Villin expression is frequently lost in poorly differentiated colon cancer

Abstract

Colorectal cancers (CRCs) are classified as having microsatellite instability (MSI) or chromosomal instability (CIN); herein termed microsatellite stable (MSS). MSI colon cancers frequently display a poorly differentiated histology for which the molecular basis is not well understood. Gene expression and immunohistochemical profiling of MSS and MSI CRC cell lines and tumors revealed significant down-regulation of the intestinal-specific cytoskeletal protein villin in MSI colon cancer, with complete absence in 62% and 17% of MSI cell lines and tumors, respectively. Investigation of 577 CRCs linked loss of villin expression to poorly differentiated histology in MSI and MSS tumors. Furthermore, mislocalization of villin from the membrane was prognostic for poorer outcome in MSS patients. Loss of villin expression was not due to coding sequence mutations, epigenetic inactivation, or promoter mutation. Conversely, in transient transfection assays villin promoter activity reflected endogenous villin expression, suggesting transcriptional control. A screen of gut-specific transcription factors revealed a significant correlation between expression of villin and the homeobox transcription factor Cdx-1. Cdx-1 overexpression induced villin promoter activity, Cdx-1 knockdown down-regulated endogenous villin expression, and deletion of a key Cdx-binding site within the villin promoter attenuated promoter activity. Loss of Cdx-1 expression in CRC lines was associated with Cdx-1 promoter methylation. These findings demonstrate that loss of villin expression due to Cdx-1 loss is a feature of poorly differentiated CRCs.

Figure 1

Villin expression is significantly down-regulated in microsatellite instability (MSI) colon cancer cell lines. A: Villin mRNA expression in five microsatellite stable (MSS) and 5 MSI colon cancer cell lines profiled using Affymetrix microarrays. B: Villin mRNA expression in a panel of 40 colon cancer cell lines (24 MSS and 16 MSI) assessed by quantitative real-time PCR. C, D: Validation of loss of villin protein expression in MSI cell lines by Western blot analysis (C) and immunohistochemistry (D). For IHC studies, five MSS and five MSI cell lines were grown as xenografts in severe combined immunodeficiency mice, and the tumors were excised and the formalin was fixed. Villin expression was determined by IHC using a commercially available anti-villin antibody.

Figure 2

A: Villin mRNA expression in primary microsatellite instability (MSI) and microsatellite stable (MSS) colon cancers. Gene expression differences between normal colonic mucosa, MSS, and MSI colon tumors were compared using Affymetrix microarrays. Villin expression was significantly down-regulated in both sporadically arising and early onset MSI tumors derived from Lynch syndrome patients. B–E: Loss of villin protein expression in MSI colon cancer. Tissue microarrays comprising 519 MSS and 58 MSI tumors were stained for villin expression using an anti-villin antibody. Villin expression was assessed in each sample by scoring of the (B) overall staining intensity and (C) the percentage of villin-positive cells. D: The percentage of MSS and MSI tumors with an overall villin staining intensity of ≤0.5. E: Representative tumors displaying various villin staining intensities of 0 to 3, as described in Materials and Methods. *P < 0.001, t-test; ^†P = 0.0002, t-test; ^‡P < 0.00001, t-test; ^§P = 0.0006, χ².

Figure 3

Loss of villin expression is most frequently observed in poorly differentiated colon cancers. Villin staining intensity and the percentage of colon cancers with villin staining intensity of ≤0.5 in well/moderately differentiated and poorly differentiated microsatellite instability (MSI) (A) and microsatellite stable (MSS) (B) colon cancers. C, D: Villin expression in representative well/moderately differentiated (C) and poorly differentiated (D) colon cancers. *P < 0.05; ^†P < 0.005, Student’s t-test; and ^§P < 0.05, χ² test.

Figure 4

Prognostic significance of villin expression and subcellular localization in Dukes B/C colon cancer. Kaplan-Meier analysis demonstrating villin staining intensity is not prognostic in microsatellite stable (MSS) (A) or microsatellite instability (MSI) (B) colon cancer. Prognostic significance of localization of villin exclusively to the cell membrane in MSS (C) and MSI (D) colon cancer. Prognostic significance of localization of villin exclusively to the cell membrane in moderately differentiated (E) and poorly differentiated (F) MSS tumors.

Figure 5

Villin promoter luciferase reporter activity reflects endogenous villin expression. A: Endogenous villin mRNA expression in four noncolon cancer cell lines, and five villin-negative and five villin-positive colon cancer cell lines. B: Villin promoter reporter activity in the same cell line panel. Each cell line was transfected with the villin promoter reporter along with TK-Renilla as a control for transfection efficiency. C: Sp-1 reporter activity in the cell line panel. Each cell line was transfected with the villin promoter luciferase reporter along with TK-Renilla as a control for transfection efficiency. Values shown in B and C are mean ± SEM of a representative experiment performed in triplicate.

Figure 6

Correlation of endogenous mRNA expression of 10 transcription factors (TCF7L2, IHH, HNF1a, HNF4a, Cdx-1, Cdx-2, KLF4, ELF3, ISX, and GATA6) implicated in the regulation of intestinal cell differentiation, and villin, in a panel of 40 colon cancer cell lines. Correlation was assessed by computing the Spearman’s correlation coefficient.

Figure 7

Cdx-1 regulates villin expression. A: Transient Cdx-1 overexpression induces villin promoter activity in RKO cells. Cells were transfected with empty vector control or a Cdx-1 expression vector in combination with the villin promoter luciferase construct, and villin reporter activity determined using the dual luciferase reporter assay. B, C: Cdx-1 knockdown represses endogenous villin expression. SW948 cells were transiently transfected with Cdx-1 targeting small-interfering RNA (siRNA) or nontargeting control siRNAs for 48 hours, and the effect on (B) Cdx-1 and (C) villin mRNA expression was determined by quantitative PCR. Values shown are mean ± SEM of a representative experiment performed in triplicate. D: Deletion of all Cdx-1 binding sites in the villin promoter construct attenuates villin promoter reporter activity. The villin promoter luciferase reporter comprising 1200 bp upstream and 500 bp downstream of the villin transcription start site (TSS) (villin 1702), or the various deletion constructs shown, were transiently transfected into SW948 cells and villin promoter activity assessed using the dual luciferase reporter assay. E: RKO cells were transiently transfected with a series of villin promoter reporter constructs and Cdx-1, and villin promoter activity assessed using the dual luciferase reporter assay. Values shown are mean ± SD of a representative experiment performed in triplicate. F: Cdx-1 promoter methylation status inversely correlates with Cdx-1 expression across a panel of 27 colon cancer cell lines (r = −0.56; P = 0.0024). EV, empty vector; NT, non-targeting.