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Regulation of transcription factors by integrin-linked kinase during oncogenesis

University of British Columbia

Integrin-linked kinase is a serine-threonine kinase that was identified by a yeast two-hybrid screen for proteins that interact with the cytoplasmic tail of Pi-integrin. Overexpression of ILK in epithelial cells promotes cell proliferation, anchorageindependent growth, epithelial to mesenchymal transformation, inhibition of anoikis, increased invasiveness and tumourgencity in nude mice. This thesis aims to identify downstream effectors of ILK, and to elucidate the pathway by which ILK regulates the activity of transcription factors. In the first part of the study, I demonstrate that ILK inhibits the activity of glycogen synthase kinase-3 (GSK-3) and that ILK can promote the phosphorylation of GSK-3 either directly or indirectly through ILK-dependent activation of Protein kinase B (PKB/Akt). Furthermore, ILK activity is stimulated upon cell adhesion to fibronectin (FN), leading to inhibition of GSK-3 activity. This in turn prevents GSK-3 from phosphorylating the DNA binding site of c-Jun, resulting in the positive regulation of the AP-1 transcriptional activity. It was previously shown that over-expression of I LK results in increased stability of P-catenin and as well as translocation of P-catenin to the nucleus. Since GSK-3 can phosphorylate P-catenin and promote its degradation, the role of ILK-mediated inhibition of GSK-3 and P-catenin stability was investigated. In the second part of this thesis, I studied the effect of inhibiting ILK activity in the human colon carcinoma cell lines, SW-480 and DLD-1, which lack functional APC. I found that ILK is able to regulate p-catenin pools, and modulate P-catenin/TCF/LEF transcription activity in an APC independent manner. It was previously observed that ILK overexpression caused a concomitant decrease in the expression of E-cadherin. This was proposed to account for the morphological changes seen upon ILK expression. The expression of E-cadherin is regulated by E-boxes in the E-cadherin promoter, which serve as regions of repressor binding. Therefore, binding of transcriptional repressor factors to the E-boxes results in repression of transcription and expression of E-cadherin. The transcriptional repressors that bind to E-boxes are members of the Snail family. This study shows that ILK upregulates the expression of the Snail transcriptional repressor, which in turn suppresses the expression of E-cadherin. Transfection with dominant negative ILK or exposure to the small molecule ILK inhibitor was able to reverse the effects of ILK. Together, these data demonstrate that ILK is involved in regulating the activity of transcription factors p-catenin/TCF/LEF, and AP-1. ILK regulates the activity of AP-1 and P-catenin-TCF/LEF through the inactivation of GSK-3 and the regulation of Pcatenin pools.

Thesis/Dissertation

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2009-08-14

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http://hdl.handle.net/2429/12253

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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