A novel pathway linking Ras oncogene activation and p53 inhibition mediated by MageC2 tumoral protein in human melanoma

PASCUCCI, FRANCO A.; SUBERBORDES MELISA; LADELFA, M FÁTIMA; MONTE, MARTÍN; ESCALADA, MICAELA

Mar del Plata

Congreso; LXV Reunión Científica de la Sociedad Argentina de Investigación Clínica (SAIC); 2020

Ras proteins (H-Ras, N-Ras and K-Ras) are small GTPases that mainly transduceproliferation signals form growth-factor activated receptor tyrosine kinases (RTKs) throughthe mitogen activated protein kinase (MAPK) pathway. MAPK is composed of a cascade ofthree kinases, Raf, MEK and ERK 1. Activating mutations in Ras or B-Raf proto-oncogenes(i.e, RasV12 or B-RafV600E) are frequent in human cancers 2,3 and can keep the MAPKsignal turned on even in the absence of growth factors or activated RTKs.It is well known that expression of oncogenic versions of Ras or B-Raf in normal cellsactivates cell proliferation that is opposed by the activation of p53 and/or pRbtumor-suppressors, in most cases triggering premature senescence 4,5. However, whentumor-suppression activity is compromised or weakened, H-RasV12 and B-RafV600Ebehave as a potent oncogene orchestrating a complex network of signaling, mainlyinvolving the hyperactivation of the MEK-ERK MAPK pathway 6.p53 and Ras/B-Raf mutations are hallmarks in cancer. Hot spot mutations have beenfound in a variety of human cancers, making them cooperate in malignancy 7. However,tumors carrying Ras/B-Raf activating mutations and wild type (wt) p53 are frequent intestis and skin cancer 3. In these cases, it has been proposed that other proteins should bedampening the p53 response.In human cutaneous melanoma, mutations in TP53 (gene encoding the p53 protein) arenot common8. Moreover, activating mutations in Ras or B-Raf proto-oncogenes (RasV12and B-RafV600E) are particularly frequent in melanoma (15-20 % and 50-70 %respectively). It has been observed that in this particular context for a tumor cell:oncogene-activated status and wt-p53, MDM2/MDM4 overexpression (negative regulatorof p53) 9,10, or CDKN2A locus deletion (which encodes for a negative regulator of MDM2)11, could explain, in a fraction of human melanoma samples, why wt-p53 response toactivated oncogenes is attenuated.Then, it is now recognized as a major goal in human melanoma research to find novelmechanisms responsible for keeping low wt-p53 response in a hyperactivated MAPKcontext 12.In the last years, we and other groups demonstrated that a selected number of proteinscodified by the MAGE-I (Melanoma Antigen GEnes-I) genes can downregulate p53 13.MAGE-I family belongs to the Cancer and Testis Antigens (CTAs) group, and thereforenormally expressed in germ line cells but aberrantly expressed in human cancer cells 14.Normal somatic cells do not express MAGE-I genes due to localized chromatinmethylation. However, epigenetic alterations frequently found in cancer cells allowsMAGE-I genes to be re-expressed in early stages of carcinogenesis. This atypical geneexpression was first observed in melanoma cells and gave the name to the gene family.Later, it was observed that MAGE-I genes are expressed in a variety of human tumors.MAGE-I genes map in discrete sectors of the X chromosome (clusters A, B, C) thus formingthe MAGE-A (11 genes), MAGE-B (10 genes), MAGE-C (3 genes) groups. Today, MAGE-Iexpression in cancer is associated with aggressiveness, increased growth, and poor clinicaloutcome 15.During the functional characterization of MAGE-A proteins, we revealed that specificmembers of the MAGE-A group are able to inhibit p53 activity, making melanoma cellsexpressing these genes, unable to execute a proper response to chemotherapeutic drugs16. From then, the p53/MAGE relationship in cancer cells is being studied by a number oflaboratories. We and others observed that MageC2, a non MAGE-A member, can beincluded as a potent regulator of p53 activity 17?19.In addition to its ability to downregulate p53, a comparison considering 31 of the mostprevalent forms of cancer profiled by The Cancer Genome Atlas (TCGA), showedcutaneous melanoma as the tumor type expressing more MageC2 mRNA.Here, we provide evidence that MageC2 could play a key role as a p53 regulator inoncogene-activated MAPKs context. We observed that H-Ras or B-Raf oncogenes enhanceMageC2 protein levels through the MEK/ERK MAPK pathway in melanoma cells. ElevatedMageC2 protein does not correlate with enhanced mRNA but with protein stabilizationand phosphorylation. To investigate the role of MageC2 in the regulation of p53 activity inactivated MEK/ERK pathway condition, we generated CRISPR/CAS9 mediated MageC2 KOin A375 melanoma cells. Results obtained from MageC2 KO melanoma cells as well asfrom gene-expression human cancer databases, support a novel role of MageC2 as adownstream target of the Ras/B-Raf oncogenic pathway able to control the p53 response.