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. 2016 Feb;10(2):317-29.
doi: 10.1016/j.molonc.2015.10.015. Epub 2015 Nov 5.

CREB-binding Protein Regulates Lung Cancer Growth by Targeting MAPK and CPSF4 Signaling Pathway

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Free PMC article

CREB-binding Protein Regulates Lung Cancer Growth by Targeting MAPK and CPSF4 Signaling Pathway

Zhipeng Tang et al. Mol Oncol. .
Free PMC article

Abstract

CBP (CREB-binding protein) is a transcriptional co-activator which possesses HAT (histone acetyltransferases) activity and participates in many biological processes, including embryonic development, growth control and homeostasis. However, its roles and the underlying mechanisms in the regulation of carcinogenesis and tumor development remain largely unknown. Here we investigated the molecular mechanisms and potential targets of CBP involved in tumor growth and survival in lung cancer cells. Elevated expression of CBP was detected in lung cancer cells and tumor tissues compared to the normal lung cells and tissues. Knockdown of CBP by siRNA or inhibition of its HAT activity using specific chemical inhibitor effectively suppressed cell proliferation, migration and colony formation and induced apoptosis in lung cancer cells by inhibiting MAPK and activating cytochrome C/caspase-dependent signaling pathways. Co-immunoprecipitation and immunofluorescence analyses revealed the co-localization and interaction between CBP and CPSF4 (cleavage and polyadenylation specific factor 4) proteins in lung cancer cells. Knockdown of CPSF4 inhibited hTERT transcription and cell growth induced by CBP, and vice versa, demonstrating the synergetic effect of CBP and CPSF4 in the regulation of lung cancer cell growth and survival. Moreover, we found that high expression of both CBP and CPSF4 predicted a poor prognosis in the patients with lung adenocarcinomas. Collectively, our results indicate that CBP regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathways.

Keywords: CBP; CPSF4; Lung cancer; hTERT.

Figures

Figure 1
Figure 1
The high expression of CBP in lung cancer cells and tissues. (A) The expression of CBP protein in various lung cancer cell lines and normal cells was determined by western blot analysis. (B) The expression and localization of CBP were detected by an immunofluorescent staining in lung cancer cell lines and normal cells. (C) The protein samples were extracted from five couple of human lung carcinoma tissues and adjacent normal tissues and the expression of CBP was examined by western blot. (D) The expression of CBP protein in tumor tissues from patients with lung adenocarcinomas and corresponding adjacent normal lung tissues was detected by immunohistochemistry analysis.
Figure 2
Figure 2
CBP promoted the proliferation and migration of lung cancer cells. (A) Cell viability measured by MTT assay in different lung cancer cell lines following CBP knockdown or HAT activity inhibition. (B) Colony formation assay of H1299 and H322 cells treated with C646 or DMSO twice a week for two weeks. The quantification assay of the number of the colonies was also shown. (C) Cell migration assay in H1299 and H322 cells following CBP knockdown or activity inhibition, and the migration rate was calculated. (D) Western blot analysis of the expression of MMP‐9 protein in H1299 and H322 cells following CBP knockdown or activity inhibition. Both the colony formation assay and migration assay were done 3 times independently, and we selected the images from one time experiment in the result part. The mean ± SD in the quantitative analysis was calculated based on different counting and measurement for the colony number and migration distance from 3 different experiments. (*P < 0.05, **P < 0.01).
Figure 3
Figure 3
CBP mediated the apoptosis of lung cancer cells through regulating Cyt C/Caspase 3/PARP pathway. (A) Apoptosis assay in H1299 and H322 cells by flow cytometry following CBP knockdown or activity inhibition. (B) Western blot analysis of the expression of Bcl‐2, cleaved caspase‐3 and cleaved PARP proteins in H1299 cells following CBP knockdown or activity inhibition. (C)The immunofluorescent assay of the distribution of Cytochrome C in H1299 and H322 cells following CBP knockdown or activity inhibition.
Figure 4
Figure 4
MAPK/ERK signaling pathway was affected by CBP in lung cancer cells. (A) Western blot analysis of the expression of the total and phosphorylated p38, ErK, MEK1/2, and C‐raf proteins in H1299 cells treated with CBP specific siRNA or its inhibitor. (B) Cell viability affected by inhibition of CBP activity or overexpression of CBP in H1299 cells treated with MEK1/2 specific inhibitor U0126. (C) Cell viability affected by inhibition of CBP activity or overexpression of CBP in H322 cells treated first with MEK1/2 specific inhibitor U0126.
Figure 5
Figure 5
CBP interacted with CPSF4 and mediated the acetylation of CPSF4 and their synergistic regulation on hTERT expression in lung cancer cells. (A) The extracted proteins from nuclear of HLF, HBE, A549 and H1299 cells were immunoprecipitated by antibody against CBP or CPSF4 or IgG as control. The complex was detected with anti‐CBP or CPSF4 antibody. In put represents the whole nuclear extracts. (B) The co‐localization of CBP and CPSF4 in human lung normal and cancer cells through immunofluorescence analysis. (C) The expression analysis of the acetylated CPSF4 in H322 and H1299 cells following CBP knock down or activity inhibition through IP assay. In put represents the whole nuclear extracts. (D) The hTERT promoter‐driven luciferase activity in H1299 cells stably expressing CPSF4 after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids and CBP siRNA or C646. (E) The hTERT promoter‐driven luciferase activity in H1299 cells after co‐transfection with hTERT promoter (−459/+9)‐driven luciferase plasmids, CBP‐overexpressing plasmids and CPSF4 siRNAs. (F) The hTERT expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646.
Figure 6
Figure 6
The synergistic regulation of lung cancer cell growth and apoptosis by CBP and CPSF4. (A, C) Cell viability analysis in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (B, D) Cell viability analysis in H322 cells after co‐transfection with CBP‐overexpressing plasmids and CPSF4 siRNAs. (E) Western blot analysis of the ErK and p‐ErK expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. (F–G) Apoptosis assay in H1299 and H322 cells after co‐treatment respectively with Lac Z plasmids and CBP siRNA, or Lac Z plasmids and C646, or CPSF4 plasmids and CBP siRNA, or CPSF4 plasmids and C646. The corresponding quantitative analysis of the apoptotic cell numbers was given below. (H) Western blot analysis of the Bcl‐2 and cleaved PARP expression in H1299 cells stably expressing CPSF4 after transfection with CBP siRNA or treatment with C646. Data In panel (A–D) are all represented as mean ± SD of three separate experiments with statistic significance calculated from the two‐tailed student's t test. (*P < 0.05, **P < 0.01).
Figure 7
Figure 7
The positive correlation between CBP and CPSF4 expression in clinical lung tumor tissue samples and their prediction for the poor prognosis of patients with lung adenocarcinoma. (A) The protein level of CPSF4 correlates positively with the protein level of CBP in lung adenocarcinoma tissues from 75 patients. (B) Cox‐regression analyses for prognosis of 75 lung carcinoma patients. (C) Correlation analyses of CBP or CPSF4 protein expression in relation to clinicopathologic variables of 75 lung carcinoma patients. (D) Correlation analyses of CBP or CPSF4 protein expression in relation to 5‐OS of 75 lung carcinoma patients. (E) Kaplan–Meier analysis of overall survival of lung cancer patients with different CBP and CPSF4 expression (P < 0.05, log‐rank test).

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