Novel snail1 target proteins in human colon cancer identified by proteomic analysis

Abstract

Background: The transcription factor Snail1 induces epithelial-to-mesenchymal transition (EMT), a process responsible for the acquisition of invasiveness during tumorigenesis. Several transcriptomic studies have reported Snail1-regulated genes in different cell types, many of them involved in cell adhesion. However, only a few studies have used proteomics as a tool for the characterization of proteins mediating EMT.

Methodology/principal findings: We identified by proteomic analysis using 2D-DIGE electrophoresis combined with MALDI-TOF-TOF and ESI-linear ion trap mass spectrometry a number of proteins with variable functions whose expression is modulated by Snail1 in SW480-ADH human colon cancer cells. Validation was performed by Western blot and immunofluorescence analyses. Snail1 repressed several members of the 14-3-3 family of phosphoserine/phosphothreonine binding proteins and also the expression of the Proliferation-associated protein 2G4 (PA2G4) that was mainly localized at the nuclear Cajal bodies. In contrast, the expression of two proteins involved in RNA processing, the Cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and the Splicing factor proline/glutamine-rich (SFPQ), was higher in Snail1-expressing cells than in controls. The regulation of 14-3-3epsilon, 14-3-3tau, 14-3-3zeta and PA2G4 by Snail1 was reproduced in HT29 colon cancer cells. In addition, we found an inverse correlation between 14-3-3sigma and Snail1 expression in human colorectal tumors.

Conclusions/significance: We have identified a set of novel Snail1 target proteins in colon cancer that expand the cellular processes affected by Snail1 and thus its relevance for cell function and phenotype.

Figure 1. Experimental workflow and characterization of SW480-ADH Mock and Snail1 cells.

(A) Scheme of the workflow followed in this study. (B) Representative phase-contrast micrographs (upper panels) and confocal laser immunofluorescence images showing F-actin staining (lower panels) of SW480-ADH Mock and Snail1 cells. Scale bar, 20 µm (upper panels) and 10 µm (lower panels). (C) Western blot analysis of whole-cell extracts from SW480-ADH Mock and Snail1 cells showing Snail1 overexpression and its effect on the expression of epithelial (E-cadherin, Occludin and Claudin-7) and mesenchymal (Lef-1) markers. β-Tubulin was used as a loading control.

Figure 2. Differential protein expression analysis of SW480-ADH Snail1 and Mock cells by 2D-DIGE electrophoresis.

Nuclear extracts from SW480-ADH Mock and Snail1 cells were differentially labeled with Cy3 and Cy5 dyes. An internal standard combining proteins from both extracts was included in all gels after labeling with Cy2 dye. 3–10 NL IPG strips were used for the IEF and standard SDS-PAGE for the second dimension. Representative 2D image shows the spot map corresponding to the internal standard, which is common to all gels analyzed. Spots indicated with arrows were informative for protein identification by mass spectrometry (see Table 1 for code assignment). Selected spots showed a statistical variance of volume ratios within the 95th confidence level (Student's t-test, p<0.05).

Figure 3. Validation of candidate Snail1 target proteins.

(A) Western blot analysis of the expression of selected proteins in cytosolic (C) and nuclear (N) extracts of SW480-ADH Mock and Snail1 cells. HNRNPH3 isoforms (1–4) are indicated. The numbers below the gels indicate the quantification of the change in expression between Snail1 and Mock cells after normalization to β-Tubulin or Lamin B (cytosolic and nuclear control, respectively). The statistical significance of the nuclear expression changes promoted by Snail1 was assessed by two-tailed unpaired Student's t-test. It was p<0.001 for 14-3-3ε, 14-3-3σ, 14-3-3ζ, CPSF6 and the isoforms 3 and 4 of HNRNPH3; p<0.01 for 14-3-3τ; and p<0.05 for 14-3-3β and PA2G4. The regulation of SFPQ (p = 0.057) and that of the isoforms 1+2 of HNRNPH3 (p = 0.058) did not reached the statistical significance, but a clear tendency was observed. (B) Western blot analysis of the expression of selected proteins in cytosolic (C) and nuclear (N) fractions of HT29 Mock and Snail1 cells. The numbers below the gels indicate the quantification of the change in expression between Snail1 and Mock cells after normalization to β-Tubulin or Lamin B (cytosolic and nuclear control, respectively). (C) Quantitative RT-PCR study of PA2G4 expression in SW480-ADH and HT29 Mock and Snail1 cells. 18S rRNA expression was used for normalization. Statistical significance was assessed by two-tailed unpaired Student's t-test, single asterisk indicates p<0.05.

Figure 4. Immunofluorescence analysis of 14-3-3 proteins in Snail1 and Mock cells.

Representative confocal microscopy images showing the expression and localization of 14-3-3 β, ε and σ (red) in SW480-ADH Mock and Snail1 cells. GFP expression (green) was unaltered by Snail1 and was used as a control. Scale bar, 10 µm.

Figure 5. Subcellular distribution of PA2G4.

(A) Representative confocal microscopy images showing the expression and localization of PA2G4 (red) in SW480-ADH Mock and Snail1 cells. GFP expression (green) was unaltered by Snail1 and was used as a control. Scale bar, 5 µm. (B) Representative confocal microscopy images showing double immunolabeling for PA2G4 (red) and for molecular markers (blue) of Cajal bodies (Coilin), Cajal bodies and nuclear speckles of splicing factors (TMG-cap), nucleolus (Fibrillarin), and PML bodies (PML) in SW480-ADH Mock cells. The pink colour in the merge images indicates the existence of colocalization. Scale bar, 5 µm.

Figure 6. Immunohistochemistry staining of 14-3-3σ and Snail1 in human colon tumors.

(A) Immunohistochemistry images showing 14-3-3σ expression in representative normal and tumor colon samples of the 46 analyzed in the tissue array. Bars indicate magnification. Sections were counterstained with haematoxylin. (B) Immunohistochemistry images showing 14-3-3σ and Snail1 expression in two consecutive slices of a representative colon cancer tumor of the five analyzed. Bars indicate magnification. Sections were counterstained with haematoxylin. Right panels are a 3X-magnification of the region indicated in left panels.