RUNX3 Transcript Variants Have Distinct Roles in Ovarian Carcinoma and Differently Influence Platinum Sensitivity and Angiogenesis

The prognosis of late-stage epithelial ovarian cancer (EOC) patients is affected by chemotherapy response and the malignant potential of the tumor cells. In earlier work, we identified hypermethylation of the runt-related transcription factor 3 gene (RUNX3) as a prognostic biomarker and contrary functions of transcript variants (TV1 and TV2) in A2780 and SKOV3 cells. The aim of the study was to further validate these results and to increase the knowledge about RUNX3 function in EOC. New RUNX3 overexpression models of high-grade serous ovarian cancer (HGSOC) were established and analyzed for phenotypic (IC50 determination, migration, proliferation and angiogenesis assay, DNA damage analysis) and transcriptomic consequences (NGS) of RUNX3 TV1 and TV2 overexpression. Platinum sensitivity was affected by a specific transcript variant depending on BRCA background. RUNX3 TV2 induced an increased sensitivity in BRCA1wt cells (OVCAR3), whereas TV1 increased the sensitivity and induced a G2/M arrest under treatment in BRCA1mut cells (A13-2-12). These different phenotypes relate to differences in DNA repair: homologous recombination deficient A13-2-12 cells show less γH2AX foci despite higher levels of Pt-DNA adducts. RNA-Seq analyses prove transcript variant and cell-line-specific RUNX3 effects. Pathway analyses revealed another clinically important function of RUNX3—regulation of angiogenesis. This was confirmed by thrombospondin1 analyses, HUVEC spheroid sprouting assays and proteomic profiling. Importantly, conditioned media (CM) from RUNX3 TV1 overexpressing A13-2-12 cells induced an increased HUVEC sprouting. Altogether, the presented data support the hypothesis of different functions of RUNX3 transcript variants related to the clinically relevant processes—platinum resistance and angiogenesis.

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Transcriptional epigenetic regulation of Fkbp1/Pax9 genes is associated with impaired sensitivity to platinum treatment in ovarian cancer

Clinical Epigenetics ◽

10.1186/s13148-021-01149-8 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Javier Andrés Soto ◽

Carlos Rodríguez-Antolín ◽

Olga Vera ◽

Olga Pernía ◽

Isabel Esteban-Rodríguez ◽

...

Keyword(s):

Gene Expression ◽

Ovarian Cancer ◽

Candidate Genes ◽

Cell Lines ◽

Clinical Data ◽

Epigenetic Regulation ◽

Chemotherapy Response ◽

Potential Candidate ◽

Platinum Sensitivity ◽

Translational Level

Abstract Background In an effort to contribute to overcoming the platinum resistance exhibited by most solid tumors, we performed an array of epigenetic approaches, integrating next-generation methodologies and public clinical data to identify new potential epi-biomarkers in ovarian cancer, which is considered the most devastating of gynecological malignancies. Methods We cross-analyzed data from methylome assessments and restoration of gene expression through microarray expression in a panel of four paired cisplatin-sensitive/cisplatin-resistant ovarian cancer cell lines, along with publicly available clinical data from selected individuals representing the state of chemoresistance. We validated the methylation state and expression levels of candidate genes in each cellular phenotype through Sanger sequencing and reverse transcription polymerase chain reaction, respectively. We tested the biological role of selected targets using an ectopic expression plasmid assay in the sensitive/resistant tumor cell lines, assessing the cell viability in the transfected groups. Epigenetic features were also assessed in 189 primary samples obtained from ovarian tumors and controls. Results We identified PAX9 and FKBP1B as potential candidate genes, which exhibited epigenetic patterns of expression regulation in the experimental approach. Re-establishment of FKBP1B expression in the resistant OVCAR3 phenotype in which this gene is hypermethylated and inhibited allowed it to achieve a degree of platinum sensitivity similar to the sensitive phenotype. The evaluation of these genes at a translational level revealed that PAX9 hypermethylation leads to a poorer prognosis in terms of overall survival. We also set a precedent for establishing a common epigenetic signature in which the validation of a single candidate, MEST, proved the accuracy of our computational pipelines. Conclusions Epigenetic regulation of PAX9 and FKBP1B genes shows that methylation in non-promoter areas has the potential to control gene expression and thus biological consequences, such as the loss of platinum sensitivity. At the translational level, PAX9 behaves as a predictor of chemotherapy response to platinum in patients with ovarian cancer. This study revealed the importance of the transcript-specific study of each gene under potential epigenetic regulation, which would favor the identification of new markers capable of predicting each patient’s progression and therapeutic response.

