a staggering 70% will become resistant to the front‐line therapy, cisplatin. RNA binding protein with multiple splicing (RBPMS) is a member of family proteins that bind to the nascent RNA transcripts regulate their processing, which includes splicing, transport, localization and stability. Evidence indicates that RBPMS interact with c‐Fos, a member of the AP‐1 family that dimerizes with c‐Jun to stimulate AP‐1 transcriptional activity. Furthermore, RBPMS interacted with transcription factor Smad3, which bind to c‐Jun increasing AP‐1 transcriptional activity. Three major RBPMS isoforms have been described, RBPMS‐A (21.8 kDa), RBPMS‐B (22.4 kDa) and RBPMS‐C (24.2 kDa). Reports have shown that each RBPMS isoform interacts with proteins and/or RNAs responsible for cell growth, proliferation, and tumor progression. Previous reports of our laboratory have found that RBPMS expression decrease in cisplatin resistance compared with cisplatin sensitive cells. However, the specific RBPMS isoform responsible of this observation has not been elucidated and less is known about its role on invasiveness ability and angiogenesis capacity. We overexpressed each RBPMS variants (A, B and C) in the cisplatin resistant ovarian cancer cells, A2780CP20, and performed cell viability, cell proliferation and invasion assays. Significant reduction in the number of colonies and their area were observed in both isoform A and isoform C clones, compared with isoform B and control empty vector clones. However, clones of isoform A were more sensitive to cisplatin treatment in comparation with isoform B and C clones. In vivo experiments showed a significant reduction in tumor growth between RBPMS A and C when compared with the controls. Tumor tissues were subjected to immunohistochemical analysis for proliferation (Ki67) and angiogenesis (CD31). Elevated signals of both antibodies were detected in tissues from control groups when compared with RBPMS A and C overexpressing tumor tissues. These studies along with the RNAseq suggest that RBPMS, regulate transcripts involved in cell proliferation and invasion capacity of ovarian cancer cells. Moreover, RBPMS A and C represses uncontrolled proliferation, invasiveness, and angiogenesis in ovarian cancer cells. However, only the isoform A contributed to the cisplatin sensitivity of ovarian cancer cells. Future efforts on this project will be focused on determining the RBPMS isoform that represses AP‐1 (c‐Fos and c‐Jun) dependent gene regulation in cisplatin sensitive ovarian cancer cells.

Overexpression of E74-like transformation-specific transcription factor 3 promotes cellular proliferation and predicts poor prognosis in ovarian cancer.

E74-like E26 transformation-specific (ETS) transcription factor 3 (ELF3), is a member of the ETS transcription factor family, and has been characterized as an epithelial cell-specific transcription factor. The role of ELF3 in tumor progression remains to be elucidated. Previous studies have indicated that loss of ELF3 mRNA and protein expression was associated with poor outcomes in ovarian cancer (OC). By contrast, the present study demonstrated that ELF3 was upregulated in OC, using data from The Cancer Genome Atlas, and elevated expression levels of ELF3 were associated with a poor prognosis. ELF3 promoted OC cell proliferation in vitro and in vivo. The present study revealed that ELF3 inhibited apoptosis and reduced the cisplatin sensitivity of OC cells. Furthermore, the mTOR pathway was found to be activated by ELF3. Collectively, the results of the present study indicated the role of ELF3 in the development and pathogenesis of OC.

Decreased RBPMS Levels Promote Cell Proliferation, Invasion, Angiogenesis, in Cisplatin Resistant Ovarian Cancer Cells

