GTPase splice variants RAC1 and RAC1B display isoform-specific differences in localization, prenylation, and interaction with the chaperone protein SmgGDS.

Identifying events that regulate the prenylation and localization of small GTPases will help define new strategies for therapeutic targeting of these proteins in disorders such as cancer, cardiovascular disease, and neurological deficits. Splice variants of the chaperone protein SmgGDS (encoded by RAP1GDS1) are known to regulate prenylation and trafficking of small GTPases. The SmgGDS-607 splice variant regulates prenylation by binding preprenylated small GTPases but the effects of SmgGDS binding to the small GTPase RAC1 versus the splice variant RAC1B are not well defined. Here we report unexpected differences in the prenylation and localization of RAC1 and RAC1B and their binding to SmgGDS. Compared to RAC1, RAC1B more stably associates with SmgGDS-607, is less prenylated, and accumulates more in the nucleus. We show that the small GTPase DIRAS1 inhibits binding of RAC1 and RAC1B to SmgGDS and reduces their prenylation. These results suggest that prenylation of RAC1 and RAC1B is facilitated by binding to SmgGDS-607 but the greater retention of RAC1B by SmgGDS-607 slows RAC1B prenylation. We show that inhibiting RAC1 prenylation by mutating the CAAX motif promotes RAC1 nuclear accumulation, suggesting that differences in prenylation contribute to the different nuclear localization of RAC1 versus RAC1B. Finally, we demonstrate RAC1 and RAC1B that cannot be prenylated bind GTP in cells, indicating that prenylation is not a prerequisite for activation. We report differential expression of RAC1 and RAC1B transcripts in tissues, consistent with these two splice variants having unique functions that might arise in part from their differences in prenylation and localization.

A Regulatory Axis between Epithelial Splicing Regulatory Proteins and Estrogen Receptor α Modulates the Alternative Transcriptome of Luminal Breast Cancer.

Epithelial splicing regulatory proteins 1 and 2 (ESRP1/2) control the splicing pattern during epithelial to mesenchymal transition (EMT) in a physiological context and in cancer, including breast cancer (BC). Here, we report that ESRP1, but not ESRP2, is overexpressed in luminal BCs of patients with poor prognosis and correlates with estrogen receptor α (ERα) levels. Analysis of ERα genome-binding profiles in cell lines and primary breast tumors showed its binding in the proximity of ESRP1 and ESRP2 genes, whose expression is strongly decreased by ERα silencing in hormone-deprived conditions. The combined knock-down of ESRP1/2 in MCF-7 cells followed by RNA-Seq, revealed the dysregulation of 754 genes, with a widespread alteration of alternative splicing events (ASEs) of genes involved in cell signaling, metabolism, cell growth, and EMT. Functional network analysis of ASEs correlated with ESRP1/2 expression in ERα+ BCs showed RAC1 as the hub node in the protein-protein interactions altered by ESRP1/2 silencing. The comparison of ERα- and ESRP-modulated ASEs revealed 63 commonly regulated events, including 27 detected in primary BCs and endocrine-resistant cell lines. Our data support a functional implication of the ERα-ESRP1/2 axis in the onset and progression of BC by controlling the splicing patterns of related genes.

The role of small GTPases of the Rho/Rac family in TGF-ß-induced EMT and cell motility in cancer.

This article focuses on the role of Rho family GTPases, particularly Rac1 and Rac1b in TGF-ß-induced epithelial-mesenchymal transition (EMT) and EMT-associated responses such as cell migration, invasion, and metastasis in cancer. EMT is considered a prerequisite for cells to adopt a motile and invasive phenotype and eventually become metastatic. A major regulator of EMT and metastasis in cancer is TGF-ß, and its specific functions on tumor cells are mediated beside Smad proteins and mitogen-activated protein kinases (MAPKs) by small GTPases of the Rho/Rac1 family. Available data point to extensive signaling crosstalk between TGF-ß and various Rho GTPases, and in particular a synergistic role of Rho and Rac1 during EMT and cell motility in normal and neoplastic epithelial cells. In contrast, the Rac1-related isoform, Rac1b, emerges as an endogenous inhibitor of Rac1 in TGF-ß signaling, at least in pancreatic carcinoma cells. Given the tumor-promoting role of TGF-ß in late-stage carcinomas and the intimate crosstalk of Rho/Rac1/Rac1b and TGF-ß signaling in various tumor cell responses, targeting specific Rho GTPases may allow for selective interference with prooncogenic TGF-ß responses to aid in anticancer treatments. Developmental Dynamics 247:451-461, 2018. © 2017 Wiley Periodicals, Inc.

