CCDC65, a Gene Knockout that leads to Early Death of Mice, acts as a potentially Novel Tumor Suppressor in Lung Adenocarcinoma

Abstract

CCDC65 is a member of the coiled-coil domain-containing protein family and was only reported in gastric cancer by our group. We first observed that it is downregulated in lung adenocarcinoma based on the TCGA database. Reduced CCDC65 protein was shown as an unfavorable factor promoting the clinical progression in lung adenocarcinoma. Subsequently, CCDC65^-/- mice were found possibly dead of hydrocephalus. Compared with the CCDC65^+/+ mice, the downregulation of CCDC65 in CCDC65^+/- mice significantly increased the formation ability of lung cancer induced by urethane. In the subsequent investigation, we observed that CCDC65 functions as a tumor suppressor repressing cell proliferation in vitro and in vivo. Molecular mechanism showed that CCDC65 recruited E3 ubiquitin ligase FBXW7 to induce the ubiquitination degradation of c-Myc, an oncogenic transcription factor in tumors, and reduced c-Myc binding to ENO1 promoter, which suppressed the transcription of ENO1. In addition, CCDC65 also recruited FBXW7 to degrade ENO1 protein by ubiquitinated modulation. The downregulated ENO1 further reduced the phosphorylation activation of AKT1, which thus inactivated the cell cycle signal. Our data demonstrated that CCDC65 is a potential tumor suppressor by recruiting FBWX7 to suppress c-Myc/ENO1-induced cell cycle signal in lung adenocarcinoma.

Keywords: CCDC65; Cell cycle; ENO1; Knockdown mice; Ubiquitin degradation; c-Myc.

Figure 1

Higher expression of CCDC65 inhibited lung adenocarcinoma and was associated with a better prognosis. (a) The heatmap of CCDC65 mRNA expression was derived based on samples from the TCGA lung cancer database (n =1299). (b) Comparison of the expression of CCDC65 mRNA in primary tumors and normal tissues. (c) Comparison of the expression of CCDC65 mRNA in primary tumors and solid normal tissues stratified according to smoking history. The data were analyzed using UCSC Xena (http://xena.ucsc.edu/). Kaplan-Meier survival curves relative to CCDC65 expression were generated from lung cancer (Kaplan-Meier plotter, http://kmplot.com/analysis). The Affymetrix IDs validated were (d) 1552321_a_at CCDC65 and (e) 1552320_a_at CCDC65. (f) qRT-PCR analysis of CCDC65 expression in lung adenocarcinoma cell lines and immortalized human bronchial epithelium cell line. (g) Expression of CCDC65 protein in 7 paired lung adenocarcinoma and adjacent nontumor tissues were detected by western blotting. P: para-carcinoma tissues C: cancer tissues. (h) The CCDC65 expression in lung cancer and para-carcinoma tissues (20 cases of para-carcinoma tissues from clinical samples and 5 cases of para-carcinoma tissues from tissue microarray, 97 cases of lung cancer from tissue microarray). (i) Tissues microarray immunohistochemical analysis of CCDC65 protein expression. (j) The survival curve was determined by Kaplan-Meier analysis based on the tissue microarray immunohistochemical score.

Figure 2

CCDC65 inhibited urethane-induced lung carcinogenesis. (a) The mice were genotyped at the age of two weeks and then CCDC65^+/+ and CCDC65^+/- groups were treated with urethane (1 g/kg in 0.9% NaCl solution) intraperitoneally for 20 weeks. (b) Lungs were collected 6 months after the last urethane treatment. (c) The number of tumor nodules. (d) The HE staining of the harvested lung samples. (e) The average area of tumor nodules.

Figure 3

CCDC65 inhibited H1975 and A549 cell proliferation and cell cycle transition, in vitro and in vivo. (a) The establishment of CCDC65 over-expressing and knocking down cell lines. The effects of CCDC65 on the cell proliferation of H1975 and A549 cells were examined by (a) the CCK8 assay, (b) the Colony formation assay. (c) Enriched pathways based on the differential gene expression of cell lines (A549, SPC-A1 and 5-8F) whit CCDC65 or empty vector by using GSEA analysis. The effects of CCDC65 on the G1 to S transition of H1975 and A549 cells were examined by (d) EdU incorporation assay and (e) cell cycle flow analysis, Mean±SD (n=3). *P<0.05; **P<0.01.; ***P<0.005. #To prevent the phenomenon that the proportion of S phase cells were too high for us to do statistical analysis, we reduced incubation time in the knockdown experiment. (f and g) ENO1, c-Myc, CCND1 and CCDC65 were detected following transfection with CCDC65 lentivirus and siRNA. β-actin or GAPDH served as a loading control. (h) Excised tumors 28 days after implantation and measured the corresponding tumor volume. (i) Representative Ki67 and PCNA IHC staining of excised tumor tissues of A549-CON and A549-CCDC65 were shown.

Figure 4

CCDC65 interacted with c-Myc. (a) Co-IP and western blotting assays indicated the interaction between CCDC65 and FBXW7. (b) Co-IP and western blotting assays indicated the interaction between CCDC65 and c-Myc. (c) The representative images of immunofluorescent staining of CCDC65 (green) and c-Myc (red). (d) Immunofluorescent staining showed CCDC65 reduced the expression and nuclear transportation of c-Myc.

Figure 5

CCDC65 mediated the ubiquitination degradation of c-Myc by recruiting FBXW7. (a, c) CHX chase analysis of c-Myc protein half-life in CCDC65 over-expressing and control group in H1975 and A549 cells. CHX (50 µg/ml). (e, g) CHX chase analysis detected the effects of FBXW7 knockdown on protein stability of c-Myc. (b,d,f,h) The half-life curves of c-Myc protein in H1975 and A549 cells. (i) The effects of DMSO or MG132 (20 µM) treatment on the stability of the c-Myc protein in the control and CCDC65 overexpression groups. (j) Co-IP and western blotting assays were used to detect the effect of CCDC65 overexpression and treated with siFBXW7 on the ubiquitination level of c-Myc.

Figure 6

c-Myc bound to the ENO1 transcriptional regulatory region and promoted its transcription. (a) Diagram of the ENO1 specific primer sequence and ENO1/MBP1 common fragment primer sequence. (b) The overexpression of CCDC65 inhibited the transcription of c-Myc and ENO1. The expression of MBP-1 was calculated by (ENO1+MBP1)/ENO1. “ns” means no statistical significance. (c) The diagram of ENO1 in Genome Browser (http://genome.ucsc.edu/). The area between the red dotted lines means the upstream 2000bp of ENO1. (d) The overexpression of c-Myc promoted the transcription of ENO1. The expression of MBP-1 was calculated by (ENO1+MBP1)/ENO1. “ns” means no statistical significance. (e) The diagram of upstream 2000bp of ENO1. Red areas: Predicted promotor. Blue arrow: Primers designed according to the predicted promoter. (f) Ch-IP-qPCR detected the binding between c-Myc and the predicted promoter of ENO1.