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Neutrophil-to-lymphocyte ratio and chemotherapy response score as prognostic markers in ovarian cancer patients treated with neoadjuvant chemotherapy

Journal of Ovarian Research ◽

10.1186/s13048-021-00902-0 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

M. Liontos ◽

A. Andrikopoulou ◽

K. Koutsoukos ◽

C. Markellos ◽

E. Skafida ◽

...

Keyword(s):

Ovarian Cancer ◽

Neoadjuvant Chemotherapy ◽

Cancer Patients ◽

Statistical Significance ◽

Univariate Analysis ◽

Neutrophil To Lymphocyte Ratio ◽

Platinum Resistance ◽

Chemotherapy Response ◽

Lymphocyte Ratio ◽

Response Score

Abstract Background Neoadjuvant chemotherapy (NACT) followed by interval debulking surgery (IDS) is the recommended approach in patients with advanced epithelial ovarian cancer (EOC). However, most patients eventually relapse despite the initial high response rate to chemotherapy. Neutrophil-to-lymphocyte ratio is a well-known biomarker that reflects severe inflammation, critical illness, and mortality in various diseases. Chemotherapy response score (CRS) and neutrophil-to-lymphocyte ratio (NLR) have been identified as potential biomarkers of platinum resistance and disease prognosis. We retrospectively evaluated 132 patients with stage IIIc or IV ovarian/fallopian tube/primary peritoneal cancer who had received NACT followed by IDS from 01/01/2003 to 31/12/2018. CRS was assessed on omental specimens collected from IDS according to ICCR guidelines. Results Median age was 64.57 years (SD: 9.72; range 39.2–87.1). Most ovarian tumors were serous epithelial (90.9%; 120/132). An elevated NLR (defined as > 3) was observed in 72% (95/132) of patients in contrast with 28% (37/132) of patients characterized by low NLR status. Median PFS (mPFS) and median overall survival (mOS) were 13.05 months (95% CI: 11.42–14.67)) and 34.69 months (95% CI: 23.26–46.12) respectively. In univariate analysis, CRS3 score was significantly associated with prolonged mPFS (CRS1/2: 12.79 months vs CRS3: 17.7 months; P = 0.008). CRS score was not associated with mOS (P = 0.876). High NLR was not significantly associated with mPFS (P = 0.128), however it was significantly associated with poor mOS (P = 0.012). In multivariate analysis, only performance of surgery maintained its statistical significance with both PFS (P = 0.001) and OS (P = 0.008). Conclusion NLR could serve as a useful predictor of OS but not PFS in ovarian cancer patients receiving NACT. In accordance with our previous study, CRS score at omentum was found to be associated with PFS but not OS in ovarian cancer patients treated with NACT and IDS.

