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

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 (m(6)A), 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 m(6)A 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 m(6)A 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 gamma H2AX 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 m(6)A writer complex.

Automated summary

The study found significant differences in the expression of various m(6)A modifiers in cell lines representing different classes of GCTs, as well as changes after treatment with all-trans retinoic acid. Knockdown of VIRMA resulted in disruption of the methyltransferase complex, decreased m(6)A abundance, reduced tumor aggressiveness, and increased sensitivity to cisplatin treatment both in vitro and in vivo. Enhanced response to cisplatin after VIRMA knockdown was associated with increased DNA damage and downregulation of specific genes related to DNA repair.