The Transcription Factor GLI1 Mediates TGFb1 Driven EMT in Hepatocellular Carcinoma via a SNAI1-Dependent Mechanism

The role of the epithelial-to-mesenchymal transition (EMT) during hepatocellular carcinoma (HCC) progression is well established, however the regulatory mechanisms modulating this phenomenon remain unclear. Here, we demonstrate that transcription factor glioma-associated oncogene 1 (GLI1) modulates EMT through direct up-regulation of SNAI1 and serves as a downstream effector of the transforming growth factor-b1 (TGFb1) pathway, a well-known regulator of EMT in cancer cells. Overexpression of GLI1 increased proliferation, viability, migration, invasion, and colony formation by HCC cells. Conversely, GLI1 knockdown led to a decrease in all the above-mentioned cancer-associated phenotypes in HCC cells. Further analysis of GLI1 regulated cellular functions showed that this transcription factor is able to induce EMT and identified SNAI1 as a transcriptional target of GLI1 mediating this cellular effect in HCC cells. Moreover, we demonstrated that an intact GLI1-SNAI1 axis is required by TGFb1 to induce EMT in these cells. Together, these findings define a novel cellular mechanism regulated by GLI1, which controls the growth and EMT phenotype in HCC.National Institutes of Health Grants CA100882 and CA128633 (to LRR) and CA165076; the Mayo Clinic Center for Cell Signaling in Gastroenterology (NIDDK P30DK084567) (to MEFZ); the Mayo Clinic Cancer Center (CA15083), the Mayo Clinic Center for Translational Science Activities (NIH/NCRR CTSA Grant Number KL2 RR024151), and an American Gastroenterological Association Foundation for Digestive Health and Nutrition Bridging Grant (to LRR)

GLI1 promotes cell proliferation, viability, migration, invasion and colony formation of HCC cells.

<p>(A) Overexpression of GLI1 increases BrdU incorporation of Huh7 cells and silencing GLI1 expression in SNU398 cells decreases BrdU incorporation. (B) As assessed by the MTT assay, cell viability is increased at 48, 72 and 96 hours by overexpression of GLI1 in Huh7 cells and decreased at 24, 48, 72 and 96 hours by knockdown of GLI1 in SNU398 cells. (C) As assessed by the wound-healing assay, at both 24 and 48 hours, cell migration rate is increased in Huh7 cells by overexpression of GLI1 and decreased in SNU398 cells by silencing GLI1 expression. (D) Cell invasion is increased by overexpression of GLI1 in Huh7 cells and decreased by knockdown of GLI1 in SNU398 cells. (E) Overexpression of GLI1 promotes colony formation of Huh7 cells; in contrast, knockdown of GLI1 represses colony formation of SNU398 cells.</p

GLI1 expression is positively associated with HCC recurrence.

<p>(A) Tumor recurrence after surgical resection in 139 patients with HCCs. Differences between the Kaplan-Meier curves of patients in the GLI1 high expresser group and those in GLI1 low/non expresser group using the log rank test. (B) Tumor recurrence after surgical resection in the 35 patients from the high GLI1 group and in the 35 patients from the low GLI1 group. Increased GLI1 expression in HCC tissues was significantly correlated with more rapid tumor recurrence after surgical resection of the primary tumor.</p

GLI1 is induced by TGFβ1 and mediates the mechanism by which TGFβ1 induces EMT in HCC cells.

<p>(A) TGFβ1 increases GLI1 expression in Huh7 cells. And TGFβ1 is also found to decrease E-cadherin expression, while increasing the expression of N-cadherin, Vimentin and SNAI1 in Huh7 cells. (B) Suppressing the TGFβ1 induced up-regulation of GLI1 in Huh7 cells by GLI1 ASO decreased the expression of both GLI1 and SNAI1 in Huh7 cells. (C) Suppressing the TGFβ1 induced up-regulation of GLI1 in Huh7 cells by GLI1 ASO increases E-cadherin expression and decreases the expression of both N-cadherin and Vimentin.</p

Knockdown of SNAI1 reverses the EMT phenotype induced by GLI1 in HCC cells.

<p>(A) SNAI1 siRNA decreases SNAI1 expression in Huh7 cells transfected stably with GLI1 expressing plasmid. SNAI1 siRNA increases E-cadherin expression (B) and decreases the expression of both N-cadherin (C) and Vimentin (D) in Huh7 cells transfected stably with GLI1 expressing plasmid.</p

Immunofluorescence studies confirm the role of GLI1 in the regulation of EMT in HCC cells.

<p>(A) As assessed by immunofluorescence staining and confocal microscopy, overexpression of GLI1 decreases E-cadherin protein expression and increases the protein expression of both N-cadherin and Vimentin in Huh7 cells. (B) As assessed by immunofluorescence staining and confocal microscopy, knockdown of GLI1 (GLI1 ASO) increases E-cadherin protein expression and decreases the protein expression of both N-cadherin and Vimentin in SNU398 cells. DAPI was used as nuclear staining.</p