RNAi Screening Of Ubiquitin Pathway Genes And Study On Functional Mechanism Of CDC34 In Lung Cancer

The Ubiquitin Proteasome System(UPS)is a well-organized destruction machine with m?ltiple protein components(ubiquitin-activating E1 enzymes,ubiquitin-conjugating E2 enzymes,ubiquitin-protein E3 ligases,and the 26S proteasome)working in concert with one another to ensure the timely and efficient proteolysis of target substrates.As such,dysregulation of any component of the UPS can significantly affect the output of any given biological process under its regulation.To systematically identify ubiquitin pathway genes that are critical to lung carcinogenesis,we used the siRNA directed gene silencing method to knockdown 696 genes in two non-small cell lung cancer(NSCLC)cell lines(A549 and H1975).Finally,We identified 42 candidates(including 31 potential oncogenes and 11 potential supressor genes)which were required for cell proliferation in NSCLC cells.Among the candidates,the five potential oncogenes(UBE2T,CBLC,CDC34,RFWD3 and TRIM17)were significantly overexpressed in lung cancer patients and their expression levels were positively correlated with overall survival of patients.Meanwhile,four of the potential tumor supressors-UBL3,TRIM22,UBE2G2 and MARCH1-were obviously down-regulated in lung cancer samples and the prognosis of these patients were poor.As one of the most highly expressed candidates,the E2 ubiquitin conjugase CDC34 was chosen to further investigate the molecular mechanism in lung carcinogenesis.CDC34 was identified as a gene essential for cell viability because of its role in regulating cell cycle associated proteins proteolysis by working together with the SKP1-CUL1-F-box protein complex(SCF)E3 ubiquitin ligase.CDC34 was reported to exert oncogenic functions in several cancers.In this study,CDC34 was elevated in tumor tissues in 67 of 102(65.7%)NSCLCs,and smokers had higher CDC34 than nonsmokers.The expression of CDC34 was inversely associated with overall survival of patients.Forced expression of CDC34 promoted,whereas knockdown of CDC34 inhibited lung cancer in vitro and in vivo.CDC34 bound EGFR and competed with E3 ligase c-Cbl to inhibit the polyubiquitination and subsequent degradation of EGFR.Besides,we explored the mechanism by which CDC34 enhances the phosphorylation activity of EGFR and observed that CDC34 was involved in SCFbeta-TrCP mediated CDC25A degradation,leading to the reduced dephosphorylation level of phosphorylated EGFR induced by CDC25A.Moreover,CDC34 promoted EGFR nuclear translocation and affected EGFR's function as a transcription factor.EGFR also positively regulated the expression of CDC34.Clinical studies have shown that more than half of NSCLC patients have EGFR overexpression or hyperactivation of EGFR by gain-of-function mutations.In tyrosine kinase inhibitor gefitinib-sensitive EGFR-L858R mutation and gefitinib-resistant EGFR-T790M/Del(exon 19)mutation-driven mouse lung cancer,knockdown of CDC34 by lentivirus mediated transfection of short hairpin RNA significantly inhibited lung carcinogenesis.These results demonstrate that an E2 enzyme is capable of competing with E3 ligase to inhibit ubiquitination and subsequent degradation of oncoprotein substrate.Silencing of CDC34 can overcome T790M mutation-caused drug resistance,therefore CDC34 represents an attractive therapeutic target in lung cancer.Preclinical and clinical studies of using CDC34 inhibitor to treat lung cancer with or without EGFR mutation warrant intensive investigation.In addition,we verified the effect of two candidate genes(RFWD3 and UBL3)on the proliferation of lung cancer cells.The data showed that knockdown of RFWD3 inhibited lung cancer cell survival and promoted apoptosis,while silencing of UBL3 promoted lung cancer cell proliferation by increasing the number of cells in S phase.In order to explore their molecular mechanisms governing the proliferation of lung cancer cells,we used the whole protein lysis of A549 and H1975 which were interfered with their specific siRNA to conduct TMT analysis and a series of proteins which were regulated by them were identifed.The mechanism remains to be further explored.