CCDC65 as a new potential tumor suppressor induced by metformin inhibits activation of AKT1 via ubiquitination of ENO1 in gastric cancer

Abstract

The coiled-coil domain containing protein members have been well documented for their roles in many diseases including cancers. However, the function of the coiled-coil domain containing 65 (CCDC65) remains unknown in tumorigenesis including gastric cancer. Methods: CCDC65 expression and its correlation with clinical features and prognosis of gastric cancer were analyzed in tissue. The biological role and molecular basis of CCDC65 were performed via in vitro and in vivo assays and a various of experimental methods including co-immunoprecipitation (Co-IP), GST-pull down and ubiquitination analysis et al. Finally, whether metformin affects the pathogenesis of gastric cancer by regulating CCDC65 and its-mediated signaling was investigated. Results: Here, we found that downregulated CCDC65 level was showed as an unfavourable factor in gastric cancer patients. Subsequently, CCDC65 or its domain (a.a. 130-484) was identified as a significant suppressor in GC growth and metastasis in vitro and in vivo. Molecular basis showed that CCDC65 bound to ENO1, an oncogenic factor has been widely reported to promote the tumor pathogenesis, by its domain (a.a. 130-484) and further promoted ubiquitylation and degradation of ENO1 by recruiting E3 ubiquitin ligase FBXW7. The downregulated ENO1 decreased the binding with AKT1 and further inactivated AKT1, which led to the loss of cell proliferation and EMT signal. Finally, we observed that metformin, a new anti-cancer drug, can significantly induce CCDC65 to suppress ENO1-AKT1 complex-mediated cell proliferation and EMT signals and finally suppresses the malignant phenotypes of gastric cancer cells. Conclusion: These results firstly highlight a critical role of CCDC65 in suppressing ENO1-AKT1 pathway to reduce the progression of gastric cancer and reveals a new molecular mechanism for metformin in suppressing gastric cancer. Our present study provides a new insight into the mechanism and therapy for gastric cancer.

Keywords: AKT1; CCDC65; ENO1; Gastric cancer; Metformin.

Figure 1

Decreased CCDC65 expression correlates with poor prognosis in gastric cancer. (A) The transcription level of CCDC65 in different types of cancers, analyzing by Oncomine. (B) The expression of CCDC65 in gastric cancer cell lines, analyzing by CCLE. (C) The expression level of CCDC65 was analyzed by qRT-PCR and WB in gastric cancer (GC) tissues and normal adjacent tissues (N). (D-E) The expression of CCDC65 in tumor and adjacent samples were monitored by immunohistochemistry (IHC) on a tissue array. The anti-CCDC65 antibody was employed for IHC. IHC scoring was performed blind, prior to association with clinical data. (F) Kaplan-Meier analysis of CCDC65 expression in 187 gastric cancer patients those were subdivided into two groups. (G-H) Stratified survival analysis of CCDC65 expression in clinical stage I-II and III-IV gastric cancer patients. (I-J) Univariate and multivariate analyses of different clinicopathological features in human gastric cancer patients.

Figure 2

CCDC65 suppresses gastric cancer growth in vitro and in vivo. (A-B) CCDC65 overexpression markedly inhibited cell viability while CCDC65 knockdown promoted cell viability in AGS and SGC7901 cells by MTT assay. Student's t test. Mean ± SD, * p < 0.05; ** p < 0.01. (C-D) EdU incorporation assays (Scale bar: 100 μm) and colony formation assays of CCDC65-overexpressing AGS and SGC7901 cells and corresponding control cells. (E) The in vivo effect of CCDC65 was evaluated in xenograft mouse model bearing tumors originating from AGS cells. Each group contained 5 mice. (F) The growth curve and weight of xenograft were obtained in nude mice using stably expressing vector control or CCDC65 cells. (G) The in vivo effect of si-CCDC65 was evaluated in xenograft mouse model bearing tumors originating from SGC7901 cells. Each group contained 5 mice. (H) The growth curve and weight of xenograft were obtained in nude mice which were injected control or si-CCDC65 every 3 days. (I-J) Representative H&E as well as IHC staining of primary tumor tissues are shown. Scale bar, 50 μm.

Figure 3

CCDC65 suppresses gastric cancer metastasis in vitro and in vivo. (A-D) Transwell assay, boyden assay and (E) wound-healing assays evaluated the migration and invasion of gastric cancer cells treated with CCDC65 plasmid, si-CCDC65, CCDC65 lentivirus and corresponding control group. (F) A pulmonary metastasis model was adopted to evaluate the effect of CCDC65 on metastasis (n = 5). (G) Representative H&E as well as IHC staining of pulmonary metastatic nodules are shown. Scale bar, 50 μm.