RBPMS is a RNA binding protein with multiple splicing encoded by the RBPMS gene located in chromosome eight in humans. Three major RBPMS isoforms have been described, RBPMS-A (21.8 kDa), RBPMS-B (22.4 kDa) and RBPMS-C (24.2 kDa). Evidence indicate that RBPMS interact with c-Fos, a member of the AP-1 family, that include also c-Jun. Furthermore, RBPMS interact with SMAD protein members to regulate gene expression. Reports have shown that each RBPMS isoform interacts with proteins and/or RNAs responsible for cell growth, proliferation, and tumor progression. Previous reports of our laboratory have found that RBPMS expression decrease in cisplatin resistance compared with cisplatin sensitive cells. However, the specific RBPMS isoform responsible of this observation has not been elucidated and less is known about its role on invasiveness ability and angiogenesis capacity. We overexpressed each RBPMS-A and RBPMS-C in the cisplatin resistant ovarian cancer cells, A2780CP20, and performed cell viability, cell proliferation, invasion assay, and tumor growth in subcutaneous mouse models. Significant reduction in the number of colonies and their area were observed in both isoform A and isoform C of RBPMS compared with control clones. However, clones of isoform A but not the isoform C were more sensitive to cisplatin treatment. Furthermore, in vivo experiments showed a significant reduction in tumor growth between RBPMS A and C when compared with the controls. Tumor tissues were subjected to immunohistochemical analysis for proliferation (Ki67) and angiogenesis (CD31). Elevated signals of both antibodies were detected in tissues from control groups when compared with RBPMS A and C overexpressing tumor tissues. These results suggest that both, isoform A and C, regulate transcripts involved in cell proliferation and invasion capacity of ovarian cancer cells. However, only the isoform A contributed to the cisplatin sensitivity of ovarian cancer cells. Moreover, RBPMS represses uncontrolled proliferation, invasiveness, and angiogenesis in ovarian cancer cells. Future efforts on this project will be focused on determining the RBPMS isoform that represses AP-1 (c-Fos and c-Jun) dependent gene regulation in cisplatin sensitive ovarian cancer cells.

Gold(III) bisdithiolate complexes: molecular conductors that also exhibit anticancer and antimicrobial activities

Introduction Au(III) complexes with dithiolene ligands have been used as molecular conductors and magnetic materials [1]. These compounds feature square planar geometries, as that for cisplatin the Pt(II)-based compound in clinical use as chemotherapeutic drug. Based on the molecular structure, a similar mechanism of action, e.g. antitumor activity and interaction with DNA should be expected for these compounds. Reports on the biological activity of similar gold(III) compounds are relatively scarce in the literature. Only recently, a Au(III) 1,2-dithiolene cyclometalated complex had proved its potential against Gram-positive bacteria [2]. In this study we evaluated two related Au(III) complexes containing N-alkyl-1,3-thiazoline-2-thione dithiolate ligand, [Au(R-thiazdt)2]−1 (R = ethyl-1; R = hydroxyethyl-2) [3a,b] as antitumor and antimicrobial agents. The compounds were assessed in vitro towards cisplatin sensitive ovarian cancer cells (A2780), bacteria and fungus of clinical importance such as Staphylococcus aureus and Candida. Spectroscopic studies were also performed to evaluate the interaction with DNA. Materials and methods The gold complexes were synthesised as previously described [3a,b]. The cytotoxic activity against the A2780 ovarian cancer cells was assessed by the IC50 determined by the MTT assay. The antimicrobial activities of complexes 1 and 2 were assessed by the MIC values towards the Gram+ S. aureus, and the fungal strains C. glabrata and C. albicans, using reported methods [4,5]. The ability of compounds to bind to DNA was assessed by fluorescence spectroscopy using ethidium bromide (EB) as the fluorescence probe. Results Complexes 1 and 2 presented high cytotoxic activity in the cisplatin sensitive A2780 cells even superior than cisplatin. Complex 1 was able to inhibit the growth of S. aureus and both Candida strains, while 2 was much less active. The fluorescence studies revealed a weak interaction of compounds with CT-DNA, in contrast with that found for cisplatin, although the interaction of 2 is somewhat stronger than that found for 1. Discussion and conclusions Results evidenced the importance in what way minor modifications of the Au dithiolate structure can result in loss of activity in particular the antimicrobial activity. In contrast with cisplatin, DNA is not the main target involved in their mode of action. Further studies are needed to explore other potential targets and the mechanism of action.

