The Function Of ETV1Regulator CBP/p300in The Tumorigenesis Of The Gastrointestinal Stromal Tumors And The Potential Mechanisms

Background:Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasm featured by activated mutations of KIT and PDGFRA. Despite overall survival rates have been improved greatly by the development of receptor tyrosine kinase (RTK) inhibitors, most patients ultimately acquire resistance mainly owing to secondary mutations of KIT or PDGFRA. Recently, inhibition of the histone acetyltransferases (HAT) CBP and p300has been shown to exhibit antiproleratvie and proapoptotic effects on several cancers. In addition, several reports have indicated that CBP/p300could stabilize ETV1, a specific survival factor for GIST, and consequently promote its transcriptional activity via direct acetylation. Therefore, antineoplastic strategies for GISTs aiming at posttranscriptional level have gained enormous attention.Objective:To determine the expression and the effect of ETV1in GIST, its expression level was detected in both vivo and vitro and siRNA targeted at ETV1was transfected into GIST882. To further illuminate the role of CBP/p300in the tumorigenesis and cell proliferation of GIST and whether they could serve as antineoplastic targets for GISTs, CBP and p300depletion were performed in GIST882cells. C646were administered to tumor cells at various doses to evaluate the therapeutic effects of the selective HAT inhibitor targeting at CBP/p300. Additionally, the possible molecular mechanisms underlying the antitumor activity were further investigated in this study.Methods:Specific short interfering RNAs (siRNA) were transfected into GIST cells to respectively downregulate the expression of ETV1, CBP and p300. The expression of KIT and ETV1within tumor and adjacent normal tissues were measure by immunohistochemical staining. GIST cells were exposure to CBP/p300inhibitor C646at various concentrations and as well as in combination with imatinib. Quantative real-time PCR and Western blot analysis were performed to detect the mRNA and protein expression of ETV1, CBP and p300following siRNA transfection.Cell viability of GIST cells was examined by CCK-8assay. Apoptotic cell death was detected by Annexin V/PI staining followed by flow cytometry analysis and caspase activity was also analyzed to demonstrate apoptosis. The cell cycle was measured by flow cytometry analysis following PI staining. The molecular changes in regard to several signaling pathways underlying these altered biological activities and functions were determined by Western blot analysis.Results:The expression of ETV1was upregulated in both tissues and cells. Downregulation of ETV1would lead to antiproliferative and proapoptotic effects in GIST cells. Transcriptional blockage of CBP, rather than p300, resulted in suppression of GIST cell proliferation. Interestingly, both CBP and p300depletion accelerated the caspase-dependent apoptosis. Lack of CBP and p300caused inhibition of KIT as well as ETV1downregulation and activated the mitochondrial apoptosis pathway in GIST882cells. Nevertheless, the absence of CBP, not p300, leads to inhibition of ERK1/2and activation of JNK, suggesting a more crucial role for CBP than p300in cell proliferation and survival. Furthermore, cell proliferation of GIST cells was reduced by administration of C646, a selective HAT inhibitor for CBP/p300. Induction of apoptosis and cell cycle arrest were detected after exposure to C646, indicating its antigrowth activities were supported by the proapoptotic and anti-cell cycle effects. Additionally, C646treatment attenuated KIT activity and consequently inactivated its downstream pathways in a similar way as CBP knockdown.Conclusions:These results demonstrate that inhibition of CBP will reduce cell viability via induction of apoptosis, whereas the same effects can’t be achieved by p300downregulation, posing some advantages of CBP as a specific target for antineoplastic therapy. The molecular mechanisms underlying this antitumor effect may be attributed to downregulation of ETV1and consequent inactivation of KIT and its downstream ERK1/2and Akt as well as activation of JNK and mitochondrial cell death pathway in GIST cells. Furthermore, antiproliferative and proapoptotic effects on GIST induced by the selective HAT inhibitor C646are dependent on CBP inhibition. Taken together, the present study suggests that CBP may serve as a better antineoplastic target for GIST therapy and the selective HAT inhibitor is a promising antitumor strategy for GISTs.