Loss of neural crest-associated gene FOXD1 impairs melanoma invasion and migration via RAC1B downregulation.

Recent studies suggest that malignant melanoma heterogeneity includes subpopulations of cells with features of multipotent neural crest (NC) cells. Zebrafish and mouse models have shown that reactivation of neural crest-specific pathways during transformation determines the invasiveness of melanoma cells. In our study, we show that the neural crest-associated transcription factor FOXD1 plays a key role in the invasion and the migration capacities of metastatic melanomas both in vivo and in vitro. Gene expression profiling analysis identified both an upregulation of FOXD1 in NC and melanoma cells, as well as a downregulation of several genes related to cell invasion in FOXD1 knockdown cells, including MMP9 and RAC1B. Furthermore, we demonstrate that knockdown of RAC1B a tumor-specific isoform of RAC1, significantly impaired melanoma cell migration and invasion and could abrogate enhanced invasiveness induced by FOXD1 overexpression. We conclude that FOXD1 may influence invasion and migration via indirect regulation of MMP9 and RAC1B alternative splicing in melanoma cells.

Blockade of ARHGAP11A reverses malignant progress via inactivating Rac1B in hepatocellular carcinoma.

BACKGROUND: The molecular signaling events involving in high malignancy and poor prognosis of hepatocellular carcinoma (HCC) are extremely complicated. Blockade of currently known targets has not yet led to successful clinical outcome. More understanding about the regulatory mechanisms in HCC is necessary for developing new effective therapeutic strategies for HCC patients. METHODS: The expression of Rho GTPase-activating protein 11A (ARHGAP11A) was examined in human normal liver and HCC tissues. The correlations between ARHGAP11A expression and clinicopathological stage or prognosis in HCC patients were analyzed. ARHGAP11A was downregulated to determine its role in the proliferation, invasion, migration, epithelial-to-mesenchymal transition (EMT) development, and regulatory signaling of HCC cells in vitro and in vivo. RESULTS: ARHGAP11A exhibited high expression in HCC, and was significantly correlated with clinicopathological stage and prognosis in HCC patients. Moreover, ARHGAP11A facilitated Hep3B and MHCC97-H cell proliferation, invasion, migration and EMT development in vitro. ARHGAP11A knockdown significantly inhibited the in vivo growth and metastasis of HCC cells. Furthermore, ARHGAP11A directly interacted with Rac1B independent of Rho GTPase- activating activity. Rac1B blockade effectively interrupted ARHGAP11A-elicited HCC malignant phenotype. Meanwhile, upregulation of Rac1B reversed ARHGAP11A knockdown mediated mesenchymal-to-epithelial transition (MET) development in HCC cells. CONCLUSION: ARHGAP11A facilitates malignant progression in HCC patients via ARHGAP11A-Rac1B interaction. The ARHGAP11A/Rac1B signaling could be a potential therapeutic target in the clinical treatment of HCC.

Targeting Colon Cancers with Mutated BRAF and Microsatellite Instability.

The subgroup of colon cancer (CRC) characterized by mutation in the BRAF gene and high mutation rate in the genomic DNA sequence, known as the microsatellite instability (MSI) phenotype, accounts for roughly 10% of the patients and derives from polyps with a serrated morphology. In this review, both features are discussed with regard to therapeutic opportunities. The most prevalent cancer-associated BRAF mutation is BRAF V600E that causes constitutive activation of the pro-proliferative MAPK pathway. Unfortunately, the available BRAF-specific inhibitors had little clinical benefit for metastatic CRC patients due to adaptive MAPK reactivation. Recent contributions for the development of new combination therapy approaches to pathway inhibition will be highlighted. In addition, we review the promising role of the recently developed immune checkpoint therapy for the treatment of this CRC subtype. The MSI phenotype of this subgroup results from an inactivated DNA mismatch repair system and leads to frameshift mutations with translation of new amino acid stretches and the generation of neo-antigens. This most likely explains the observed high degree of infiltration by tumour-associated lymphocytes. As cytotoxic lymphocytes are already part of the tumour environment, their activation by immune checkpoint therapy approaches is highly promising.

Targeting the serrated pathway of colorectal cancer with mutation in BRAF.

