Background: Colon cancer can be classified into those with microsatellite instability (MSI) and those with chromosomal instability (CIN or microsatellite stable (MSS)). MSI colon cancers frequently display poorly differentiated histology, the mechanistic basis for which is not well understood. We recently demonstrated that poorly differentiated MSI colon cancer cell lines and primary tumours display loss of expression of the cytoskeletal protein villin. Furthermore, we observed that villin promoter activity reflects endogenous villin expression suggesting villin loss is transcriptionally mediated. The goal of this study was to identify the transcription factors which regulate villin expression in colon cancer cells, as a means of providing insight into the mechanistic basis for the poorly differentiated histology of MSI colon cancer.
Methods: Affymatrix microarrays were used to identify genes differentially expressed between 5 well differentiated MSS and 5 poorly differentiated MSI colon cancer cell lines. The MSS colon cancer cell lines SW948 and SW403 were transfected with a Non Targeting siRNA or siRNAs targeting CDX-1, GATA-6, ISX and ELF3. Cells were collected 48 hours post transfection and levels of CDX1, ELF3, ISX, GATA-6 and villin determined by qRT-PCR.
Results: Microarray analysis of well and poorly differentiated colon cancer cell lines identified 4 gut-specific transcription factors with significantly reduced expression in poorly differentiated lines - CDX-1, GATA-6, ISX and ELF3. While knockdown of ISX and GATA-6 had no effect on endogenous villin expression, knockdown of CDX1 and ELF3 reduced villin expression by 65% and 55% respectively. Similar results were observed in a second colon cancer cell line, SW403. Notably, knockdown of ELF3 also resulted in reduced endogenous CDX1 expression suggesting ELF3 is an upstream regulator of CDX-1.
Conclusion: This study begins to identify a transcription-mediated regulatory hierarchy involving ELF3 and CDX1 which mediates expression of the cell differentiation marker villin, in colon cancer.