The small Rho family GTPase Rac1 plays an important role in pre-metastatic events like cell scattering, migration and invasion in several tumor types. Alternative splicing leads to the generation of the splice variant Rac1b, which includes the alternative exon 3b and was characterized as a constitutive active Rac1 isoform. Previous work of our group and collaboration partners revealed antagonistic activities of Rac1 and Rac1b in transforming growth factor beta (TGFbeta)-induced epithelial-to-mesenchymal transition (EMT) and tumor cell motility in pancreatic carcinoma cell lines. The studies of other groups have shown, that EMT is also fine-tuned by various splicing factors. Especially, the epithelial splicing regulatory proteins 1 (ESRP1) and 2 (ESRP2) were shown to regulate the alternative splicing of epithelialspecific proteins and were observed to be downregulated at the onset of EMT.In the course of this work, functional differences between the two GTPase isoforms Rac1 and Rac1b and their differential role in tumor cell invasion as well as EMT were analyzed. Initial experiments revealed substantial differences in Rac1b mRNA and protein amounts between various pancreatic and lung carcinoma cell lines, whereas Rac1 mRNA and proteins were expressed in comparable amounts. Subcellular fractionation and fluorescence microscopy analyses to determine the subcellular localization of Rac1 and Rac1b wildtype and mutant proteins revealed a predominantly membranous localization of wildtype Rac1b, which was comparable to constitutive active Rac1. In contrast, wildtype Rac1 showed a comparable cytoplasmic and membranous distribution. By applying the chorioallantoic membrane (CAM) model to study carcinoma cell invasion, a high heterogeneity in the invasive behaviors between all analyzed cell lines with different Rac1 and Rac1b protein expression levels was observed, indicating a more complex network of proteins and factors involved. Furthermore, CAM assays with H23 cell clones stably expressing EGFP-Rac1 and EGFP-Rac1b revealed an enhanced tumor cell scattering of invading H23/EGFP-Rac1 in comparison to H23/EGFP-Rac1b cells.Analysis of the mRNA and protein levels of different putative RAC1 splicing factors, both Rac1 isoforms and EMT-related factors revealed a relation in the mRNA and protein expression of Rac1b and ESRP1, a factor shown to be involved in exon 3b out-splicing, as well as ESRP2 and epithelial marker proteins. Furthermore, the siRNA-mediated depletion of Rac1b, ESRP1 and ESRP2 pointed toward a cooperation in the expression of ESRP2 and Rac1b. The TGFbeta1 stimulation of H358 lung carcinoma cells to induce EMT revealed a concomitant downregulation of Rac1b, ESRP1 and ESRP2 protein levels, thereby indicating a possible role of these factors in maintaining an epithelial phenotype.
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