A recently acknowledged morphological pathway to colorectal cancer originates from precursor polyps with a serrated appearance due to branching and folding of the colon epithelium. This serrated origin accounts for up to 30% of all colorectal tumors but these are heterogeneous regarding molecular characteristics and patient outcome. Here we review the current knowledge about the classification of this tumor subtype and its association with five key features: mutation status of the BRAF or KRAS genes, the CpG island methylation phenotype, microsatellite instability, immune cell infiltration, and overexpression of GTPase RAC1b. Subsequently, available therapeutic approaches for targeting these molecular characteristics are presented and critically discussed.

Epithelial splicing regulatory proteins 1 (ESRP1) and 2 (ESRP2) suppress cancer cell motility via different mechanisms.

ESRP1 (epithelial splicing regulatory protein 1) and ESRP2 regulate alternative splicing events associated with epithelial phenotypes of cells, and both are down-regulated during the epithelial-mesenchymal transition. However, little is known about their expression and functions during carcinogenesis. In this study, we found that expression of both ESRP1 and ESRP2 is plastic: during oral squamous cell carcinogenesis, these proteins are up-regulated relative to their levels in normal epithelium but down-regulated in invasive fronts. Importantly, ESRP1 and ESRP2 are re-expressed in the lymph nodes, where carcinoma cells metastasize and colonize. In head and neck carcinoma cell lines, ESRP1 and ESRP2 suppress cancer cell motility through distinct mechanisms: knockdown of ESRP1 affects the dynamics of the actin cytoskeleton through induction of Rac1b, whereas knockdown of ESRP2 attenuates cell-cell adhesion through increased expression of epithelial-mesenchymal transition-associated transcription factors. Down-regulation of ESRP1 and ESRP2 is thus closely associated with a motile phenotype of cancer cells.

The Roles of RAC1 and RAC1B in Colorectal Cancer and Their Potential Contribution to Cetuximab Resistance.

Colorectal cancer (CRC) is one of the most diagnosed cancers and a leading contributor to cancer-related deaths in the United States. Clinically, standard treatment regimens include surgery, radiation, and chemotherapy; however, there has been increasing development and clinical use of targeted therapies for CRC. Unfortunately, many patients develop resistance to these treatments. Cetuximab, the first targeted therapy approved to treat advanced CRC, is a monoclonal antibody that targets the epidermal growth factor receptor and inhibits downstream pathway activation to restrict tumor cell growth and proliferation. CRC resistance to cetuximab has been well studied, and common resistance mechanisms include constitutive signal transduction through downstream protein mutations and promotion of the epithelial-to-mesenchymal transition. While the most common resistance mechanisms are known, a proportion of patients develop resistance through unknown mechanisms. One protein predicted to contribute to therapy resistance is RAC1, a small GTPase that is involved in cytoskeleton rearrangement, cell migration, motility, and proliferation. RAC1 has also been shown to be overexpressed in CRC. Despite evidence that RAC1 and its alternative splice isoform RAC1B play important roles in CRC and the pathways known to contribute to cetuximab resistance, there is a need to directly study the relationship between RAC1 and RAC1B and cetuximab resistance. This review highlights the recent studies investigating RAC1 and RAC1B in the context of CRC and suggests that these proteins could play a role in resistance to cetuximab.

RAC1b Collaborates with TAp73α-SMAD4 Signaling to Induce Biglycan Expression and Inhibit Basal and TGF-ß-Driven Cell Motility in Human Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer type characterized by a marked desmoplastic tumor stroma that is formed under the influence of transforming growth factor (TGF)-ß. Data from mouse models of pancreatic cancer have revealed that transcriptionally active p73 (TAp73) impacts the TGF-ß pathway through activation of Smad4 and secretion of biglycan (Bgn). However, whether this pathway also functions in human PDAC cells has not yet been studied. Here, we show that RNA interference-mediated silencing of TAp73 in PANC-1 cells strongly reduced the stimulatory effect of TGF-ß1 on BGN. TAp73-mediated regulation of BGN, and inhibition of TGF-ß signaling through a (Smad-independent) ERK pathway, are reminiscent of what we previously observed for the small GTPase, RAC1b, prompting us to hypothesize that in human PDAC cells TAp73 and RAC1b are part of the same tumor-suppressive pathway. Like TAp73, RAC1b induced SMAD4 protein and mRNA expression. Moreover, siRNA-mediated knockdown of RAC1b reduced TAp73 mRNA levels, while ectopic expression of RAC1b increased them. Inhibition of BGN synthesis or depletion of secreted BGN from the culture medium reproduced the promigratory effect of RAC1b or TAp73 silencing and was associated with increased basal and TGF-ß1-dependent ERK activation. BGN also phenocopied the effects of RAC1b or TAp73 on the expression of downstream effectors, like the EMT markers E-cadherin, Vimentin and SNAIL, as well as on negative regulation of the ALK2-SMAD1/5 arm of TGF-ß signaling. Collectively, we showed that tumor-suppressive TAp73-Smad4-Bgn signaling also operates in human cells and that RAC1b likely acts as an upstream activator of this pathway.