Abstract 4320: Ovarian cancer persister cells are characterized by enhanced ER stress gene expression correlating with poor survival

Abstract 75% of women suffering from ovarian cancer are diagnosed at late stage of the disease often associated with cancer cell infiltration into the peritoneal cavity. Standard therapy is tumor resection and subsequent platin-based chemotherapy. Relapse is frequently observed caused by persisting cancer cells. Persister cells comprise a therapy-tolerant subpopulation of cancer cells and repeated therapies presumably select for increased tolerance. The present project aims to find molecular markers for the identification and targeted eradication of persister ovarian cancer cells. Persister cells of the cisplatin-sensitive high-grade serous ovarian cancer cell line, OVCAR-3, were selected after cisplatin incubation with clinically relevant doses. Individual clones were isolated from control or cisplatin challenged OVCAR-3 cells. A subpopulation of the resulting clonal persister cells showed a distinct morphological phenotype characterized by increased migration and high viability in short- and long-term survival after cisplatin treatment. A significant upregulation (p<0.001) of the endoplasmic reticulum (ER) stress marker, ATF3, was identified by RNA sequencing and this ER stress phenotype is maintained for more than 20 passages. Additionally, the ovarian cancer OVCAR-8 luciferase reporter cell line was transduced with the “Watermelon” (WM) library [1]. The WM library is a complex barcode library that enables simultaneous tracing of cell lineage and the cellular transcriptional and proliferative states. To study cancer relapse in a CDX model, WM-labelled OVCAR-8-Luc cells were injected into mice and pulse-treated with carboplatin. Single cell RNA sequencing of 900 cells isolated from solid metastases, ascites and treatment-naïve mice revealed an increase in multiple ER stress markers including ATF3, ATF4, JUN and XBP1 in the most abundant solid relapse lineages. Furthermore, analysis of clinical data from GEO, EGA and TCGA showed that low ATF3 expression is associated with increased 5-year-survival of patients with high-grade serous ovarian cancer (Logrank p=6.3e-6). We found that increased ER stress correlates with survival of cancer persister cells and incidence of metastases. This phenomenon was observed in cell lines, a CDX model and clinical data from human ovarian cancer samples. We propose ER stress response, exemplified by ATF3, as a molecular marker to identify and therapeutic targeting of ovarian cancer persister cells. [1] Metabolic switching underlies the ability of cancer persister cells to cycle under drug treatment, Oren Y et al., unpublished Citation Format: Kathrin Böpple, Meng Dong, Markus Kleih, Andrea Gaißler, Yaara Oren, Whitney S. Henry, Moshe Oren, Yael Aylon, Bernd Winkler, Aviv Regev, Robert A. Weinberg, Hans-Georg Kopp, Walter E. Aulitzky, Frank Essmann. Ovarian cancer persister cells are characterized by enhanced ER stress gene expression correlating with poor survival [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4320.

Abstract A57: Identification of novel targets for ovarian cancer treatment

Abstract Ovarian cancer remains one of the most lethal diseases for the female reproductive system with 22,530 new cases and more than 13,980 deaths predicted for 2019 in the United States alone, according to the American Cancer Society. Currently, there is no effective therapy for ovarian cancer patients, in particular for those who become resistant to chemotherapy (cisplatin). We need to identify novel targets for ovarian cancer therapy. We performed RNA deep-sequencing studies in cisplatin-sensitive and cisplatin-resistant ovarian cancer cells. Several RNA (messengers and long noncoding) were differentially abundant in cisplatin-resistant versus cisplatin-sensitive cells. Many of these RNAs have not been previously studied in ovarian cancer. Therefore, we performed rigorous bioinformatic studies that included data filtering and survival rates (using Kaplan-Meier plot analysis) and IPA (Ingenuity Pathway Analysis) to select key candidate genes that can be used as potential targets for ovarian cancer therapy. Additional data filtering was used to select a list of candidate genes whose expression correlates with the overall survival and progression-free survival of ovarian cancer patients. Future experiments will investigate the biologic consequence of targeting each of these candidate genes in ovarian cancer cells and ovarian cancer mouse models. Citation Format: Ricardo Noriega, Jeyshka Reyes, Blanca Quiñones, Joseline Serrano, Pablo Vivas, Josué Pérez. Identification of novel targets for ovarian cancer treatment [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A57.

