COL4A1 promotes the growth and metastasis of hepatocellular carcinoma cells by activating FAK-Src signaling

Abstract

Background: Collagens are the most abundant proteins in extra cellular matrix and important components of tumor microenvironment. Recent studies have showed that aberrant expression of collagens can influence tumor cell behaviors. However, their roles in hepatocellular carcinoma (HCC) are poorly understood.

Methods: In this study, we screened all 44 collagen members in HCC using whole transcriptome sequencing data from the public datasets, and collagen type IV alpha1 chain (COL4A1) was identified as most significantly differential expressed gene. Expression of COL4A1 was detected in HCC samples by quantitative real-time polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry (IHC). Finally, functions and potential mechanisms of COL4A1 were explored in HCC progression.

Results: COL4A1 is the most significantly overexpressed collagen gene in HCC. Upregulation of COL4A1 facilitates the proliferation, migration and invasion of HCC cells through FAK-Src signaling. Expression of COL4A1 is upregulated by RUNX1 in HCC. HCC cells with high COL4A1 expression are sensitive to the treatment with FAK or Src inhibitor.

Conclusion: COL4A1 facilitates growth and metastasis in HCC via activation of FAK-Src signaling. High level of COL4A1 may be a potential biomarker for diagnosis and treatment with FAK or Src inhibitor for HCC.

Keywords: COL4A1; FAK; HCC; RUNX1; Src.

Fig. 1

Upregulation of COL4A1 in HCC. a Box-whisker Plot indicated the mRNA expression profiles of 44 collagen genes in TCGA dataset. The gene name shown in red and blue illustrates the 27 upregulation genes and 4 downregulation genes in tumor tissues compared with normal liver tissues, respectively. b Expression of COL4A1 was significantly upregulated in HCC compared with normal liver tissues in 4 datasets, including Roessler Liver Statistics, Roessler Liver 2 Statistics, Wurmbach Liver Statistics, and Mas Liver Statistics. c mRNA levels of COL4A1 in paired HCC samples were detected by qRT-PCR (n = 89). Fold changes (C/N) were presented. C, cancerous tissues; N, noncancerous liver tissues. d The protein levels of COL4A1 in 10 paired cancerous tissues (C) and the matched adjacent noncancerous liver tissues (N) from HCC patients were analyzed by western blot. e COL4A1 proteins were highly expressed in HCC tissues. Immunohistochemistry staining of COL4A1 was performed in paired HCC samples and normal liver tissues (n = 24). The representative images were shown. f Expression levels of COL4A1 were measured by qRT-PCR and western blot in indicated HCC cell lines

Fig. 2

COL4A1 knockdown inhibits the proliferation, migration and invasion of HCC cells. a Knockdown efficiency of COL4A1 using shRNA was confirmed by qRT-PCR and western blot in SMMC7721 cells and SK-Hep1 cells. b-d COL4A1 knockdown inhibited cell proliferation (b), migration (c) and invasion(d) by Real-time cell analyzer. e Downregulation of COL4A1 inhibited cell migration by wound healing assay. f COL4A1 knockdown inhibited cell proliferation by colony formation assay. g COL4A1 knockdown suppressed tumor growth in vivo. n = 7/group. Tumor volume was measured, and photographs of tumors were taken. Data are presented as means ± standard deviation. Student t test and Two-way ANOVA followed by Tukey’s multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

COL4A1 overexpression promotes the abilities of cell proliferation, migration and invasion in HCC cells. a CRISPR/Cas9/Synergistic Activation Mediator (SAM) - mediated overexpression of COL4A1 in HepG2 cells and PLC/PRF/5 cells were confirmed by qRT-PCR and western blot. b-d Stable overexpression of COL4A1 promoted cell proliferation (b), migration (c) and invasion (d). Data are presented as means ± standard deviation. Student t test and Two-way ANOVA followed by Tukey’s multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

RUNX1 activates the transcription of COL4A1 in HCC. a Correlation between mRNA levels of COL4A1 and RUNX1. Linear regression analysis showed the positive correlation between mRNA levels of RUNX1 and COL4A1 from HCC samples in TCGA database (r = 0.5800, P < 0.0001). b mRNA level of RUNX1 was upregulated in cancerous tissues (C, n = 374) compared with noncancerous liver tissues (N, n = 50) from TCGA datasets. c&d RUNX1 regulates COL4A1 expression. Protein levels of COL4A1 and RUNX1 were detected by western blot analysis in indicated HCC cell lines after transfected with either overexpression vector (HA-RUNX1) (c) or si-RUNX1 (d). e&f RUNX1 activates transcription of COL4A1 by dual luciferase reporter assay. Overexpression of RUNX1 activated the transcription of COL4A1 (e) and knockdown of RUNX1 inhibited the transcription of COL4A1 (f). Student t test, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Overexpression of COL4A1 activates FAK-Src signaling. a Phosphorylation of FAK, Src, and AKT were analyzed by western blot in COL4A1 overexpressed HCC cells. b Phosphorylation of FAK, Src, and AKT were analyzed by western blot in COL4A1 knockdown HCC cells. Quantification of western blots were analyzed by Image J. Student t test, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 6

FAK or Src inhibitor selectively inhibits the cell viability of HCC cells with high expression of COL4A1. a&b Cell viability was tested after treatment with inhibitors. HCC cells were treated with Defactinib (FAK inhibitor) or Saracatinib (Src inhibitor) at the indicated concentrations for 48 h. HCC cells with high expression level of COL4A1 (a) were sensitive to Defactinib or Saracatinib treatment, but HCC cells with low expression level of COL4A1 (b) were resistance to Defactinib or Saracatinib treatment. c&d Knockdown of COL4A1 reduced the sensitive to Defactinib or Saracatinib treatment in SMMC7721 (c) and SK-Hep1 (d). Indicated cells were treated with Defactinib (FAK inhibitor) or Saracatinib (Src inhibitor) at the indicated concentrations for 48 h. e&f Overexpression of COL4A1 increased the sensitive to Defactinib or Saracatinib treatment in HepG2 (e) and PLC/PRF/5 (f). Indicated cells were treated with Defactinib (FAK inhibitor) or Saracatinib (Src inhibitor) at the indicated concentrations for 48 h. Cell viability was analyzed by crystal violet staining assay (Left) and CCK8 assay (Right), respectively. Data are presented as means ± standard deviation. Student t test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant

Fig. 7

Schematic diagram of COL4A1 promoting the growth and metastasis of HCC cells. COL4A1 promotes the growth, migration and invasion of HCC cells by activating FAK-Src signaling. Col IV, Collagen type IV