SYTL5 Promotes Papillary Thyroid Carcinoma Progression by Enhancing Activation of the NF-κB Signaling Pathway.

The function and mechanism of SYTL5 in papillary thyroid carcinoma (PTC) are still unclear. In this research, we found that SYTL5 was significantly overexpressed in PTC tissues compared with normal thyroid tissues. SYTL5 downregulation significantly weakened the proliferative, migratory, and invasive abilities of PTC cells. In addition, upregulated SYTL5 could promote cancer progression by activating the NF-κB signaling pathway. RAC1b expression is positively associated with SYTL5, and overexpressed RAC1b abrogated the antitumor effect after SYTL5 inhibition. In conclusion, our findings identify the oncogenic role of SYTL5 in PTC by activation of the NF-κB signaling pathway, thus facilitating PTC development and progression.

Activation of a Ductal-to-Endocrine Transdifferentiation Transcriptional Program in the Pancreatic Cancer Cell Line PANC-1 Is Controlled by RAC1 and RAC1b through Antagonistic Regulation of Stemness Factors.

Epithelial-mesenchymal transition (EMT) is a driving force for tumor growth, metastatic spread, therapy resistance, and the generation of cancer stem cells (CSCs). However, the regained stem cell character may also be exploited for therapeutic conversion of aggressive tumor cells to benign, highly differentiated cells. The PDAC-derived quasimesenchymal-type cell lines PANC-1 and MIA PaCa-2 have been successfully transdifferentiated to endocrine precursors or insulin-producing cells; however, the underlying mechanism of this increased plasticity remains elusive. Given its crucial role in normal pancreatic endocrine development and tumor progression, both of which involve EMT, we analyzed here the role of the small GTPase RAC1. Ectopic expression in PANC-1 cells of dominant negative or constitutively active mutants of RAC1 activation blocked or enhanced, respectively, the cytokine-induced activation of a ductal-to-endocrine transdifferentiation transcriptional program (deTDtP) as revealed by induction of the NEUROG3, INS, SLC2A2, and MAFA genes. Conversely, ectopic expression of RAC1b, a RAC1 splice isoform and functional antagonist of RAC1-driven EMT, decreased the deTDtP, while genetic knockout of RAC1b dramatically increased it. We further show that inhibition of RAC1 activation attenuated pluripotency marker expression and self-renewal ability, while depletion of RAC1b dramatically enhanced stemness features and clonogenic potential. Finally, rescue experiments involving pharmacological or RNA interference-mediated inhibition of RAC1 or RAC1b, respectively, confirmed that both RAC1 isoforms control the deTDtP in an opposite manner. We conclude that RAC1 and RAC1b antagonistically control growth factor-induced activation of an endocrine transcriptional program and the generation of CSCs in quasimesenchymal PDAC cells. Our results have clinical implications for PDAC patients, who in addition to eradication of tumor cells have a need for replacement of insulin-producing cells.

The RNA-Binding Protein ESRP1 Modulates the Expression of RAC1b in Colorectal Cancer Cells.

RNA binding proteins are well recognized as critical regulators of tumorigenic processes through their capacity to modulate RNA biogenesis, including alternative splicing, RNA stability and mRNA translation. The RNA binding protein Epithelial Splicing Regulatory Protein 1 (ESRP1) can act as a tumor suppressor or promoter in a cell type- and disease context-dependent manner. We have previously shown that elevated expression of ESRP1 in colorectal cancer cells can drive tumor progression. To gain further insights into the pro-tumorigenic mechanism of action of ESRP1, we performed cDNA microarray analysis on two colorectal cells lines modulated for ESRP1 expression. Intriguingly, RAC1b was highly expressed, both at mRNA and protein levels, in ESRP1-overexpressing cells, while the opposite trend was observed in ESRP1-silenced CRC cells. Moreover, RAC1 and RAC1b mRNA co-immunoprecipitate with ESRP1 protein. Silencing of RAC1b expression significantly reduced the number of soft agar colonies formed by ESRP1-overexpressing cells, suggesting that ESRP1 acted, at least partially, through RAC1b in its tumor-promoting activities in CRC cells. Thus, our data provide molecular cues on targetable candidates in CRC cases with high ESRP1 expression.

