S100A4 regulation and function

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S100A4 is a calcium binding and tumor associated protein. Despite its critical roles in multiple aspects of cancer progression and non-malignant disease development, the investigations on the regulation of S100A4 are limited. This dissertation focuses on exploring the mechanisms of S100A4 regulation by integrin α6β4 in breast cancer cells, and hyperosmotic stress response in colon cancer cells, as well as the functional aspects of S100A4 in these two models. Using breast cancer cell lines, I found that S100A4 is upregulated by integrin α6β4 and its expression correlates well with integrin α6β4 expression. Using siRNA, promoter analysis and chromatin immunoprecipitation (ChIP), I demonstrate that S100A4 is regulated by NFAT5 in breast cancer cells. To study the regulation of S100A4 by NFAT5 in a more readily inducible model, I used colon cancer cells under hyperosmotic stress as the model. I found that the osmotic stress response elements are located in the first intron region of S100A4 by luciferase reporter assays. Inhibition of Src kinase pathways reduced S100A4 induction by affecting NFAT5 transactivation and protein levels. Inhibition of DNA methyltransferases stimulated expression of S100A4 in cells lacking the α6β4 integrin, whereas demethylation inhibitors suppressed expression in α6β4 integrin expressing cells. Alterations in DNA methylation were confirmed by bisulfate sequencing, thus suggesting that integrin α6β4 signaling can lead to the demethylation of select promoters. Suppression of S100A4 by siRNA leads to decreased chemoinvasion of breast cancer cells and cell viability of colon cancer cells under hyperosmotic stress. The data suggest that NFAT5 in conjunction with DNA demethylation are important components of S100A4 regulation. The interaction of S100A4 with cytoskeleton proteins suggests that S100A4 could be a potential regulator of actin cytoskeleton reorganization. Using GST pull-down and immunoprecipitation assays, I demonstrate that Rhotekin is a direct and specific partner of S100A4. Confocal microscopy showed that S100A4 co-localizes with Rhotekin. Using RNAi, I found that suppression of both S100A4 and Rhotekin leads to the loss of Rho-dependent membrane ruffling and a reduction in invasive growth in three-dimensional culture. My data suggest that interaction of S100A4 and Rhotekin alters the functional output of Rho signaling to confer an invasive phenotype in breast cancer cells. In summary, this dissertation highlights the novel regulation and signaling of S100A4. Further elucidating the mechanism of the underlying function will help us to develop a potential therapeutic target for cancer metastasis.

S100A4, NFAT, osmotic stress, cancer metastasis, RhoGTPase, Rhotekin, actin reorganization,breast cancer, colon cancer, epigenetic regulation, DNA methylation,migration, invasion