Abstract 5060: Myosin-IIA heavy chain phosphorylation on S1943 regulates tumor cell invasion

Abstract

Abstract Nonmuscle myosin IIA (NMHC-IIA) heavy chain phosphorylation has been shown to regulate motility and chemotaxis in invasive breast cancer cells; phosphorylation on S1943 promotes myosin-IIA filament disassembly in vitro and enhances two-dimensional cell migration. To test the role of NMHC-IIA S1943 phosphorylation in regulating the invasive properties of tumor cells, we produced MDA-MB-231 breast cancer cells in which the endogenous NMHC-IIA was stably knocked down via shRNA and replaced with exogenously expressed murine GFP-tagged wild type, phosphomimetic (S1943E) or non-phosphorylatable (S1943A) NMHC-IIA. Since previous studies have shown that the matrix-degrading activity of invadopodia is regulated via a myosin II-FAK-Cas pathway (Alexander et al, Curr Biol 18:1295, 2009), we examined the ability of MDA-MB-231 cells expressing wild type, S1943E or S1943A NMHC-IIA to form invadopodia and degrade matrix. Only 39.4% of S1943A NMHC-IIA expressing cells exhibited matrix degradation as compared to wild type NMHC-IIA (90.5%) and S1943E NMHC-IIA (90.3%). In addition, the average percent degradation area for cells expressing S1943A NMHC-IIA was 15-fold lower than for cells expressing wild type NMHC-IIA, whereas S1943E NMHC-IIA cells exhibited a 2-fold increase in degradation area. There was no significant difference in the numbers of immature invadopodia between the three cell lines. However, S1943A NMHC-IIA cells had significantly fewer mature invadopodia, while S1943E NMHC-IIA cells had increased numbers of mature invadopodia compared to wild type NMHC-IIA cells. To investigate the consequences of altered invadopodia maturation and activity, we examined the secretion of MMP-9 from cells expressing wild type, S1943E or S1943A NMHC-IIA. Gelatin zymography and MMP-9 activity assays of conditioned medium showed that S1943E NMHC-IIA expression enhanced MMP-9 secretion by 2-fold, whereas S1943A NMHC-IIA expression reduced MMP-9 secretion by 4-fold. In experimental metastasis assays in SCID mice, S1943A NMHC-IIA cells rarely formed lung metastases as compared to wild type NMHC-IIA cells, which formed poorly differentiated metastatic foci with some pleural localization. The S1943E NMHC-IIA cells also produced poorly differentiated carcinomas, which primarily localized along the pleural lining. Overall, S1943A NMHC-IIA cells produced approximately 2-fold fewer and S1943E NMHC-IIA cells produced 2-fold greater metastases than wild type NMHC-IIA cells. Together, these observations indicate that NMHC-IIA S1943 phosphorylation contributes to tumor cell invasion and metastasis via the regulation of extracellular matrix degradation. Citation Format: Laura E. Norwood Toro, Jonathan M. Backer, Anne R. Bresnick. Myosin-IIA heavy chain phosphorylation on S1943 regulates tumor cell invasion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5060.