DNA Methylation of miR-7 is a Mechanism Involved in Platinum Response through MAFG Overexpression in Cancer Cells

Abstract

One of the major limitations associated with platinum use is the resistance that almost invariably develops in different tumor types. In the current study, we sought to identify epigenetically regulated microRNAs as novel biomarkers of platinum resistance in lung and ovarian cancers, the ones with highest ratios of associated chemo-resistance.Methods: We combined transcriptomic data from microRNA and mRNA under the influence of an epigenetic reactivation treatment in a panel of four paired cisplatin -sensitive and -resistant cell lines, followed by real-time expression and epigenetic validations for accurate candidate selection in 19 human cancer cell lines. To identify specific candidate genes under miRNA regulation, we assembled “in silico” miRNAs and mRNAs sequences by using ten different algorithms followed by qRT-PCR validation. Functional assays of site-directed mutagenesis and luciferase activity, miRNAs precursor overexpression, silencing by antago-miR and cell viability were performed to confirm their specificity in gene regulation. Results were further explored in 187 primary samples obtained from ovarian tumors and controls.Results: We identified 4 candidates, miR-7, miR-132, miR-335 and miR-148a, which deregulation seems to be a common event in the development of resistance to cisplatin in both tumor types. miR-7 presented specific methylation in resistant cell lines, and was associated with poorer prognosis in ovarian cancer patients. Our experimental results strongly support the direct regulation of MAFG through miR-7 and their involvement in the development of CDDP resistance in human tumor cells.Conclusion: The basal methylation status of miR-7 before treatment may be a potential clinical epigenetic biomarker, predictor of the chemotherapy outcome to CDDP in ovarian cancer patients. To the best of our knowledge, this is the first report linking the regulation of MAFG by miRNA-7 and its role in chemotherapy response to CDDP. Furthermore, this data highlights the possible role of MAFG as a novel therapeutic target for platinum resistant tumors.

A novel role for the tumor suppressor geneITF2in lung tumorigenesis and chemotherapy response

“…The NSCLC and ovarian cancer cell lines H23, H460, OVCAR3 and A2780 were purchased from the ATCC (Manassas, Virginia, USA) and ECACC (Sigma-Aldrich, Madrid, Spain) and cultured as recommended. Their CDDP-resistant variants H23R, H460R, OVCAR3R and A2780R were previously established in our laboratory 13,37 . Cisplatin (Farma Ferrer, Barcelona, Spain) was used for CDDP-viability assays.…”

“…We selected a representative number of fresh frozen surgical specimens from University Hospital La Paz (HULP)-Hospital Biobank, totaling 25 NSCLC and 10 ovarian cancer samples, belonging to previously reported cohorts of patients 37…”

“…Total RNA from human cancer cell lines and surgical samples was isolated, reverse transcribed and quantitative RT-PCR analysis was performed as previously described 37 GADPH: Hs03929097_g1). Primers for DKK1, HOXD9, CLDN6, XIST and RIOX1 for RT-PCR assays were designed, when possible, to analyze the specific transcript that showed significant changes in the RNA-seq; all primers and specific amplification conditions are listed in Table S6.…”

“…In this study, we performed high resolution million feature array-Comparative Genomic Hybridization (aCGH) with four NSCLC and ovarian cancer sensitive/resistant paired cell lines previously reported by our group 37 to explore the chromosomic deletions that differ in the resistant subtypes. We found a common deletion that includes the Transcription Factor 4, TCF4 (hereafter called ITF2).…”

MAPK-induced miR-29 targets MAFG and suppresses melanoma development

“…Thus, MAFG is deregulated in melanoma through multiple mechanisms, and our in vitro data further suggest that MAFG plays an important role in melanoma progression and maintenance. Oncogenic roles for MAFG have so far been described in lung, ovarian, colorectal, and liver cancer (Fang et al 2016;Vera et al 2017;Liu et al 2018), and, interestingly, MAFG is a binding partner of CNC and BACH protein families, including NRF2 (reviewed in (Katsuoka and Yamamoto 2016)). NRF2 is a critical regulator of redox biology and cellular metabolism (Hirotsu et al 2012;Hayes and Dinkova-Kostova 2014), and mutations in NRF2 and its negative regulator KEAP1 occur frequently in lung and upper airway cancers (Cloer et al 2019).…”