Abstract
Human pancreatic ductal adenocarcinoma (PDAC) is characterized by early systemic dissemination. Although RhoC has been implicated in cancer cell migration, the relevant underlying molecular mechanisms remain unknown. RhoC has been implicated in the enhancement of cancer cell migration and invasion, with actions which are distinct from RhoA (84% homology), and are possibly attributed to the divergent C-terminus domain. Here, we confirm that RhoC significantly enhances the migratory and invasive properties of pancreatic carcinoma cells. In addition, we show that RhoC over-expression decreases cancer cell adhesion and, in turn, accelerates cellular body movement and focal adhesion turnover, especially, on fibronectin-coated surfaces. Whilst RhoC over-expression did not alter integrin expression patterns, we show that it enhanced integrin α5β1 internalization and re-cycling (trafficking), an effect that was dependent specifically on the C-terminus (180-193 amino acids) of RhoC protein. We also report that RhoC and integrin α5β1 co-localize within the peri-nuclear region of pancreatic tumor cells, and by masking the CAAX motif at the C-terminal of RhoC protein, we were able to abolish this interaction in vitro and in vivo. Co-localization of integrin α5β1 and RhoC was demonstrable in invading cancer cells in 3D-organotypic cultures, and further mimicked in vivo analyses of, spontaneous human, (two distinct sources: operated patients and rapid autopsy programme) and transgenic murine (LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre), pancreatic cancers. In both cases, co-localization of integrin α5β1 and RhoC correlated with poor differentiation status and metastatic potential. We propose that RhoC facilitates tumor cell invasion and promotes subsequent metastasis, in part, by enhancing integrin α5β1 trafficking. Thus, RhoC may serve as a biomarker and a therapeutic target.
Highlights
The RhoA-like sub-family molecules of small GTPases (RhoA, RhoB and RhoC) share nearly 84% amino acid sequence homology, differing predominantly in their C-terminus domain [1]
When endogenous RhoC expression was downregulated, by small hairpin RNA interference (shRNA), in high expressors Panc0403 and HPAF (Figure S2D-F), we were able to diminish the migratory capacity of the cells, while re-introduction of wild-type RhoC partially restored this phenotype (Figure 3). These data demonstrate that the levels of endogenous RhoC protein can significantly influence the capacity of pancreatic cancer cells to migrate, which is in agreement with the reported role of RhoC in promoting migration in different types of cancers [25,26]
We observed that RhoC protein levels were dramatically elevated in cells which had migrated through the Transwell insert, regardless of whether RhoC was ectopically expressed, or its endogenous expression was knocked-down by shRNA (Panc0403 cells, Figure S4), suggesting that elevated RhoC protein levels enhanced cell migration
Summary
The RhoA-like sub-family molecules of small GTPases (RhoA, RhoB and RhoC) share nearly 84% amino acid sequence homology, differing predominantly in their C-terminus domain [1]. Until recently, this sequence homology has prevented specific causality being attributed to each Rho GTPase for their explicit roles in distinct biological functions [2,3,4]. We over-expressed wildtype full-length human RhoC cDNA (nRhoC), as well as its Cterminus-deleted (nDCT) or tagged (cRhoC) forms, in human pancreatic ductal adenocarcinoma (PDAC) cell lines to investigate the intracellular localization and downstream mechanisms during cell migration and invasion. In this report, a direct involvement of RhoC with trafficking and signaling of integrin α5β1 in invading pancreatic cancer cells
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