YAP promotes the proliferation and migration of colorectal cancer cells through the Glut3/AMPK signaling pathway

Abstract

Yes-associated protein (YAP), as a major downstream effector in the Hippo signaling pathway, is considered as an oncogene in cancer. The present study aimed to investigate the potential role of YAP in the development and progression of colorectal cancer (CRC). The mRNA and protein expression levels of YAP in human CRC tissue samples and adjacent normal tissue were analyzed using public databases, as well as clinical samples. The potential roles of YAP and the underlying mechanism regulating the proliferation and migration of CRC cells were examined using genetic manipulation in vitro. The correlation between the expression of the YAP gene and epithelial-to-mesenchymal transition (EMT) markers was investigated in order to determine the mechanism underlying the observed effects of YAP. YAP mRNA expression levels were significantly upregulated in CRC tissue compared with in normal tissue, as determined using datasets obtained from Oncomine. Similarly, in clinical samples, the protein expression levels of YAP were significantly upregulated in CRC tissue samples compared with in normal tissue samples. YAP knockdown inhibited the proliferation and migration of CRC cells in vitro, whereas its overexpression resulted in the opposite effect. The expression levels of the YAP gene were positively correlated with those of EMT markers (such as vimentin and N-cadherin) and EMT-inducing transcription factors (such as Snail1, Slug and zinc finger E-box binding homeobox 1 and 2) in CRC samples from Gene Expression Profiling Interactive Analysis. Furthermore, YAP silencing increased the protein expression of E-cadherin and decreased that of vimentin in CRC cells. By contrast, the overexpression of YAP had the opposite effect. YAP promoted the glucose transporter 3 (Glut3)/AMP-activated protein kinase (AMPK) signaling pathway in CRC cells. In conclusion, YAP promoted the proliferation and migration of CRC cells, as well as the expression of EMT markers, possibly by regulating the Glut3/AMPK signaling pathway.

Keywords: Yes-associated protein; colorectal cancer; epithelial-to-mesenchymal-transition; migration; proliferation.

Figure 1.

YAP is upregulated in human CRC. (A) Column dot plots indicating a difference in YAP mRNA expression between human CRC tissue and normal tissue based on two public microarray datasets (accession nos. GSE74602 and GSE44067). (B and C) Relative YAP mRNA expression in (B) normal colon and cecum adenocarcinoma tissue or (C) normal colon and colon adenocarcinoma tissue in the Oncomine dataset by Kaiser et al (21). (D) Relative YAP mRNA expression in normal colon or colorectal carcinoma tissue in the Oncomine dataset by Hong et al (22). (E) Relative YAP mRNA expression in normal ascending colon, descending colon, rectum, sigmoid colon, transverse colon or rectal adenoma tissue in the Oncomine dataset by Sabates-Bellver et al (23). (F) Western blot analysis of YAP protein expression levels in 18 pairs of CRC tumor and normal adjacent tissue samples. (G) Semi-quantitative analysis of YAP expression levels (relative to GAPDH) in 18 pairs of CRC tumor and normal adjacent tissue samples. ***P<0.001, two-tailed paired Student's t-test. (H) YAP expression in human colorectal tumor and normal tissue. Images were taken from the Human Protein Atlas online database (magnification, ×100 and ×400) and are available from https://www.proteinatlas.org/ENSG00000137693-YAP1/tissue/colon#img and https://www.proteinatlas.org/ENSG00000137693-YAP1/pathology/colorectal+cancer#img. CRC, colorectal cancer; GEPIA, Gene Expression Profiling Interactive Analysis; N, normal adjacent tissue; T, tumor tissue; YAP, Yes-associated protein.

Figure 2.

YAP protein expression levels in human CRC cells. (A) Western blot analysis of YAP protein levels in six CRC cell lines (HCT116, CCL244, CaCo2, SW480, LoVo and RKO) performed in triplicate. (B) Western blot analysis of YAP protein expression in SW480 cells transfected with NC or KD. The bands were semi-quantified and the results are presented as the mean ± SEM. n=3. (C) Western blot analysis of YAP in HCT116 cells transfected with VEC or YAP-OE. The bands were semi-quantified and the results are presented as the mean ± SEM. n=3. ***P<0.001, two-tailed unpaired Student's t-test. CRC, colorectal cancer; KD, YAP siRNA knockdown; NC, siRNA negative control; OE, overexpression; VEC, empty vector; YAP, Yes-associated protein; siRNA, small interfering RNA.