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Frequently deleted genes in ovarian cancer as indicators of platinum response.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.5583 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 5583-5583

Author(s):

Scooter Willis ◽

Victor Manuel Villalobos ◽

Brandon Michael Young ◽

Branimir I. Sikic ◽

Brian Leyland-Jones

Keyword(s):

Ovarian Cancer ◽

Gene Signature ◽

Replication Study ◽

Rank Test ◽

Outcome Analysis ◽

Platinum Resistance ◽

P Value ◽

Full Cohort ◽

Gene Signatures ◽

Platinum Sensitivity

5583 Background: Integrating chromosomal deletions/amplifications with sequencing alterations is increasingly important in the determination of key drivers of outcome in cancer (Leary 2008, Curtis 2012). We introduce a novel computational approach, Gene Set Outcome Analysis, to determine gene signatures constrained to regions with frequent deletion or amplification events in ovarian cancer identified by TCGA. Differential expressions of mRNA from these genes are used as a proxy for loss of function from deletions or amplifications. Methods: Gene signatures from each region were evaluated using log-rank test comparing high and low gene expression groups split by cohort mean. In total, 30,119,708 signatures constrained to 47 deleted and 36 amplified regions were tested for PFS for first line platinum treatment in a random 2/3 split of TCGA ovarian cohort (n=262) resulting in 26,271 gene signatures with p < .001. The remaining 1/3 cohort (n=135) and full cohort (n=397) were used as a replication study where 111 signatures have a p < .01 located in 2 deletion and 1 amplification region. The signature with the lowest p-value from each region that validated in the replication study, were evaluated in GSE9891 (n=179) (Tothill 2008) for PFS when treated with platinum and taxol resulting in gene signatures from 2 deletion regions with p<.05. Results: Greater than mean expression in this gene signature [OXTR, SATB1, WNT7A, SH3BP5, CRBN, ATG7, CRELD1, TMEM43] located at 3p23-p26.2 predicts poor response to platinum chemotherapy in TCGA full ovarian cohort (17.9 vs 14 months p = 1.4E-7) and validates in GSE9891 (19.6 vs 13.3 months p = .014). Using a separate, compact gene signature [CAAP1, LRRC19, IFNA8] located at 9p21.2, samples with lower than mean expression demonstrate increased platinum sensitivity in TCGA full ovarian cohort (12.2 vs 18.2 months p = 1.0E-6) and in GSE9891 (15.5 vs 18.6 p = .055). Conclusions: Response to platinum therapy is an important predictor of survival in high-risk ovarian cancer patients. These signatures arising from common deletion and amplification events in ovarian cancer can anticipate platinum sensitivity and merits further study for use in choosing optimal therapies studying platinum resistance mechanisms.

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Promoter Methylation of the MGRN1 Gene Response to Chemotherapy of Epithelial Ovarian Cancer Patients

10.21203/rs.3.rs-298962/v1 ◽

2021 ◽

Author(s):

Xiaofei Li ◽

Haiyan Sun ◽

Hua Tian ◽

Haibo Zhang ◽

Yunjie Tian ◽

...

Keyword(s):

Mass Spectrometry ◽

Ovarian Cancer ◽

Epithelial Ovarian Cancer ◽

Mrna Expression ◽

Methylation Level ◽

Promoter Region ◽

Upstream Region ◽

Platinum Resistance ◽

Maldi Tof Mass Spectrometry ◽

Skov3 Cells

Abstract Objective: Aberrant DNA methylation is considered to play a critical role in the chemoresistance of epithelial ovarian cancer (EOC). In this study, we explored the relationship between hypermethylation of the Mahogunin Ring Finger 1 (MGRN1) gene promoter and primary chemoresistance in EOC patients.Methods: Hypermethylation of the MGRN1 promoter region in the cancer tissues of platinum-resistant EOC patients was observed by genome-wide methylation level analysis. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used to analyze the methylation level of the MGRN1 promoter region. MGRN1 mRNA and protein expression were examined using RT-qPCR and IHC assays, respectively. The effect of MGRN1 methylation on the cellular response to cisplatin was detected by knockdown assays in SKOV3 cells. Additionally, we performed transcriptome analysis using RNA-seq and explored the possible mechanism by which MGRN1 expression affects the resistance of ovarian cancer cells to platinum. Results: The RRBS assay showed that the upstream region of MGRN1 from -1148 to -1064 was significantly hypermethylated in chemoresistant EOC patients (P=1.78×10−7). The MALDI-TOF mass spectrometry assays revealed a strong association between hypermethylation of the MGRN1 upstream region and platinum resistance in patients with EOC. Spearman’s correlation analysis revealed a significantly negative connection between the methylation level of MGRN1 and its expression in EOC. In vitro analysis demonstrated that knockdown of MGRN1 reduced the sensitivity of cells to cisplatin and that expression of EGR1 was significantly decreased in SKOV3 cells with low levels of MGRN1 expression. Similarly, EGR1 mRNA expression was lower in platinum-resistant EOC patients and was positively correlated with MGRN1 mRNA expression. Conclusion: The hypermethylation of the MGRN1 promoter region and low expression of MGRN1 were associated with platinum resistance in EOC patients.

