An Important Role Of The ROCK-c-Myc Signaling In Modulating Tumor Growth

ObjectiveTo reveal the mechanism of ROCK-c-Myc signaling in modulating the tumor growth.MethodsThe in vivo and in vitro experiments were carried out to evaluate the oncobiological effect of ROCK-c-Myc signaling in tumor cells. The activity of ROCK, c-Myc and RhoA were inhibitor by Y27632,10085-F4and CT04, respectively. The proliferation of cells were assessed by the cells counting, MTT and BrdU assay. Tranwell and Cytodex-1beads were used to assess to migration and invation of the cells. Co-IP and GST-pulldown were used to evaluate the interaction between ROCK and c-Myc. HSPC111-specific shRNA was used to conduct the stable low expressed cell line.. Co-IP and Mass spectrometry were used to screen the proteins interacting with HSPC111. RT-qPCR and Western blot analysis were used to assess the mRNA and protein level of genes and proteins. And The6-yr survival rates (for72months) were estimated with the Kaplan-Meier method, and the threshold was determined as previously described.ResultsROCK could increase the transcriptional activity of c-Myc by promoting c-Myc protein stability and subsequent nuclear transportation. ROCK inhibition reduced c-Myc-mediated expression of mRNA targets (such as HSPC111) and miRNA targets (such as the miR-17-92cluster). Importantly, we demonstrated that ROCK1directly interacted with c-Myc to phosphorylate it at S62and then stabilized its protein, but ROCK2may not. Suppression on ROCK-c-Myc downstream molecules, such as c-Myc-regulated miR-17, also impaired PC3cell growth. Additionally, c-Myc was proved to exert a positive feedback regulation on ROCK by increasing RhoA mRNA expression. These data together suggest that ROCK signaling facilitates PC3cell growth by enhancing c-Myc activity. HSPC111knockdown largely suppressed cell growth of MDA-MB-231breast cancer cells in vitro and in vivo. HSPC111protein was recognized to be predominantly localized in nucleus, and was speculated to modulating ribosomal biosynthesis and assembly by interacting with RNA3’-phosphate cyclase (RTCD1), and the low expression of RTCD1could inhibite the proliferation of tumor cells. Additionally, the synergistic upregulation of HSPC111and RTCD1exerted more robust impact on tumor progression than one individual gene upregulation only.ConclusionInhibition of ROCK and its stimulated signaling might prove to be a promising strategy for restraining tumor progression in tumors.