Figure 3.

Effects of YAP on CRC cell proliferation and migration in vitro. Colony formation assays were performed in (A) SW480 cells transfected with NC or KD and (B) HCT116 cells transfected with VEC or YAP-OE. Representative images of the wells are presented (left). The number of colony-forming units (right) is presented as the mean ± SEM. n=3. Migration assays were conducted in (C) SW480 cells transfected with NC or KD and (D) HCT116 cells transfected with VEC or YAP-OE. Representative images are presented (left). Magnification, ×200. The number of migratory cells (right) is presented as the mean ± SEM. n=3. *P<0.05, **P<0.01, two-tailed unpaired Student's t-test. CRC, colorectal cancer; KD, YAP siRNA knockdown; NC, siRNA negative control; OE, overexpression; VEC, empty vector; YAP, Yes-associated protein; siRNA, small interfering RNA.

Figure 4.

YAP is associated with EMT. (A) Correlation analysis of YAP1 and VIM gene expression levels in patients with CRC using GEPIA datasets (COAD and READ). (B) Correlation analysis of YAP1 and CDH2 gene expression levels using CRC datasets from GEPIA. (C) Correlation analysis between YAP1 and SNAI1 gene expression in patients with CRC using GEPIA datasets. (D) Correlation analysis between YAP1 and SNAI2 gene expression in patients with CRC using GEPIA datasets. (E) Correlation analysis between YAP1 and ZEB1 gene expression in patients with CRC using GEPIA datasets. (F) Correlation analysis between YAP1 and ZEB2 gene expression in patients with CRC using GEPIA datasets. Western blot analysis of E-cadherin and vimentin protein expression in (G) SW480 cells transfected with NC or KD and (H) HCT116 transfected with VEC or YAP-OE. GAPDH was used as a loading control. The bands were semi-quantified and the results are presented as the mean ± SEM (right). n=3. **P<0.01, two-tailed unpaired Student's t-test. CRC, colorectal cancer, CDH2, N-cadherin; GEPIA, Gene Expression Profiling Interactive Analysis; SNAI1/2, Snail family transcriptional repressor 1/2; ZEB1/2, zinc finger E-box binding homeobox 1/2; TPM, transcripts per million; KD, YAP siRNA knockdown; NC, siRNA negative control; OE, overexpression; VEC, empty vector; YAP, Yes-associated protein; siRNA, small interfering RNA; COAD, colon adenocarcinoma; READ, rectum adenocarcinoma.

Figure 5.

YAP regulates the Glut3/AMPK pathway in CRC cells. (A) Correlation analysis between YAP1 and SLC2A3 (Glut3) gene expression levels in patients with CRC using GEPIA datasets. Western blot analysis of Glut3, p-AMPK and AMPK in (B) SW480 cells transfected NC or KD and (C) HCT116 cells transfected with VEC or YAP-OE. GAPDH was used as a loading control. The bands were semi-quantified and the results are presented as the mean ± SEM (right). n=3. (D) Relative ATP levels in SW480 cells transfected with NC or KD and HCT116 cells transfected with VEC or YAP-OE. (E) Western blot analysis of Glut3 protein expression in HCT116 cells transfected with siGlut3 or NC. (F) Western blot analysis of Glut3 expression in HCT116 cells co-transfected with siGlut3 or NC together with YAP-OE or VEC. (G) Migration ability of HCT116 cells in HCT116 cells co-transfected with siGlut3 or NC together with YAP-OE or VEC. Representative images are presented (left). Magnification, ×200. The number of migratory cells (right) is presented as the mean ± SEM. n=3. *P<0.05; **P<0.01; NS, not significant, two-tailed unpaired Student's t-test. AMPK, AMP-activated protein kinase; CRC, colorectal cancer; Glut3, glucose transporter 3; KD, YAP siRNA knockdown; NC, siRNA negative control; p-; phosphorylated; OE, overexpression; SLC2A3, solute carrier family 2 member 3; VEC, empty vector; YAP, Yes-associated protein; siRNA, small interfering RNA.