RING-finger protein 6 amplification activates JAK / STAT 3 pathway by modifying SHP-1 ubiquitylation and associates with poor outcome in colorectal cancer Running Title : RNF 6 amplification contributes to CRC by regulating SHP-1

Objective The E3 ubiquitin ligase RNF6 (RING-finger protein 6) plays a crucial role in carcinogenesis. However, the copy number and expression of RNF6 were rarely reported in colorectal cancer (CRC). We aimed to explore the mechanical, biological and clinical role of RNF6 in CRC initiation and progression. Design The copy number and expression of RNF6 were analyzed from Tumorscape and The Cancer Genome Atlas (TCGA) datasets. Gene expressions were examined by real time PCR, western blot and immunohistochemical staining. Gene expression profiling studies were performed to identify pivotal genes regulated by RNF6. Biological function of RNF6 on tumor growth and metastasis was detected in vivo and vitro. Role of RNF6 in modulating SHP-1 expression was examined by co-immunoprecipitation and confocal microscopy respectively. Results The copy number of RNF6 was significantly amplified in CRC and the amplification was associated with RNF6 expression level. Amplification and overexpression of RNF6 positively correlated with CRC patients with poor prognosis. GSEA analysis revealed cell proliferation and invasion-related genes were enriched in RNF6 high-expressed CRC cells as well as in patients from TCGA dataset. Down-regulation of RNF6 impaired the CRC cell proliferation and invasion in vitro and vivo. RNF6 may activate JAK/STAT3 pathway and increase pSTAT3 levels by inducing the ubiquitination and degradation of SHP-1. Conclusions Genomic amplification drives RNF6 overexpression in CRC. RNF6 may be a novel biomarker in colorectal carcinogenesis, and RNF6 may increase pSTAT3 level via promoting SHP-1 ubiquitylation and degradation. Targeting the RNF6/SHP-1/STAT3 axis provides a potential therapeutic option for RNF6-amplified tumors. Cancer Research. on December 31, 2017. © 2017 American Association for clincancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on December 29, 2017; DOI: 10.1158/1078-0432.CCR-17-2133