Abstract A68: Increasing intracellular glucose levels decreased expression of Enolase-I promoting cisplatin resistance in ovarian cancer cells

Abstract Cytoreductive surgery in combination with platinum-based chemotherapy has been used for more than four decades for treat ovarian cancer. Although initially most ovarian tumors respond well to cisplatin treatment, many of those tumors become resistant to chemotherapy. Although many mechanisms of cisplatin resistance have been proposed, the molecular mechanism leading to the cisplatin resistance in ovarian cancer tumors has not been fully understood. In this study, we sought to identify novel proteins associated with the cisplatin resistance of ovarian cancer cells. A proteomic analysis showed 48 proteins differentially abundant in cisplatin-resistant (A2780CP20) compared to their counterpart cisplatin-sensitive (A2780) ovarian cancer cells. The quantitative analysis revealed low protein levels of Enolase-1 (ENO1) in A2780CP20 cells. Small-interference RNA (siRNA)-targeted ENO1 showed a reduction in the sensitivity of ovarian cancer cells to cisplatin treatment. In contrast, ectopic expression of ENO1 in cisplatin-resistant ovarian cancer cells reduced the sensitivity of these cells to cisplatin treatment. Since ENO1 is an enzyme involved in glucose metabolism, we evaluated ovarian cancer cells’ metabolic state by measuring their glucose consumption. Intracellular glucose uptake was measured in pairs of cisplatin-sensitive and cisplatin-resistant cells. The glucose content levels were higher in all cisplatin-resistant cells compared with their -sensitive counterparts. Overall, these results suggest that low levels of ENO1 are associated with the cisplatin resistance of ovarian cancer cells. Therefore, targeting the glycolytic pathways could be a strategy to overcome the cisplatin resistance of women with advanced and drug-resistant ovarian cancer. Citation Format: Robert J. Rabelo-Fernandez, Yasmarie Santana, Nilmary Grafals, Blanca Quiñones, Ginette Santiago, Eunice Lozada, Jeyshka Reyes, Pablo E. Vivas-Mejía. Increasing intracellular glucose levels decreased expression of Enolase-I promoting cisplatin resistance in ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A68.

MiR-194-5p inhibits SLC40A1 expression to induce cisplatin resistance in ovarian cancer

BackgroundmiR-194-5p has been associated with drug resistance in many cancers. However, the role of miR-194-5p in cisplatin resistance in ovarian cancer is still unclear. Materials and methodsTo study the role and mechanism of miR-194-5p in cisplatin resistance, qRT-PCR was performed to determine the expression of miR-194-5p and SLC40A1 in ovarian cancer. Cell Counting Kit-8 (CCK8) assay was used to analyse cell viability after cisplatin treatment. Dual-luciferase reporter gene assay was performed to examine the relationship between miR-194-5p and SLC40A1. The genes downstream of SLC40A1 were investigated through bioinformatics analysis. ResultsCompared to cisplatin-sensitive ovarian cancer cells, higher miR-194-5p expression and lower SLC40A1 expression were found in cisplatin-resistant ovarian cancer cells. Moreover, this study demonstrated that over-expression of miR-194-5p inhibited SLC40A1 expression, and knockdown of miR-194-5p promoted SLC40A1 expression. In addition, dual-luciferase reporter gene assay further confirmed the negative correlation between miR-194-5p and SLC40A1. Furthermore, we found that over-expression of miR-194-5p resulted in cisplatin resistance. When miR-194-5p and SLC40A1 were simultaneously up-regulated, cisplatin sensitivity increased, while down-regulation of miR-194-5p sensitised ovarian cancer cells to cisplatin. However, when miR-194-5p and SLC40A1 were simultaneously down-regulated, cisplatin sensitivity was decreased. These data suggested that miR-194-5p inhibited SLC40A1 expression to induce cisplatin resistance. In addition, bioinformatics analysis indicated a positive correlation of SLC40A1 with hephaestin (HEPH), and homeostatic iron regulator (HFE). However, we found that HEPH and HFE were associated with cisplatin resistance, suggesting that their role in drug resistance is induced by miR-194-5p/SLC40A1. ConclusionIn conclusion, we found that miR-194-5p inhibited SLC40A1 expression to induce cisplatin resistance in ovarian cancer. This study suggests that miR-194-5p could be a potential therapeutic target and a prognostic biomarker for ovarian cancer, with important implications for future research.

Downstream Effectors of ILK in Cisplatin-Resistant Ovarian Cancer.

