The Tumor-promoting Role Of TRIP4 In Melanoma Progression And Its Involvement In Response To BRAF-targeted Therapy

Objective: Thyroid Hormone Receptor Interactor 4(TRIP4)was identified with anti-proliferative effect in melanoma based on siRNA library screening,but its precise function in melanoma progression is completely unknown.Here we explored the carcinogenic role of TRIP4 in melanoma.We investigated the expression of TRIP4 in different melanoma cells and patients with melanoma to definite the mechanism and biological function of TRIP4 in the regulation of melanoma growth.Moreover,we studied the regulation of TRIP4 in melanoma growth by establishment of xenograft mouse model.Collectively,these results demonstrate the protumorigenic role of TRIP4 in melanoma,provide novel insight into the mechanism of TRIP4 as a potential prognostic biomarker and therapeutic target,and also suggest the potential of TRIP4 and BRAF dual targeting as a new therapeutic strategy for melanoma treatment.Methods:(1)SiRNA library screen technology was used to discover and identify potential target genes that affect melanoma cells.(2)Genetransfer technology was applied to specifically overexpress or inhibit TRIP4 expression for the study of melanoma cell growth,cell proliferation,migration,invasion,apoptosis,and other biological function study in vivo and in vitro.(3)Westernblot and immunohistochemistry techniques were used to detect the expression levels of TRIP4 in melanoma tumor cells and the expression levels of TRIP4 in melanoma patients to analyzed clinical significance of TRIP4,such as diagnostic,prognostic value,recurrence and therapeutic targets.(4)We further verified the binding of TRIP4 on COX-2 promoter and iNOS promoter by Ch IP and pulldown assay.(5)We used Co-IP to verify TRIP interacted with p300 directly.(6)Sensitivity to anti-BRAF agents was measured by MTT assay.(7)Xenograft mouse model was established to determine the influence of TRIP4 on tumor growth and detect the expression of COX-2 and iNOS by IHC and Western blot.Results:(1)TRIP4 was identified as the potential target genes that affect melanoma cells using siRNA library screen technology.(2)TRIP4 silencing led to melanoma cell growth delay.(3)The regulation of melanoma growth by Trip4 was COX-2 and iNOS activity-dependent in vitro and in vivo.(4)Trip4 regulated COX-2 and iNOS expression through activating NF-κ B signaling in melanoma cells.(5)Trip4 regulated COX-2/iNOS transcription directly by binding to their promoters.(6)TRIP4 cooperated with p300 to promote melanoma progression by targeting inflammatory factors.(7)TRIP4 knockdown sensitized the anti-tumor effect of BRAF targeting agents in melanoma cells.(8)TRIP4 expression was positively correlated with COX-2/iNOS expression and predicted poor prognosis in patients with melanoma.Conclusion: TRIP4 overexpression predicted poor survival in melanoma patients,and was positively correlated with the up-regulation of COX-2 and iNOS in melanoma carcinogenesis.TRIP4 was identified to cooperate with P300 through the HAT domain of P300 to co-regulate the COX-2 and iNOS protein level in melanoma cells.Furthermore,the regulation of sensitivity to anti-BRAF targeted agents by TRIP4 was confirmed in BRAF-mutant human melanoma cells and xenograft.These results demonstrate the protumorigenic role of TRIP4,provide novel insight into the mechanism of TRIP4 as a candidate target,and also suggest the potential of TRIP4 and BRAF dual targeting as a new therapeutic strategy for melanomas.