Carcinoembryonic antigen related cell adhesion molecule 6 promotes the proliferation and migration of renal cancer cells through the ERK/AKT signaling pathway

Abstract

Background: Carcinoembryonic antigen related cell adhesion molecule 6 (CEACAM6) is a versatile glycoprotein and a member of the CEACAM family. Studies suggested that it served as a diagnostic and prognostic biomarker in some malignancies. In addition, it is involved in tumorigenesis by stimulating proliferation, suppressing apoptosis, facilitating migration and invasion, promoting angiogenesis, and inducing drug resistance. In the present study, we demonstrated the oncogenic effects of CEACAM6 in clear cell renal cell carcinoma (ccRCC).

Methods: CEACAM6 expression was detected by quantitative real-time PCR (qRT-PCR), immunohistochemical staining and western blot in ccRCC tumor tissues and cell lines. Survival analysis was performed using the data of TCGA database. Cell proliferation and migration were detected by CCK-8 and transwell assays with the overexpression or silencing of CEACAM6. LY294002 was used to block the activation of PI3K/AKT pathway. Associated pathway proteins were detected by western blot.

Results: CEACAM6 was upregulated in ccRCC cell lines and tumor tissues. Longer overall survival was observed in patients with relatively low CEACAM6 levels. Furthermore, overexpression of CEACAM6 promoted the proliferation and migration of ccRCC cells. Conversely, shRNA-mediated CEACAM6 depletion modulated those changes. Further investigation demonstrated that the ERK/AKT signaling pathway activation played a pivotal role. In addition, PI3K/AKT pathway blockade abrogated the effects of CEACAM6 overexpression.

Conclusions: Aberrantly high expression of CEACAM6 is a stimulus for the formation and progression of ccRCC.

Keywords: Carcinoembryonic antigen related cell adhesion molecule 6 (CEACAM6); clear cell renal cell carcinoma (ccRCC); migration; proliferation.

Figure 1

CEACAM6 was upregulated in both ccRCC tissues and cell lines and was associated with overall survival. ccRCC tumor tissues (n=15) and normal kidney tissues (n=15) were collected. (A) The relative mRNA levels of CEACAM6 were detected by qRT-PCR. P<0.05; (B) the increased expression of CEACAM6 was confirmed by immunohistochemical staining. The images were taken under a microscope with magnifications of ×100 and ×400; (C) the protein levels of CEACAM6 in several RCC cell lines and HK-2 cells were detected by Western blot; (D) Kaplan-Meier analysis of the correlation between CEACAM6 expression and overall survival for RCC patients was performed. P value was calculated by log-rank test. P<0.01. CEACAM6, carcinoembryonic antigen related cell adhesion molecule 6; ccRCC, clear cell renal cell carcinoma; qRT-PCR, quantitative real-time PCR.

Figure 2

CEACAM6 overexpression facilitated the proliferation and migration of RCC cells. 786-O and A498 cells were transfected with GV230-CEACAM6 or GV230-NC (NC) to elevate CEACAM6 expression. (A) Transfection efficiency was confirmed by Western blot; (B) cell proliferation was detected by CCK-8 assay. The absorbances at 450 nm were examined after transfection for 0, 24, 48, 72, or 96 h; (C) cell migration was detected by transwell assay. Cells were stained by crystal violet, ×200. Data were analyzed by Student’s t-test. **, P<0.01; ***, P<0.001. CEACAM6, carcinoembryonic antigen related cell adhesion molecule 6.

Figure 3

CEACAM6 silencing suppressed the proliferation and migration of RCC cells. CEACAM6-specific shRNAs were used to induce gene silencing in 786-O and A498 cells. (A) CEACAM6 silencing was confirmed by Western blot; (B) after transfection for 0, 24, 48, 72, or 96 h, cell viability was assessed by CCK-8 assay; (C) cell migration was detected by transwell assay, crystal violet staining, ×200. Data were analyzed by Student’s t-test. **, P<0.01; ***, P<0.001. CEACAM6, carcinoembryonic antigen related cell adhesion molecule 6.

Figure 4

CEACAM6 exerts oncogenic effects in RCC through the ERK/AKT signaling pathway. (A) The protein levels of several pivotal molecules were detected by Western blot following CEACAM6 silencing; (B) the expression of the above molecules following CEACAM6 overexpression. After transfection with GV230-CEACAM6 plasmids, 786-O cells were treated with LY294002 for 24 h. Cell proliferation and migration was detected by CCK-8 assay (C) and transwell assay (D) respectively. Cells were stained by crystal violet, ×200. Data were analyzed by one-way ANOVA. *, P<0.05; **, P<0.01; ***, P<0.001. CEACAM6, carcinoembryonic antigen related cell adhesion molecule 6; ns, no significant difference; RCC, renal cell carcinoma.