Despite good responses to first-line treatment with platinum-based combination chemotherapy, most ovarian cancer patients will relapse and eventually develop platinum-resistant disease with poor prognosis. Although reports suggest that integrin-linked kinase (ILK) is a potential target for ovarian cancer treatment, identification of ILK downstream effectors has not been fully explored. The purpose of this study was to investigate the molecular and biological effects of targeting ILK in cisplatin-resistant ovarian cancer. Western blot analysis showed that phosphorylation levels of ILK were higher in cisplatin-resistant compared with cisplatin-sensitive ovarian cancer cells. Further immunohistochemical analysis of ovarian cancer patient samples showed a significant increase in phosphorylated ILK levels in the tumor tissue when compared to normal ovarian epithelium. Targeting ILK by small-interfering RNA (siRNA) treatment reduced cisplatin-resistant cell growth and invasion ability, and increased apoptosis. Differential gene expression analysis by RNA sequencing (RNA-Seq) upon ILK-siRNA transfection followed by Ingenuity Pathway Analysis (IPA) and survival analysis using the Kaplan–Meier plotter database identified multiple target genes involved in cell growth, apoptosis, invasion, and metastasis, including several non-coding RNAs. Taken together, results from this study support ILK as an attractive target for ovarian cancer and provide potential ILK downstream effectors with prognostic and therapeutic value.

Ubiquitin-specific protease 14 regulates ovarian cancer cisplatin-resistance by stabilizing BCL6 oncoprotein

Ubiquitin-specific protease 14 (USP14) is one of the three proteasome-associated deubiquitinating enzymes and implicated in the progression of various cancers. However, the role of USP14 in ovarian cancer remains unknown. By using an unbiased qRT-PCR screen, here we show that USP14 is considerably increased in cisplatin-resistant ovarian cancer cells. Overexpression of USP14 confers resistance to cisplatin-sensitive ovarian cancer cells. Genetic or pharmacological inhibition of USP14 is able to reverse cisplatin-resistance of ovarian cancer cells, which was accompanied by decreased protein expression of BCL6. Besides, BCL6 protein level was also increased in cisplatin-resistant ovarian cancer cells and silencing of BCL6 in these cells restored their sensitivity to cisplatin. At the molecular lever, we found that USP14 interacted with BCL6 and prevented it from proteasomal-dependent degradation. Thus, our results provide a rationale to target USP14-BCL6 axis in ovarian cancer that may be therapeutically beneficial.

Nrf2 induced cisplatin resistance in ovarian cancer by promoting CD99 expression

Cisplatin resistance is a vital obstacle for the prognosis of ovarian cancer. However, the mechanism of cisplatin resistance is still unknown. This research was performed to explore the role of Nrf2 (nuclear factor, erythroid 2 like 2) and CD99 (CD99 molecule) in cisplatin resistance in ovarian cancer. QRT-PCR and Western blot were used to detect the expression of CD99 in ovarian cancer cells and tissues with different cisplatin sensitivities. Cell viability was analyzed by the Cell Counting Kit-8 (CCK8). The relationship of Nrf2 and CD99 was assessed by dual-luciferase reporter gene assay and chromatin immunoprecipitation (ChIP). Bioinformatics analysis was performed to search for the downstream gene of CD99. In this study, it was revealed that CD99 was highly expressed in cisplatin-resistant ovarian cancer cells and tissues, while lower CD99 expression was found in cisplatin-sensitive ovarian cancer cells and tissues. In addition, the overexpression of CD99 resulted in cisplatin resistance; on the other hand, knockdown of CD99 sensitized ovarian cancer to cisplatin. Furthermore, survival analysis indicated that overall survival (OS) and progression-free survival (PFS) of patients with higher CD99 expression were shorter than those with lower CD99 expression. It was also found that when Nrf2 was upregulated in cisplatin-sensitive ovarian cells, CD99 expression and cell viability increased after cisplatin treatment. Knockdown of CD99 could reverse cisplatin resistance induced by Nrf2. Conversely, when Nrf2 was knocked down in cisplatin-resistant ovarian cancer cells, CD99 expression and cell viability with cisplatin treatment decreased, while simultaneously upregulating CD99 reactivated cisplatin resistance in ovarian cancer cells. The dual-luciferase reporter gene assay and ChIP analysis suggested CD99 was a downstream gene of Nrf2, and Nrf2 positively regulated the expression of CD99 at the transcriptional level. In conclusion, Nrf2 induced cisplatin resistance in ovarian cancer cells by promoting CD99 expression. Targeted CD99 might be an effective way to reverse cisplatin resistance in ovarian cancer.

