Abstract 3160: ROCK inhibition promotes microtentacle formation and reattachment of breast cancer cells

Abstract

Abstract Circulating Tumor Cells (CTCs) predict poor patient outcome and increased CTC detection is correlated with higher risk of metastasis. The efficiency of cancers to metastasize depends on the survival and reattachment of CTCs in distant tissues. Our lab has demonstrated the significance of microtubule based cellular extensions, termed microtentacles, in enhancing CTC aggregation and reattachment to the endothelial wall. Microtentacles (McTNs) are highly dynamic protrusions formed in free-floating suspended cells and occur at higher frequencies in more metastatic cell lines. Recently, there has been interest generated in using ROCK as a therapeutic target in cancers based on emerging data that over-activation of Rho/ROCK pathway plays a role in tumor development and metastasis. ROCK inhibitors are being considered for clinical trials because of their pre-clinical success in reducing tumor cell migration, invasion and metastasis. However, the effect of Rho/ROCK inhibition on CTC reattachment and metastasis has not been well established. ROCK promotes cellular contraction by phosphorylating non-muscle myosin II (NMII), allowing it to crosslink actin, as well as by phosphorylating and inhibiting the activity of Myosin Light Chain Phosphatase (MLCP). In a normal, noncancerous cell, the outward expansion of microtubules is balanced by the inward contraction of the actin cortex. ROCK inhibition decreases cell contractility and shifts the balance towards a looser, more relaxed actin cortex that is unable to contain the outward growth of microtubules. Western blot analysis of attached and suspended cells treated with a ROCK inhibitor showed a decrease in phosphorylated MLCP and NMII in the breast cancer cell lines, BT549 and Hs578T. Treatment with ROCK inhibitor significantly increased the number of cells expressing McTNs. The increase in McTNs corresponded with increased in vitro cell reattachment, measured by the xCelligence, Real-Time Cell Analyser. Knocking down ROCK1 and ROCK2 with silencing RNAs also resulted in increased McTN counts. Conversely, increasing cell contractility by treating cells with a Rho pathway activator led to a reduction in McTNs and cell attachment. These results show that ROCK inhibitors could actually promote metastasis by increasing attachment of CTCs to the vasculature. A better understanding of the effect of cancer therapies on all stages of metastasis will prevent the use of drugs that could reduce tumor growth and motility in the short-term but result in increasing circulating tumor cells and elevating metastatic potential in the long-term. Citation Format: Lekhana Bhandary, Michele I. Vitolo, Rebecca A. Bettes, Monica S. Charpentier, Amanda E. Boggs, Jana Slovic, Keyata Thompson, Stuart S. Martin. ROCK inhibition promotes microtentacle formation and reattachment of breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3160. doi:10.1158/1538-7445.AM2014-3160