CREB-binding Protein Regulates Lung Cancer Growth By Targeting MAPK And CPSF4 Signaling Pathway

Objective: CBP,a CREB-binding protein,has been reported to be participated in many biological processes,including embryonic development,growth control,and homeostasis.It shares regions of very high-sequence similarity with protein p300 and is involved in the transcriptional coactivation of many different transcriptional factors by interacting with them and increasing the expression of their target genes.Meanwhile,as a histone acetyltransferase,CBP is also involved in gene transactivation or repression by mediating the acetylation of both histone and nonhistone proteins.Together with p300,gene mutation or chromosomal translocation within CBP gene or its aberrant recruitment at chromatin structure has been identified to be associated with several types of cancer,including tumors arising from colon and breast,pancreas cancers,acute myeloid leukemia.Moreover,the inhibition of histone acetyltransferase activity of CBP/p300 or the inhibition of CBP's activity as transcriptional co-activator have been found to be able to block cancer cell growth in vitro and in vivo in neuroblastoma,pancreatic cancer,acute myeloid leukemia.In lung cancer,the patients with CBP-positive expression had shown significantly lower OS(Overall Survival)and DFS(Disease Free Survival)than those with CBP-negative tumors Furthermore,the high expression of CBP was found in different lung carcinoma cell lines and was positively correlated with the expression of h TERT in lung tumor cells and tissues.Here weinvestigated the molecular mechanisms and potential targets of CBP involved in tumor growth and survival in lung cancer cells.Methods: In the present study,the effect of silencing of CBP expression or its activity inhibition on cell viabilities were detected in human lung cancer A549,H1299 and H322 cells by MTT assay.The cell viability of human normal lung cell line HLF cells was also evaluated at the same way.The role of CBP in lung cancer progression was initially assessed by evaluating its effects on lung cancer cell proliferation,migration and apoptosis.Immunoblotting was used to identify the underlying molecular mechanisms by which CBP promoted lung cancer cell growth.We also examined the translocation of cytochrome C mediated by CBP in lung cancer cells using confocal immunofluorescence assay.Co-Immunoprecipitation and confocal immunofluorescence assay were used to evaluated CBP interacted with CPSF4 and mediated the acetylation of CPSF4.The synergistic effects of CBP and CPSF4 on lung cancer cell growth were then determined using FACS assay,immunoblotting and MTT assay.To further confirm the involvement of CPSF4 in CBP mediated lung cancer survival,we examined the expression of CBP and CPSF4 in clinical lung tumor tissue samples and analyzed their relationship with the prognosis of patients with lung adenocarcinomas.Results:(1)CBP was highly expressed in lung cancer cells and tumor tissues;(2)CBP regulated the proliferation and migration of lung cancer cells;(3)CBP regulated apoptosis of lung cancer cells by regulating Bcl-2 and cytochrome C/caspase pathway;(4)MAPK pathway was involved in the proliferation regulation mediated by CBP;(5)CBP interacted with CPSF4 and mediated the acetylation of CPSF4;(6)CBP and CPSF4 synergistically regulated the transcription and expression of h TERT;(7)CBP and CPSF4 synergistically regulated lung cancer cell growth and apoptosis;(8)CBP and CPSF4 overexpression was positively correlated with poor prognosis of patients with lung adenocarcinomas.Conclusion: In summary,we found that CBP induced proliferative growth of lung tumor cells by affecting C-Raf/MEK/Erk and cytochrome C/caspase signaling pathways.In addition,we showed that such induction happened in a process requiring CPSF4,whereby CBP recruited,interacted with and acetylated CPSF4 at gene promoter regions to synergistically regulate downstream gene transcription and tumor cell proliferation.This association between CBP and CPSF4 was synergistically responsible for the activation of h TERT expression and,in part,contributed to the mechanisms by which CBP was involved in growth promotion of lung cancer.Since both CBP and CPSF4 are highly expressed in lung cancer tissues,our study might provide a very potential therapeutic strategy to treat this cancer by dual blocking these two proteins simultaneously.