LncRNA ANRIL affects the sensitivity of ovarian cancer to cisplatin via regulation of let-7a/HMGA2 axis.

This paper tried to explore ANRIL expression in ovarian cancer and how it affects cisplatin-sensitivity of ovarian cancer cells via regulation of let-7a/high-mobility group protein A2 (HMGA2) axis. qRT-PCR was used to detect ANRIL and let-7a levels in ovarian cancer tissues and cell lines (SKOV3 and SKOV3/DDP). Then cells were randomly assigned into Blank, negative control siRNA, ANRIL siRNA, let-7a inhibitor, and ANRIL siRNA+let-7a-inhibitor groups. CCK-8 assay was applied for assessing cell viability of cells treated with different concentrations of cisplatin. Flow cytometry was employed to test cell apoptosis rate. qRT-PCR and Western blot were performed for related molecules detection. Nude mice transplanted with SKOV3/DDP cells were used to confirm the effects of ANRIL siRNA on the cisplatin-sensitivity. Ovarian cancer tissues and cisplatin-resistant cells had increased ANRIL expression and decreased let-7a expression, and those patients with higher clinical stage and pathological grade showed higher ANRIL and lower let-7a. Dual-luciferase reporter-gene assay confirmed the targeting relationship between ANRIL and let-7a, and between let-7a and HMGA2. The cell viability and cisplatin IC50 were decreased in ANRIL siRNA group exposed to different concentrations of cisplatin, with enhanced apoptosis, as well as elevated let-7a and declined HMGA2, which would be reversed by let-7a inhibitor. Meanwhile, ANRIL down-regulation enhanced the inhibitory effect of cisplatin on tumor growth of nude mice and reduced tumor weight. Silencing ANRIL expression reduced HMGA2 expression to promote the apoptosis and improve cisplatin-sensitivity of ovarian cancer cells via up-regulating let-7a expression.

Platinum sensitivity of ovarian cancer cells does not influence their ability to induce M2-type macrophage polarization.

Development of platinum resistance in ovarian cancer is mediated by both cancer cells and tumor microenvironment. Activation of epithelial-mesenchymal transition program in cancer cells may lead to enrichment for resistant clones. These processes can be affected by tumor-associated macrophages, a highly plastic population of cells that participate in tumor progression and response to treatment by shaping the microenvironment. We aimed to study how platinum resistance influences the crosstalk between macrophages and ovarian cancer cells. Using cisplatin-sensitive ovarian cancer cell line A2780, we developed and characterized cisplatin-resistant A2780Cis and cisplatin and doxorubicin co-resistant A2780Dox cell lines. Next, we set up an indirect coculture system with THP-1 cell line-derived M0-type-, M1-type- and M2-type-like polarized macrophages. We monitored the expression of genes associated with cellular stemness, multidrug resistance, and epithelial-mesenchymal transition in cancer cells, and expression profile of M1/M2 markers in macrophages. Development of drug resistance in ovarian cancer cell lines was accompanied by increased migration, clonogenicity, and upregulated expression of transcription factors, associated with cellular stemness and epithelial-mesenchymal transition. Upon coculture, we noted that the most relevant changes in gene expression profile occurred in A2780 cells. Moreover, M0- and M1-type macrophages, but not M2-type macrophages, showed significant transcriptional alterations. Our results provide the evidence for bidirectional interplay between cancer cells and macrophages. Independent of platinum resistance status, ovarian cancer cells polarize macrophages toward M2-like type, whereas macrophages induce epithelial-mesenchymal transition and stemness-related gene expression profile in cisplatin-sensitive, but not cisplatin-resistant cancer cells.

SREBP2 contributes to cisplatin resistance in ovarian cancer cells.

