Involvement of CYR61 and CTGF in the fascin-mediated proliferation and invasiveness of esophageal squamous cell carcinomas cells

Abstract

Fascin is overexpressed in esophageal squamous cell [corrected] carcinoma (ESCC) and involved in the proliferation and invasiveness of ESCC cells. In this study, we retrospectively examined the expression of fascin in ESCC samples by immunohistochemistry and revealed that overexpression of fascin was related to poor patient survival. RNAi-mediated knockdown of fascin in ESCC cells significantly inhibited cell proliferation and invasiveness, whereas forced expression of fascin in immortalized esophageal epithelial cells accelerated cell proliferation and invasiveness. To explore the underlying mechanism, cDNA microarray was performed to identify the differential gene expression profiles between a fascin-depleted cell line by RNAi and the corresponding control ESCC cells. Results showed that 296 genes were differentially expressed on fascin depletion. In this study, we focused on two down-regulated genes: CYR61 and CTGF. We found that restored expression of either CYR61 or CTGF led to a recovery of the suppression of cellular proliferation and invasiveness induced by down-regulation of fascin expression; the protein level of CYR61 and CTGF were up-regulated in ESCCs and their expression pattern correlated with fascin overexpression. Finally, analysis of signal transduction revealed that fascin affected the expressions of CYR61 and CTGF through transforming growth factor (TGF)-beta pathway. Taken together, we propose that fascin regulates the proliferation and invasiveness of ESCC cells by modulating the expression of CTGF and CYR61 via TGF-beta pathway.

Figure 1-8375

Fascin expression in ESCCs and its impact on patient survival. A through D: Immunohistochemical staining of fascin in ESCCs (400×). A: A negative-staining case. B: A case with 1+ staining. C and D: Two cases with 2+ staining. E: Kaplan–Meier estimate of the survival by fascin expression status.

Figure 2-8375

Inhibition of cell proliferation and invasiveness by RNAi-mediated knockdown of fascin in KYSE150 cells. KYSE150 cells were transfected with fascin siRNA-expressing plasmid (KYSE150/siRNA) or empty vector (KYSE150/V). A: Western blot analysis was used to show RNAi-mediated knockdown of fascin. Equal loading was ascertained using β-actin as an internal control. B: Cell proliferation rate was determined by BrdU incorporation assay. Modified Boyden chamber assay (C) and invasiveness assay (D) were used to determine the effects of fascin knockdown on cell migration and invasiveness. Migrated and invasive cells were fixed and stained, and representative fields were photographed (bar, 100 μm). For quantification, the cells were counted in 10 random fields under a light microscope (400×). Each experiment was performed in triplicate, and results represent the mean ± SD of three experiments. *P < 0.05.

Figure 3-8375

Forced expression of fascin in SHEE cells accelerated cell proliferation and invasiveness. SHEE cells were transfected with either the empty pcDNA3 vector (SHEE/V) or the fascin expression vector (SHEE/Fascin). A: Two SHEE/Fascin clones were selected for their high expression of fascin as shown by Western blot analysis. BrdU incorporation assay (B), modified Boyden chamber assay (C), and invasiveness assay (D) were used to determine the effects of fascin on cell proliferation, migration, and invasiveness. Representative fields were photographed (scale bar, 100 μm). *P < 0.05.

Figure 4-8375

Real-time RT-PCR, Western blotting, and immunofluorescence analysis validate the microarray data. A: Real-time RT-PCR analysis was performed to analyze the expression levels of five genes: CTGF, CYR61, THBS1, ATF3, and DSC2. The expression levels were normalized by the level of GAPDH and represented as fold change over PSC cells. B: Western blot analysis was performed to determine the protein levels of CTGF, CYR61, and ATF3. C: Immunofluorescence analysis was performed to compare THBS1 and DSC2 expression. Representative images show localization of THBS1 and DSC2. Scale bar, 10 μm. EC109 is a human ESCC cell line that expresses high levels of fascin (control). PSC is an EC109 cell line transfected with nonspecific siRNA (control). PSF8 and PSF10 are two different PSC-based fascin-depleted cell lines prepared using RNAi method.

Figure 5-8375

Increased expressions of CYR61 and CTGF in ESCC tissues. Photomicrographs are shown to compare immunohistochemical staining of (A) CTGF, (B) CYR61, and (C) fascin in normal esophageal epithelium (normal) and ESCC tissues (cancer). Scale bars, 20 μm.

Figure 6-8375

Restored expression of CTGF or CYR61 resulted in a recovery of the suppression of cellular proliferation and invasiveness induced by down-regulation of fascin expression. A and B: PSF8 cells (fascin-depleted) were transfected with either the empty pcDNA3 vector (PSF8/V), the CTGF expression vector (PSF8/CTGF), or the CYR61 expression vector (PSF8/CYR61). Restored expression of CTGF (A) or CYR61 (B) was shown by real-time RT-PCR analysis. C and D: Effect of CTGF or CYR61 restored-expression on cell proliferation and invasiveness assay was determined by using BrdU incorporation assay (C) and invasiveness assay (D). Invasive cells were fixed and stained, and representative fields were photographed (Scale bar, 100 μm). *P < 0.05. Cell lines were as described in Figure 4.

Figure 7-8375

Fascin affected the expressions of CTGF and CYR61 via TGF-β pathway. Induced expressions of p-Smad2/3 (A), CTGF, and CYR61 (B) by addition of exogenous TGF-β1 protein in EC109 cells were addressed by Western blotting or real-time RT-PCR. C: Smad2/3 phosphorylation in whole-cell extracts was determined by Western blotting to evaluate the TGF-β signaling pathway. D: TGF-β1 treatment led to a recovery of the fascin-mediated alteration of cell invasiveness. *P < 0.05. Cell lines were as described in Figure 4.

Figure 8-8375

Impact of fascin on the TGF-β pathway was possibly mediated by the expression of THBS1. Induced expressions of p-Smad2/3 (A), CTGF, and CYR61 (B) by addition of exogenous THBS1 protein were determined by Western blotting or real-time RT-PCR. Proliferation (BrdU incorporation assay, C) and invasiveness (invasiveness assay, D) of EC109 and PSF8 cells were addressed under control conditions or pretreated with THBS1. *P < 0.05. Cell lines were as described in Figure 4.