The Role of ROCK in Colon Cancer Invasion Using Three-Dimensional Collagen I Microenvironments
thesisposted on 10.12.2012, 00:00 by Ramana V. Vishnubhotla
Colon cancer is the third most commonly diagnosed cancer and the third leading cause of cancer deaths in the US. Survival rates greatly decrease as the cancer progresses and invades (metastasis) into other portions on the body. Preventing cancer cell metastasis is essential for treating colon cancer. Actin stress fiber formation is critical for cell motility and thus plays a significant role in cancer cell metastasis. Several cellular factors including RhoA and Rho-kinases (ROCK-I and ROCK-II) are involved in actin stress fiber formation and thus affects cell motility. We had previously observed ROCK-II is localized to the leading edge of colon cancer cells in histological samples. Since colon cancer cells invade through tissues composed of an extracellular matrix (ECM), we used type I collagen scaffolds to study how i)ROCK and ii) tissue density impacts spread of colon cancer cells. My results show that both ROCK-I and ROCK-II affect colon cancer invasion in different manners. ROCK-II is heavily expressed in colon cancer cells in ECM depleted regions of collagen. Much of ROCK-II was localized to structures consistent with invadopodia - a matrix degrading structure which aids cell invasion. ROCK-II knockdown reduced invasion of metastatic cells significantly by decreasing the expression of ECM-degrading proteins such as matrix-metalloproteinase-2 (MMP-2) and MMP-13 and reducing invadopodia. ROCK-II promotes epithelial-to-mesenchymal transition (EMT) by disrupting cell-cell junctions in malignant cells. In contrast, ROCK-I knockdown increased invasiveness of colon cancer cells in collagen I scaffolds. This observation can be explained by the fact that ROCK-I inhibition reduces focal adhesion formation. Additionally my results highlight the impact of tissue density on colon cancer progression. I show that an increase in cell density resulted in increased cell invasion and proliferation of metastatic colon cancer cells. Increasing ECM density also significantly increased cell proliferation. However, ROCK inhibition attenuated the effect of increasing ECM which resulted in a decrease in cell proliferation. My research has shown the different roles of each ROCK isoform in colon cancer. New therapies can be developed specifically targeting ROCK-II in order to reduce the impact of EMT and eventually, reduce colon cancer metastasis.