The progression to invasive and metastatic carcinoma involves profound alterations in epithelial structure and function. We show here that the EMT of colon carcinoma is coincident with an increase in αvβ6 integrin expression, and the culmination of our observations is that high expression of β6 in primary colon carcinomas is a prognostic marker for aggressive disease and patient mortality. This integrin is expressed primarily during development, and high αvβ6 expression in adults is limited only to a few epithelial tissues (18
). However, αvβ6 can be reexpressed in parallel with specific morphogenetic events such as inflammation and wound healing (19
). Wound healing, in particular, is believed to involve EMT processes, and the induction of αvβ6 implies a central role for the receptor (19
). In addition, several studies have shown neo-expression of αvβ6 in oral squamous cell carcinomas (19
), although a link between expression and patient outcome has not been established. Nonetheless, these studies are consistent with our observation that αvβ6 expression is induced as a consequence of EMT. Interestingly, studies on the phenotypic changes induced by the EMT have traditionally focused on the transcriptional repression of epithelial markers that maintain tissue polarity, such as E-cadherin, and the functional consequences of induced mesenchymal markers. Our discovery that an epithelial integrin is dynamically regulated during the EMT of colon carcinoma, and specifically one that is upregulated during development, is consistent with the long-standing hypothesis that colorectal carcinogenesis more accurately reflects a dedifferentiation of epithelia to a more embryonic phenotype.
The induction of αvβ6 expression that we observed during the EMT is dependent on the transcriptional upregulation of the β6 integrin subunit. The transcriptional regulation of β6 had not been investigated previously to our knowledge, and it now appears that Ets-1 may be a key transcription factor that influences EMT. In fact, we have recently reported that Ets-1 expression is upregulated as a consequence of the EMT, and that it is responsible for the induction of the VEGF receptor Flt-1 (12
). A functional role for Ets-1 in β6 regulation was evidenced by its ability to bind and transactivate the human β6 promoter. Our screening identified 4 putative consensus sites within the first kilobase of the promoter sequence, but only the first site appeared to be of importance. Ets-1 bound preferentially to this site in gel shift assays, and, most significantly, site-directed mutagenesis of this region completely abrogated transactivation activity. Since Ets-1 typically exerts its effects in cooperation with other transcriptional factors and cofactors, we are currently seeking to identify other regulators of β6 transcription. The role, if any, of the other 3 consensus sites also awaits further characterization. Although they appear dispensable for β6 regulation during the EMT process, the possibility exists that alternate triggers, or tissue-specific factors, might act to influence β6 gene regulation via these sites.
We define 2 key functions of αvβ6 that can be linked to the biology of aggressive colon carcinoma: activation of autocrine TGF-β, and promotion of migration on fibronectin matrices. Firstly, TGF-β has been proposed to act as a cell-autonomous promoter of late-stage human tumor development because of its ability to regulate EMT (5
). In this context, the autocrine TGF-β production we observed would provide the means to stabilize and sustain the EMT by continuous TGF-β receptor signaling in a cell-autonomous fashion. However, it is the ability of αvβ6 to activate autocrine TGF-β in post-EMT cells that is most novel with respect to colon carcinoma progression. The recognition that this integrin can bind and activate latent TGF-β (16
) identified a likely role for this receptor in contributing to the onset of TGF-β–mediated diseases, such as inflammation and fibrosis (24
). From our perspective, induced expression of αvβ6 in colon carcinoma would provide a mechanism to locally regulate TGF-β function in vivo, to provide a feedback loop to perpetuate the EMT process, and, in turn, to create a tumor microenvironment more amenable to progression. Indeed, the morphological analysis of the xenografts (Figure ) provides in vivo evidence for this concept.
Secondly, having a specific fibronectin receptor elicited as a consequence of the EMT also has selective advantages for tumor cells. The colonic epithelium resides upon a basement membrane, composed primarily of laminins, collagen IV, and proteoglycans, which provides an interface with the lamina propria beneath (26
). The structural composition of this zone differs in its high fibrillar collagen content, and also in enrichment with fibronectin and tenascin, both ligands for αvβ6 (14
). Thus, an enhanced migratory capacity on fibronectin would facilitate the escape and dissemination of invading colon carcinoma cells into the lamina propria. Moreover, αvβ6 expression and fibronectin recognition may also be important for colonization of the liver, the primary site for metastases derived from colorectal tumors. In addition to the anatomical consideration that portal blood flow from the colon goes directly to this organ, our data suggest that there may be an additional dimension to colonization that specifically involves αvβ6. Since there is no basement membrane under the endothelium of liver microvessels as in other tissues, it has been suggested that adhesion to fibronectin may be of central importance for liver metastasis, because it is abundant on the surface of hepatocytes (28
). We propose, therefore, that an increased propensity to adhere to fibronectin would promote colon carcinoma metastasis to the liver by aiding extravasation.
The key conclusion of this study relates to the expression of αvβ6 in human colorectal cancer. We identified this receptor as a marker of aggressive carcinoma, in part based on its high frequency of expression (almost 40%) in malignant tumors. Additional compelling evidence for the involvement of αvβ6 in tumor progression is provided by our demonstration of β6 expression in both lymphatic and hepatic metastases derived from human colorectal carcinomas. However, the result of greatest consequence is that analysis of the clinical data revealed that elevated β6 expression, rather than the simple presence or absence of the receptor, is linked to significantly reduced survival times. Even more striking was the discovery that its value as a prognostic marker is more significant for early-stage tumors. It is likely that the effects of β6 expression on late-stage tumors are masked by additional genetic and epigenetic alterations. However, based on our results, it is tempting to speculate that the EMT process can contribute directly to tumor progression and development of metastatic disease, and that αvβ6 represents a novel marker for this event. Moreover, the hazard ratio analysis confirmed that this receptor is a stand-alone variable, similar to those of stage and age at diagnosis, for colorectal tumors, and indicated that high expression of this integrin is an independent and important risk factor for patient survival. Overall, these findings establish αvβ6 as a novel prognostic indicator for human colon cancer and validate our EMT model as a valuable tool for the identification of clinically relevant markers.
Finally, a potentially important consequence of our work is that αvβ6 may be an attractive therapeutic candidate for colon cancer and, in particular, that it may be possible to selectively target invading and metastasizing cells. Furthermore, because elevated β6 expression is predictive of outcome in early-stage disease, it may also be a feasible target for earlier intervention and treatment. Targeting integrin function has emerged as a legitimate therapeutic strategy for several disease types. In this regard, the current development and characterization of function-blocking anti-β6 antibodies (29
) offer the promise of new reagents and novel treatments not only for diseases such as fibrosis but also, in light of the findings presented here, for aggressive colorectal cancer.