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.
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.
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.
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.
Journal of experimental & clinical cancer research : CR. 2021 Aug 25*** epublish ***
Vera Miranda-Gonçalves, João Lobo, Catarina Guimarães-Teixeira, Daniela Barros-Silva, Rita Guimarães, Mariana Cantante, Isaac Braga, Joaquina Maurício, Christoph Oing, Friedemann Honecker, Daniel Nettersheim, Leendert H J Looijenga, Rui Henrique, Carmen Jerónimo
Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP) / RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto) / Porto Comprehensive Cancer Center (Porto.CCC), R. Dr. António Bernardino de Almeida, 4200-072, Porto, Portugal., Department of Urology, Portuguese Oncology Institute of Porto (IPOP), R. Dr. António Bernardino de Almeida, 4200-072, Porto, Portugal., Department of Medical Oncology, Portuguese Oncology Institute of Porto (IPOP), R. Dr. António Bernardino de Almeida, 4200-072, Porto, Portugal., Department of Oncology, Hematology and Bone Marrow Transplantation with Section of Pneumology, Mildred Scheel Cancer Career Center HaTriCs4, University Cancer Center Hamburg, University Medical Center Hamburg-Eppendorf, Martinistraße 52, 20246, Hamburg, Germany., Tumour and Breast Center ZeTuP St. Gallen, Rorschacher Strasse 150, 9006, St. Gallen, Switzerland., Department of Urology, Urological Research Lab, Translational UroOncology, University Hospital Düsseldorf, 40225, Düsseldorf, Germany., Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584, CS, Utrecht, The Netherlands., Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP) / RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto) / Porto Comprehensive Cancer Center (Porto.CCC), R. Dr. António Bernardino de Almeida, 4200-072, Porto, Portugal. ., Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP) / RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto) / Porto Comprehensive Cancer Center (Porto.CCC), R. Dr. António Bernardino de Almeida, 4200-072, Porto, Portugal. .