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Treatment of chemoresistant ovarian cancer with sigma2/SMAC and cisplatin.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.e16508 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. e16508-e16508

Author(s):

Ivy Wilkinson-Ryan ◽

Dirk Spitzer ◽

Robert Mach ◽

Suwanna Vangveravong ◽

Peter S Goedegebuure ◽

...

Keyword(s):

Ovarian Cancer ◽

Combination Therapy ◽

Cancer Cells ◽

Caspase 3 ◽

Platinum Resistance ◽

Apoptotic Pathway ◽

Major Barrier ◽

Skov3 Cells

e16508 Background: Platinum resistance continues to be a major barrier to the successful treatment of ovarian cancer. Overexpression of the X-linked inhibitor of apoptosis proteins (XIAP) contribute to platinum resistance in ovarian cancer through inhibition of caspases and up regulation of Akt activity. Second mitochondrial-derived activators of caspases (SMAC) is an endogenous protein that binds to and reverses XIAP-mediated inhibition of caspases. In order to exploit the SMAC-mediated pro-apoptotic pathway pharmacologically, SMAC mimetics have been developed and shown to induce apoptosis in cancer cells in vitro and in vivo. Untargeted cytotoxic cancer drugs bind to both malignant and normal tissue leading to significant toxicity. We have shown previously that solid tumors upregulate the sigma-2 receptor. We have also shown that sigma-2 ligands are internalized into cancer cells and are therefore an appealing vehicle for tumor targeted therapy. The goal of this study is to test if a conjugate drug of sigma-2 ligand and a SMAC mimetic (sigma-2/SMAC) in combination with chemotherapy is capable of overcoming chemoresistance in ovarian cancer. Methods: SKOV3 and OVCAR3 ovarian cancer cell lines were treated with sigma2/SMAC (1-16μM) and/or cisplatin (.5-10μg/mL). Viability assays were used to detect cell death. Luminescence-based caspase assays were used to compare the activity of caspase-3, -7, and -9 between treatment groups to document involvement of the XIAP survival pathway. Results: We found that sigma2-SMAC is synergistic when used in combination with cisplatin. Compared to untreated cells, SKOV3 cells treated with sigma/2SMAC (4uM), cisplatin .5ug/mL, or combination therapy showed 52.6%, 117.7%, and 34.8% viability respectively (p<.05). Cisplatin and sigma2/SMAC remained synergistic at increasing doses. Similar results were observed in OVCAR3 cells. Caspase-3 and -7 increased in combination therapy 1.2-fold over Sigma/2SMAC alone (4uM) and 7-fold over cisplatin alone (.5ug/mL) in SKOV3 cells (p<.05). Conclusions: This study suggests that the sigma2/SMAC conjugate provides a targeted means for overcoming chemoresistance in ovarian cancer through inhibition of XIAP and activation of caspases.

