SATB1 expression is associated with biologic behavior in colorectal carcinoma in vitro and in vivo

Abstract

There is increasing evidence that Special AT-rich sequence-binding protein 1 (SATB1) is aberrantly expressed in several cancers and is correlated with clinicopathologic parameters in these tumors. In this study, we showed over-expression of SATB1 in 80 cases of colorectal cancer and in 3 colorectal cancer cell lines and found expression levels were strongly associated with tumor differentiation and stage. Expression levels of SATB1 protein were higher in poorly-differentiated as compared with well-differentiated cell lines, and both quantity and distribution patterns of SATB1 were associated with tumor differentiation and pTNM stage. Strikingly, we further investigated the effect of down regulation of SATB1 expression on malignant phenotypic features in colorectal cancer cells in vitro, and showed that SABT1 down-regulation negatively affected growth potential, anchorage-independent colony formation and cancer cell invasion, and resulted in increased apoptosis. SATB1 expression was positively associated with the expression of various biological and genetic markers, including Cyclin D1, MMP-2, NF-κB, and PCNA, and was associated with loss of APC and BRAF(V600E). These findings suggest that SATB1 is involved in the carcinogenesis, development and progression of colorectal cancer.

Figure 1. SATB1 expression in CRC tissues and cell lines.

A–E: SATB1 protein expression (positive signals show red staining with AEC) in matched normal colorectal epithelium (A); and well (B), moderately (C) and poorly differentiated (D, E) CRC tissues. In normal colon tissue (A) positive staining is seldom found in the normal glandular epithelium. T lymphocytes in the lamina propria are positive. In CRC tissues (B–E) positive signals for SATB1 are distributed in the cytoplasm or in nuclei or both in carcinoma epithelial cells. F: Expression of SATB1 mRNA in cytoplasm of CRC cells (arrows, purple signals stained with NBT/BCIP). G: SATB1 protein expression in one sample of CRC tissue and in the peri-tumoral tissue (red signals stained with AEC). a, b and c are higher magnifications of the corresponding regions in the upper left panel. SATB1 is negative in the peri-tumoral normal glandular epithelium (a). More cancer cells are positive for SATB1 in the central zone of the neoplasm (c) than in the periphery (b) of the cancer and normal tissue. H: Agarose gel electrophoresis of RT-PCR amplification products following mRNA extraction from three cell lines and a positive control. I: Relative levels of SATB1 in CRC cell lines using qRT-PCR. J–M: Cyclin D1(J), MMP-2(K), NF-κB (L) and PCNA(M) expression in CRC tissues (J, L, M: purple signals stained with NBT/BCIP; K: red signals stained with AEC). Bars = 50 µm.

Figure 2. Reduction of SATB1 expression by siRNA affects the biologic behavior of CRC cells.

A: Effect of SATB1 down-regulation. B: Evalaution of cell proliferation with the MTS essay shows that SATB1 down-regulation significantly inhibits growth of LOVO cells. C: Results of soft agar colony formation essay in LOVO cells with SATB1 down regulation. D: AnnexinV-PI analysis shows that the percentage of apoptotic LOVO cells is higher after SATB1 down-regulation as compared with the empty vector control. E: SATB1 down-regulation suppresses the invasion potential of CRC cells. Bars = 50 µm.