Figure 4

CCDC65 involves in regulating AKT1 signaling pathway. (A) Schematic diagram of the potential downstream pathways of CCDC65 with the TCGA gastric cancer RNAseq (IlluminaHiSeq; n = 415) data set. (B-C) Expression levels of CCDC65, AKT1, p-AKT1 (ser473), E-cadherin, N-cadherin, Vimentin, CCND1, P21 were detected following transfection with CCDC65 lentivirus and siRNA. GAPDH was used as a loading control. (D) IHC staining of p-AKT1 in primary tumor tissues. Scale bar, 50 μm. (E) Expression levels of p-AKT1 (Ser473), N-cadherin, E-cadherin, Vimentin, CCND1 and P21 in si-NC, si-NC+MK-2206, si-CCDC65, si-CCDC65+MK-2206 groups. (F-G) MTT assays, transwell and boyden assays evaluated the cell viability and metastasis of si-NC, si-NC+MK-2206, si-CCDC65, si-CCDC65+MK-2206 groups.

Figure 5

CCDC65 interacts with ENO1. (A) Coomassie brilliant blue staining showed the proteins that interacted with CCDC65 in AGS cells and the molecular weight of CCDC65 and ENO1. (B) Co-IP detected the interaction of exogenous CCDC65 and ENO1. (C) Co-IP detected the interaction of endogenous CCDC65 and ENO1. (D) Colocalization of CCDC65 and ENO1 in HEK293T cells by immunofluorescence staining. (E) N-terminal deletion mutant domain (Flag-CCDC65-130-484) of CCDC65 is important for interaction with ENO1. The mutants of CCDC65 were transfected into HEK293T cells and analyzed by immunoprecipitation using anti-Flag antibody. (F) The C-terminal TIM barrel domain (Myc-ENO1-140-434) of ENO1 is required for its interaction with CCDC65. The mutants of ENO1 were transfected into HEK293T cells and analyzed by immunoprecipitation using anti-Myc antibody.

Figure 6

CCDC65 recruits FBXW7 to promotes the degradation of ENO1. (A) The expression of ENO1 mRNA was detected by qRT-PCR after CCDC65 overexpression, normalized to GAPDH. Student's t test, mean ± SD, * p < 0.05; ** p < 0.01. (B) The protein expression of ENO1 were detected by WB after CCDC65 overexpression. (C) IHC staining of ENO1 in primary tumor tissues and pulmonary metastatic nodules were shown. Scale bar, 50 μm. (D) WB was used to detect the effects of DMSO or MG132 treatment and CHX treatment for different duration on the stability of ENO1 protein in the control and CCDC65 overexpression groups. (E) Co-IP detected the effects of CCDC65 overexpression on protein stability of ENO1 in GC cells. (F) Co-IP detected the interaction of CCDC65 and ENO1 with FBXW7. (G) GST-CCDC65 interacts with His-ENO1 in vitro by GST pull-down assay. (H) Co-IP detected the effects of FBXW7 knockdown on protein stability of ENO1 in CCDC65 overexpression GC cells. (I) Co-IP was conducted to identify the function of CCDC65 overexpression on the interplay among ENO1, FBXW7 and ubiquitin in GC cells.

Figure 7

ENO1 interacts with AKT1. (A) Co-IP detected the interaction of exogenous ENO1 and AKT1. (B) Colocalization of ENO1 and AKT1 in HEK293T cells was detected by immunofluorescence staining. (C) Co-IP detected the interaction of endogenous ENO1 and CCDC65 with AKT1. (D) Schematic representations of AKT1 and its mutants. The full-length of AKT1 is important for interaction with ENO1. The mutants of AKT1 were transfected into HEK293T cells and analyzed by immunoprecipitation using anti-Myc antibody. (E) Co-IP detected the effects of si-CCDC65 on the interaction of ENO1 and AKT1 in GC cells.

Figure 8

Metformin potentiates tumor suppression of CCDC65 in vitro and in vivo. (A) Metformin up-regulated CCDC65 expression in a dose-dependent manner in AGS and SGC7901 cells. (B) The expression of ENO1, p-AKT1 (ser473), N-cadherin, E-cadherin, Vimentin, CCND1 and P21 in si-NC, si-CCDC65, si-NC+metformin, si-CCDC65+metformin groups. (C) The in vivo effect of normal saline (NS), metformin (met) and metformin+si-CCDC65 groups was evaluated in xenograft mouse model bearing tumors originating from SGC7901 cells. Each group contained 5 mice. (D-E) The weight and growth curve of xenograft were obtained in nude mice treated with NS, met and metformin+si-CCDC65. (F-G) Comparisons of CCDC65, ENO1 and p-AKT1 in xenografts of NS, metformin and metformin+si-CCDC65 groups by western blot and IHC assays. (H) Working model of CCDC65 induced by metformin in inactivating AKT1 via the ubiquitination of ENO1 in gastric cancer.