Ann. N.Y. Acad. Sci. ISSN 0077-8923 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Issue: Thymosins in Health and Disease Thymosin β4 regulates migration of colon cancer cells by a pathway involving interaction with Ku80 Czeslaw S. Cierniewski, 1,2 Izabela Papiewska-Pajak, 2 Mariusz Malinowski, 1 Izabela Sacewicz-Hofman, 2 Magdalena Wiktorska, 2 Jakub Kryczka, 1 Tomasz Wysocki, 1 Jolanta Niewiarowska, 2 and Radoslaw Bednarek 1 1 Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland. 2 Department of Molecular and Medical Biophysics, Medical University of Lodz, Poland Address for correspondence: Czeslaw S. Cierniewski, Department of Molecular and Medical Biophysics, Medical University of Lodz, 6/8 Mazowiecka Street, Lodz, Poland. cciern@zdn.am.lodz.pl Aberrant expression of thymosin β4 (Tβ4) has recently been found to be associated with colorectal carcinoma (CRC) progression evidently due to an increase of the motility and invasion of tumor cells and the induction of a proangiogenic phenotype of endothelial cells. Both mechanisms depend upon matrix-degrading proteases, particularly plasmin and matrix metalloproteinases (MMPs) that are responsible for extensive tissue remodeling. Cleavage of ECM macromolecules weakens the structural integrity of tissues and exposes cryptic domains of ex- tracellular components, which elicit biological responses distinct from intact molecules. Interestingly, signaling via integrins (αVβ3, α5β1) in CRC cells (HT29, CX1.1) is induced by Tβ4 and VEGF-A only when they grow in 3D fibrin gels but not in 2D ones. The cells growing in 3D fibrin gels release upon Tβ4 significant amounts of active MMPs (MMP-2, MMP-9, and MMP-7) that cause extensive proteolysis in their close vicinity. As evidenced by a variety of approaches (transfection experiments, coimmunoprecipitation, gene silencing with siRNA), we found that this involves interaction of Tβ4 with Ku80, which has recently been described by us to mediate Tβ4 intracellular activity. Keywords: thymosin 4; matrix metalloproteinases; colorectal carcinoma cells; invasiveness Introduction Colon carcinogenesis, the result of a progressive transformation of colorectal epithelial cells, is pri- marily due to the accumulation of mutations in a number of oncogenes as well as tumor suppressor genes. 1 Thus epithelial cells lose their polarized ep- ithelial structures and concomitantly acquire a mi- gratory or mesenchymal phenotype, 2 which is es- sential for normal embryonic development and for progression of noninvasive adenomas into malig- nant, metastatic carcinomas. The “gate-keeping” event for initiation of col- orectal neoplasia appears to be the inactivation of the adenomatous polyposis coli (APC) gene. 3 APC functions as a scaffolding protein that promotes complex formation between GSK-3, -catenin, axin, and various kinases and phosphatases, thereby playing a crucial role in Wnt signaling. 4 APC loss re- sults in impaired association between catenins and E-cadherin, thus disrupting normal adherens junc- tions and polarized phenotype of epithelial cells. 5 When released from the complex with E-cadherin on the cell membrane, -catenin is rapidly phos- phorylated by glycogen synthetase kinase-3 in the APC/axin/GSK-3 complex and is subsequently de- graded by the ubiquitin–proteosome pathway. APC loss also results in aberrant cytoskeletal regulation that affects both microtubules 6 and F-actin, 7 lead- ing to defects in directed cell migration, and an in- creased incidence of mitotic errors. Early mutation of K-ras causes the next stage of transformation. 8 K-ras is the best studied and the most common oncogene involved in this process. 9 It suppresses expression of GSK-3, and thus stimulates Wnt sig- naling. 10 About 15% of sporadic colorectal carci- noma (CRC) is caused by somatic inactivation of “mismatch repair” (MMR) genes such as hMSH2 doi: 10.1111/j.1749-6632.2010.05480.x 60 Ann. N.Y. Acad. Sci. 1194 (2010) 60–71 c  2010 New York Academy of Sciences.