The component of the m6A writer complex VIRMA is implicated in aggressive tumor phenotype, DNA damage response and cisplatin resistance in germ cell tumors

Abstract

Background Germ cell tumors (GCTs) are developmental cancers, tightly linked to embryogenesis and germ cell development. The recent and expanding field of RNA modifications is being increasingly implicated in such molecular events, as well as in tumor progression and resistance to therapy, but still rarely explored in GCTs. In this work, and as a follow-up of our recent study on this topic in TGCT tissue samples, we aim to investigate the role of N6-methyladenosine (m6A), the most abundant of such modifications in mRNA, in in vitro and in vivo models representative of such tumors. Methods Four cell lines representative of GCTs (three testicular and one mediastinal), including an isogenic cisplatin resistant subline, were used. CRISPR/Cas9-mediated knockdown of VIRMA was established and the chorioallantoic membrane assay was used to study its phenotypic effect in vivo. Results We demonstrated the differential expression of the various m6A writers, readers and erasers in GCT cell lines representative of the major classes of these tumors, seminomas and non-seminomas, and we evidenced changes occurring upon differentiation with all-trans retinoic acid treatment. We showed differential expression also among cells sensitive and resistant to cisplatin treatment, implicating these players in acquisition of cisplatin resistant phenotype. Knockdown of VIRMA led to disruption of the remaining methyltransferase complex and decrease in m6A abundance, as well as overall reduced tumor aggressiveness (with decreased cell viability, tumor cell proliferation, migration, and invasion) and increased sensitivity to cisplatin treatment, both in vitro and confirmed in vivo. Enhanced response to cisplatin after VIRMA knockdown was related to significant increase in DNA damage (with higher γH2AX and GADD45B levels) and downregulation of XLF and MRE11. Conclusions VIRMA has an oncogenic role in GCTs confirming our previous tissue-based study and is further involved in response to cisplatin by interfering with DNA repair. These data contribute to our better understanding of the emergence of cisplatin resistance in GCTs and support recent attempts to therapeutically target elements of the m6A writer complex.

Ki67 and LSD1 Expression in Testicular Germ Cell Tumors Is Not Associated with Patient Outcome: Investigation Using a Digital Pathology Algorithm

“…Briefly, cells were seeded on T25 culture flasks, left to adhere for 24 h, and then treated with 10 µM of the drug for 10 days, with drug renewal every two days. Morphological evidence of differentiation has been confirmed (for details, see [33,34]).…”

“…NCCIT and NT2 cells were treated with differentiating agent all-trans retinoic acid-ATRA (STEMCELL TM Technologies, Vancouver, BC, Canada) to generate differentiated cell lines from the parental ones, as described in [34]. Briefly, cells were seeded on T25 culture flasks, left to adhere for 24 h, and then treated with 10 µM of the drug for 10 days, with drug renewal every two days.…”

The Role of N6-Methyladenosine (m6A) Methylation Modifications in Hematological Malignancies

“…In addition, there are METTL5 (methyltransferase-like 5), ZCCHC4 (zinc finger CCHC-type containing 4), etc., which also have some catalytic functions for m 6 A on rRNAs [ 48 ]. In the methyltransferase complex, some new components including VIRMA (vir-Like m 6 A methyltransferase associated) [ 49 , 50 , 51 ], RBM15/15B(RNA binding motif protein 15/15B) [ 52 ], ZC3H13 (zinc finger CCCH-type containing 13) [ 53 , 54 ], etc. can interact with WTAP directly or indirectly as regulatory subunits, which together with WTAP form the MACOM complex and direct the localization of the whole complex in the cell [ 55 , 56 ].…”

RNA m6A Modification in Immunocytes and DNA Repair: The Biological Functions and Prospects in Clinical Application

“…Recent studies even clarified the correlation between DNA damage repair and m 6 A-modified retrotransposable element (RTE) RNAs, in which intronic Long Interspersed Element-1 (LINE-1) interacts with the hosting gene transcription, resulting in the downregulation of its expression (Xiong et al, 2021). KIAA1429 was also recently discovered to have close links with the modulation of response to cisplatin in germ cell tumor by interfering with DNA damage response (Miranda-Gonçalves et al, 2021).…”