This study is to investigate transcription factors involved in cisplatin resistance in ovarian cancer cells. The transcriptome of cisplatin resistant and sensitive A2780 epithelial ovarian cancer cells was obtained from GSE15372. Ovarian transcriptome data GSE62944 was downloaded from TCGA and applied for transcription regulatory network analysis. The analysis results were confirmed using quantitative polymerase chain reaction. The roles of SREBP2 in cisplatin-resistant cells were investigated by RNA inference and cell viability analysis. Transcription regulatory network analysis found that 12 transcription factors and their targets were involved in cisplatin resistant in A2780 cells. Among these factors, the targets of EZH2 and SREBP2 revealed by Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining were also enriched in differentially expressed genes between cisplatin resistant and cisplatin sensitive cells. Their targets were enriched mainly in cell cycle and cholesterol metabolic process, respectively. Bioinformatic analysis illustrated three known targets of SREBP2, namely LDLR, FDFT1, and HMGCR were increased in A2780-resistant cell lines. Additionally, the three genes and SREBP2 were also elevated in live cells after cisplatin treatment via quantitative polymerase chain reaction. Importantly, RNA inference of SREBP2 in A2780 cell line resulted in a decrease of cell viability after cisplatin treatment. SREBP2 played important roles in cisplatin resistance and cholesterol metabolic process might be a novel target for cancer therapy. Impact statement Transcriptome of cisplatin resistant and sensitive A2780 epithelial ovarian cancer cells was obtained from GSE15372 and TCGA. Twelve transcription factors and their targets were involved in cisplatin resistant. Among these factors, the targets of EZH2 and SREBP2 revealed by Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining were also enriched in differentially expressed genes. Their targets were enriched mainly in cell cycle and cholesterol metabolic process. Three targets of SREBP2, namely LDLR, FDFT1, and HMGCR were increased in A2780-resistant cell lines and were found elevated in live cells after cisplatin treatment via qPCR. RNAi of SREBP2 in A2780 cell line resulted in a decrease of cell viability after cisplatin treatment. SREBP2 played important roles in cisplatin resistance and might be a novel target for cancer therapy.

MiRNA-409-3p enhances cisplatin-sensitivity of ovarian cancer cells by blocking the autophagy mediated by Fip200.

Background: Efficacy of chemotherapy for ovarian cancer (OC) is usually affected by various factors,especially the chemoresistance of ovarian cancer cells. Downregulation of miR-409-3p has been found in a variety of tumors. However, the role of miR-409-3p in chemo-resistant OC cells still remains unknown. The aim of this study was to investigate the effect of miR-409-3p on cisplatin-resistant OC cells and to elucidate the mechanism by which enhances cisplatin-sensitivity of OC cells. Methods: Expression of miR-409-3p in OC cells was assessed by qRT-PCR. MTS and clone formation assays were used to validate cell proliferation. Flow cytometry was performed for apoptosis analysis. Western blot assay was used to assess the alterations of signaling pathway related proteins. BALB/c nude mouse xenograft model was used to evaluate the role of miR-409-3p in cisplatin-resistant OC cells in vivo. Results: We found that miR-409-3p was apparently downregulated in the OC cells compared with normal ovarian cells. Results also showed that compared with the cisplatinsensitive cells, in cisplatin-resistant cells, miR-409-3p was decreased with an obvious increasing autophagic activity. In addition, based on the bioinformatics analysis, miR-409-3p was supposed to bind with Fip200. Our results demonstrated that in miR-409-3p overexpression cells, significant decreases were seen in protein expression of Fip200 and autophagic activity, which might be caused by conjugation between overexpressed miR-409-3p and 3'-UTR in Fip200 mRNA. Moreover, under the miR-409-3p overexpression, we found that cisplatin-sensitive and cisplatin-resistant ovarian cancer cells were more sensitive to the chemotherapeutics, which could be reversed by Fip2000. Further, we found that chemosensitivity in tumor cells was augmented by miR-409-3p overexpression, and Fip200 acted as a key link in interrupting the chemotherapy-induced autophagy. Conclusions: Results mentioned above revealed that miR-409-3p can ameliorate cisplatin-sensitivity of ovarian cancer cells through blocking the autophagy mediated by Fip200.