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A Retrospective Cross-Sectional Cohort Trial Assessing the Prevalence of MTHFR Polymorphisms and the Influence of Diet on Platinum Resistance in Ovarian Cancer Patients

Cancers ◽

10.3390/cancers13205215 ◽

2021 ◽

Vol 13 (20) ◽

pp. 5215

Author(s):

Caitlin Phillips-Chavez ◽

Jermaine Coward ◽

Michael Watson ◽

Janet Schloss

Keyword(s):

Ovarian Cancer ◽

Survival Rate ◽

Serum Folate ◽

Platinum Resistance ◽

Aberrant Methylation ◽

Dietary Zinc ◽

Mthfr Polymorphism ◽

Mthfr Polymorphisms ◽

Platinum Sensitivity

Ovarian cancer has the lowest survival rate in gynaecologic malignancies with a 5-year survival rate of 43%. Platinum resistance is one of the main drivers of ovarian cancer mortality, of which aberrant methylation has been cited as a significant contributor. Understanding the essential role of the methylenetetrahydrofolate reductase enzyme (MTHFR) on DNA synthesis and repair, and how nutrient status can vastly affect its performance, led to the investigation of MTHFR status and dietary influence on platinum response in epithelial ovarian cancer (EOC) patients. Twenty-five adult female patients who completed first-line platinum-based chemotherapy for primary ovarian cancer were selected from Icon Cancer Centres in Australia. Participants were grouped based on platinum response. A full medical and family history, food frequency questionnaire and single blood test were completed, testing for MTHFR polymorphisms, serum folate, serum and active B12 and homocysteine levels. Nineteen of twenty-five participants had an MTHFR polymorphism. Of those, 20% were compound heterozygous, 12% were heterozygous C677T (CT), 4% homozygous C677T, 12% homozygous A1298C and 28% were heterozygous A1298C (AC). Statistically significant associations were found between dietary zinc (p = 0.0086; 0.0030; 0.0189) and B12 intakes in CT genotypes (p = 0.0157; 0.0030; 0.0068) indicating that zinc or vitamin B12 intakes below RDI were associated with this genotype. There were strong associations of vitamin B6 intakes in AC genotypes (p = 0.0597; 0.0547; 0.0610), and dietary folate in compound heterozygotes with sensitive and partially sensitive disease (p = 0.0627; 0.0510). There were also significant associations between serum folate (p = 0.0478) and dietary B12 (p = 0.0350) intakes above RDI and platinum sensitivity in wild-types as well as strong associations with homocysteine levels (p = 0.0886) and zinc intake (p = 0.0514). Associations with dietary B12 (p = 0.0514) and zinc intakes (p = 0.0731) were also strong in resistant wild types. Results indicate that dietary zinc, B12 and B6 intakes may be associated with platinum sensitivity dependent on MTHFR genotype. These results require further research to clarify the dosages necessary to elicit a response; however, they provide a novel foundation for acknowledging the role of diet on treatment response in EOC.

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Neutrophil-to-Lymphocyte Ratio and Chemotherapy Response Score as prognostic markers in ovarian cancer patients treated with Neoadjuvant Chemotherapy

10.21203/rs.3.rs-667991/v1 ◽

2021 ◽

Author(s):

Michalis Liontos ◽

Angeliki Andrikopoulou ◽

Konstantinos Koutsoukos ◽

Christos Markellos ◽

Efthymia Skafida ◽

...

Keyword(s):

