Using single-cell tracing technique to elucidate SLK-ROCK interaction in cancer cell migration

• Main Authors: Cheng-Yu Fan, 范誠祐
• Other Authors: 蔡丰喬
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/3vkr6h

碩士 === 國立臺灣大學 === 藥理學研究所 === 106 === Cell migration is a fundamental process in embryonic development, tissue repair and cancer metastasis. Previous studies have identified many genes regulating cell migration, but how these genes interact with each other during cell migration remains unclear. We therefore conducted a “two-hit” screen using shRNAs combined with small molecule inhibitors and scratch wound healing assays in endothelial cells, to identify potential synergistic or antagonistic effects among signaling molecules during cell migration. The screen identified Ste20-like kinase (SLK), a serine-threonine kinase. Its knockdown together with ROCK inhibitor Y27632 drastically increased HUVEC migration. We therefore hypothesized that SLK interacts with ROCK signaling during cell migration. To test if the SLK-ROCK interaction is an universal phenomenon across cell types, we tested the head and neck cancer cells (SAS cell line) on the above platform. In contrast to HUVEC cells, knockdown of SLK in cancer cell line SAS decreased its migration speed, suggesting that SLK might not primarily affect cell motility to change cell migration. We thus performed single-cell tracing techniques to analyze traces of individual moving cell in scratch wound healing assays, showing that SLK knockdown disrupted the migration polarity of SAS cells while SLK over-expression moderately enhanced cell polarity. Together with our observation and previous reports that SLK was accumulated at the migrating cell front, we propose that SLK accumulates at the leading edge of the migrating cell to regulate cell polarity. We further explored the interaction between SLK and ROCK using single-cell tracing. Interestingly, ROCK inhibition reversed the decreased motility caused by SLK knockdown, supporting the idea of interaction between these two molecules. We also noticed that SLK knockdown reversed the effect of RhoA knockdown on cell motility and focal adhesion, and that ROCK inhibition totally abolished focal adhesion formation. All above indicate a pivotal role of SLK on the RhoA-ROCK axis during cell migration. Moreover, we found that ROCK inhibition significantly reduced cell coordination, which probably proceed through the regulation of adherens junctions during cell migration. Hence, SLK and ROCK may also interact with each other by regulating different cell migration modules (polarity vs. coordination.) Taken together, our results indicate that (1) SLK accumulates at the leading edge of migrating cells regulating cell migration polarity, that (2) SLK regulates the RhoA-ROCK axis during cell migration, and that (3) SLK may also interact with ROCK via modular collaboration (polarity vs. coordination.) Detailed mechanistic investigation is still under the way. Through these work we will elucidate how SLK interacts with ROCK signaling during cell migration and develop new pharmacological strategies to treat cell migration-related diseases.