Integrin [alpha]6A[béta]4 in colon cancer
Dydensborg, Anders Bondo
The expression level of the [alpha]6A[béta]4 integrin has been reported to be elevated in a number of human cancers in accordance with its known stimulatory role in several aspects of cancer progression such as promoting invasion and stimulating intracellular signaling of importance for cellular proliferation and survival. In the present work the expression levels of [béta]4 in adenocarcinomas of the human colon were evaluated at the transcript level in comparison with the expression levels in patient matched healthy colonic tissue. Not only were the expression level of the [béta]4 subunit found to be upregulated in cancerous tissue, but levels of expression of [béta]4 were found to be closely correlated to levels of expression of those of the oncogenic transcription factor MYC. This correlation was demonstrated to be of functional importance for the expression level of [béta]4, since forced expression of MYC in a colon cancer cell line stimulated transcriptional activity of various [béta]4 promoter constructs.The pre-mRNA of the [alpha]6 subunit undergo alternative splicing yielding two different variants: the A and the B variants. Their distinct intracellular domains and their distinct biochemical properties characterize these variants. As part of the characterization of the expression of the [alpha]6A[béta]4 integrin in colon cancers, the expression level of total [alpha]6 as well as the two different variants were investigated. As for the [béta]4 subunit the total level of [alpha]6 were found to be elevated in cancers as compared to their corresponding healthy resection margins. Interestingly, an alteration of the ratio of expression of two splice variants of the [alpha]6 subunit was found to be significantly altered in 81% of the cancers investigated. Thus, the expression ratio shifted from a predominant expression of [alpha]6B in the healthy resection margins to a predominant expression of [alpha]6A in the primary adenocarcinomas. Such differential expression of splice variants was also found in the normal human small intestine. Indeed, the proliferative crypts were found to express the [alpha]6A variant, while the differentiated compartments of the crypt-villus axis were found to express the [alpha]6B variant.The functional importance of this was confirmed when over-expression of the two different splice variants were demonstrated to differentially activate the [béta]-catenin/TCF4 and MYC transcriptional complexes, two well known players in intestinal proliferation and adenocarcinomas formation. On the other hand the two splice variants were not found to differentially affect differentiation of human enterocytes as assayed by monitoring the transcriptional activity from promoter constructs of sucrase-isomaltase, lactasephlorizin hydrolase, DPPIV and ALPP. Finally, it has become increasingly clear that the classic normalizing genes such as GAPD and [béta]-actin can display modulated expression levels across tissue types and during cellular differentiation. Therefore, experiments aimed at identifying and validating normalizing genes for transcript measurements during the complex cellular processes of differentiation and cancer progression in the human gut were carried out. Previously generated microarray data were screened for candidate genes of which two were selected for further analyses along with seven commonly used normalizing genes. Real time RT-PCR amplifying the nine putative normalizing genes were performed on various samples representing different differentiation stages of human enterocytes as well as on patient matched primary tumors and healthy resection margins. Using several statistical approaches, RPLP0 and B2M were found to be the two best normalizing genes for experiments aimed at assessing transcript during differentiation and in cancers respectively. These studies should serve as a paradigm for other studies aimed at validating other normalizing genes under other experimental settings.