Ovarian Cancer ◽

Cancer Patients ◽

Statistical Significance ◽

Univariate Analysis ◽

High Response Rate ◽

Neutrophil To Lymphocyte Ratio ◽

Platinum Resistance ◽

Chemotherapy Response ◽

Lymphocyte Ratio ◽

Response Score

Abstract BackgroundNeoadjuvant chemotherapy (NACT) followed by interval debulking surgery (IDS) is the recommended approach in patients with advanced epithelial ovarian cancer (EOC). However, most patients eventually relapse despite the initial high response rate to chemotherapy. Chemotherapy response score (CRS) and neutrophil-to-lymphocyte ratio (NLR) have been identified as potential biomarkers of platinum resistance and disease prognosis. We retrospectively evaluated 132 patients with stage IIIc or IV ovarian/fallopian tube/primary peritoneal cancer who had received NACT followed by IDS from 01/01/2003 to 31/12/2018. CRS was assessed on omental specimens collected from IDS according to ICCR guidelines. ResultsMedian age was 64.57 years (SD: 9.72; range 39.2 - 87.1). Most ovarian tumors were serous epithelial (90.9%; 120/132). An elevated NLR (defined as > 3) was observed in 72% (95/132) of patients in contrast with 28% (37/132) of patients characterized by low NLR status. Median PFS (mPFS) and median overall survival (mOS) were 13.05 months (95% CI: 11.42-14.67)) and 34.69 months (95% CI: 23.26 - 46.12) respectively. In univariate analysis, CRS3 score was significantly associated with prolonged mPFS (CRS1/2: 12.79 months vs CRS3: 17.7 months; P= 0.008). CRS score was not associated with mOS (P=0.876). High NLR was not significantly associated with mPFS (P=0.128), however it was significantly associated with poor mOS (P=0.012). In multivariate analysis, only performance of surgery maintained its statistical significance with both PFS (P=0.001) and OS (P=0.008).ConclusionNLR could serve as a useful predictor of OS but not PFS in ovarian cancer patients receiving NACT. In accordance with our previous study, CRS score at omentum was found to be associated with PFS but not OS in ovarian cancer patients treated with NACT and IDS.

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CHD4 regulates platinum sensitivity through MDR1 expression in ovarian cancer: A potential role of CHD4 inhibition as a combination therapy with platinum agents

PLoS ONE ◽

10.1371/journal.pone.0251079 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0251079

Author(s):

Yoshiko Oyama ◽

Shogo Shigeta ◽

Hideki Tokunaga ◽

Keita Tsuji ◽

Masumi Ishibashi ◽

...

Keyword(s):

Ovarian Cancer ◽

Cancer Cells ◽

The Cancer Genome Atlas ◽

Ovarian Cancer Cells ◽

Platinum Resistance ◽

Nucleosome Remodeling ◽

Platinum Sensitivity ◽

Mdr1 Expression ◽

Platinum Agents

Platinum sensitivity is an important prognostic factor in patients with ovarian cancer. Chromodomain-helicase-DNA-binding protein 4 (CHD4) is a core member of the nucleosome remodeling and deacetylase complex, which functions as a chromatin remodeler. Emerging evidence indicates that CHD4 could be a potential therapeutic target for cancer therapy. The purpose of this study was to clarify the role of CHD4 in ovarian cancer and investigate its therapeutic potential focusing on platinum sensitivity. In an analysis of the Cancer Genome Atlas ovarian cancer dataset, CHD4 gene amplification was associated with worse overall survival. CHD4 mRNA expression was significantly higher in platinum-resistant samples in a subsequent clinical sample analysis, suggesting that CHD4 overexpression conferred platinum resistance to ovarian cancer cells, resulting in poor patient survival. In concordance with these findings, CHD4 knockdown enhanced the induction of apoptosis mediated by cisplatin in ovarian cancer cells TOV21G and increased cisplatin sensitivity in multiple ovarian cancer cells derived from different subtypes. However, CHD4 knockdown did not affect the expression of RAD51 or p21, the known targets of CHD4 in other cancer types that can modulate platinum sensitivity. Knockdown and overexpression assays revealed that CHD4 positively regulated the expression of multi-drug transporter MDR1 and its coding protein p-glycoprotein. In addition, a first-in-class CHD4/SMARCA5 inhibitor ED2-AD101 showed synergistic interactions with cisplatin. Our findings suggest that CHD4 mediates platinum sensitivity by modulating MDR1 expression in ovarian cancer. Further, CHD4 suppression has a potential to be a novel therapeutic strategy in combination with platinum agents.

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