Identification of RNF4 dependent gene signature in melanoma tumors

Among hallmarks of cancer is impaired proteostasis, that is characterized by increased oncoprotein stability. Here we demonstrate that RNF4, a ubiquitin ligase implicated in the stabilization of short-lived oncoproteins, is important for melanoma tumorigenesis. High levels of RNF4 in melanoma patient-derived specimens coincide with poor prognosis. Accordingly, RNF4, is essential for the growth and migration and survival of melanoma cells, both in culture and in xenograft models. RNA-seq analyses of RNF4-expressing tumors identified a gene signature related to melanoma growth and migration. Among the upstream regulators of RNF4-dependent gene signature are c-Myc and HIF1a which exhibit elevated protein levels that also coincided with stabilization of the translation initiation regulator p-eIF2a. Correspondingly, the effects of RNF4 on melanomagenesis are p-eIF2a-dependent, thus highlighting an unexpected regulatory axis in melanoma via RNF4-and ubiquitin-dependent protein stabilization we injected human A375 cells with stable expression of either GFP or Doxycycline (Dox)-inducible WT or catalytic inactive RING mutant RNF4C159A (termed RNF4RM) into immunocompromised nude mice. RNF4 expression was induced 3 days post injection by the addition of Dox to drinking water. Dox-induced expression of RNF4, but not of RNF4RM, accelerated tumor growth compared with GFP-expressing tumors (control) Therefore, to determine changes in gene signature upon RNF4 expression we performed RNA-seq analysis of the above xenograft-derived tumors . Each two similar tumors were pooled to extract RNA, thus 3 samples containing six tumors were analyzed from each group