The Ratio of RAC1B to RAC1 Expression in Breast Cancer Cell Lines as a Determinant of Epithelial/Mesenchymal Differentiation and Migratory Potential.

Breast cancer (BC) is a heterogenous disease encompassing tumors with different histomorphological phenotypes and transcriptionally defined subtypes. However, the non-mutational/epigenetic alterations that are associated with or causally involved in phenotype diversity or conversion remain to be elucidated. Data from the pancreatic cancer model have shown that the small GTPase RAC1 and its alternatively spliced isoform, RAC1B, antagonistically control epithelial-mesenchymal transition and cell motility induced by transforming growth factor ß. Using a battery of established BC cell lines with either a well-differentiated epithelial or poorly differentiated mesenchymal phenotype, we observed subtype-specific protein expression of RAC1B and RAC1. While epithelial BC lines were RAC1Bhigh and RAC1low, mesenchymal lines exhibited the reverse expression pattern. High RAC1B and/or low RAC1 abundance also correlated closely with a poor invasion potential, and vice versa, as revealed by measuring random cell migration (chemokinesis), the preferred mode of cellular movement in cells that have undergone mesenchymal transdifferentiation. We propose that a high RAC1B:RAC1 ratio in BC cells is predictive of an epithelial phenotype, while low RAC1B along with high RAC1 is a distinguishing feature of the mesenchymal state. The combined quantitative assessment of RAC1B and RAC1 in tumor biopsies of BC patients may represent a novel diagnostic tool for probing molecular subtype and eventually predict malignant potential of breast tumors.

Ibuprofen disrupts a WNK1/GSK3ß/SRPK1 protein complex required for expression of tumor-related splicing variant RAC1B in colorectal cells.

A major risk factor promoting tumor development is chronic inflammation and the use of nonsteroidal anti-inflammatory drugs (NSAID), including ibuprofen, can decrease the risk of developing various types of cancer, including colorectal cancer (CRC). Although the molecular mechanism behind the antitumor properties of NSAIDs has been largely attributed to inhibition of cyclooxygenases (COXs), several studies have shown that the chemopreventive properties of ibuprofen also involve multiple COX-independent effects. One example is its ability to inhibit the alternative splicing event generating RAC1B, which is overexpressed in a specific subset of BRAF-mutated colorectal tumors and sustains cell survival. Here we describe the mechanism by which ibuprofen prevents RAC1B alternative splicing in a BRAF mutant CRC cell line: it leads to decreased translocation of SRPK1 and SRSF1 to the nucleus and is regulated by a WNK1/GSK3ß/SRPK1 protein kinase complex. Surprisingly, we demonstrate that ibuprofen does not inhibit the activity of any of the involved kinases but rather promotes disassembly of this regulatory complex, exposing GSK3ß serine 9 to inhibitory phosphorylation, namely by AKT, which results in nuclear exclusion of SRPK1 and SRSF1 hypophosphorylation. The data shed new light on the biochemical mechanisms behind ibuprofen's action on alternative spliced RAC1B and may support its use in personalized approaches to CRC therapy or chemoprevention regimens.

RAC1B Regulation of TGFB1 Reveals an Unexpected Role of Autocrine TGFß1 in the Suppression of Cell Motility.

Autocrine transforming growth factor (TGF)ß has been implicated in epithelial-mesenchymal transition (EMT) and invasion of several cancers including pancreatic ductal adenocarcinoma (PDAC) as well as triple-negative breast cancer (TNBC). However, the precise mechanism and the upstream inducers or downstream effectors of endogenous TGFB1 remain poorly characterized. In both cancer types, the small GTPase RAC1B inhibits cell motility induced by recombinant human TGFß1 via downregulation of the TGFß type I receptor, ALK5, but whether RAC1B also impacts autocrine TGFß signaling has not yet been studied. Intriguingly, RNA interference-mediated knockdown (RNAi-KD) or CRISPR/Cas-mediated knockout of RAC1B in TGFß1-secreting PDAC-derived Panc1 cells resulted in a dramatic decrease in secreted bioactive TGFß1 in the culture supernatants and TGFB1 mRNA expression, while the reverse was true for TNBC-derived MDA-MB-231 cells ectopically expressing RAC1B. Surprisingly, the antibody-mediated neutralization of secreted bioactive TGFß or RNAi-KD of the endogenous TGFB1 gene, was associated with increased rather than decreased migratory activities of Panc1 and MDA-MB-231 cells, upregulation of the promigratory genes SNAI1, SNAI2 and RAC1, and downregulation of the invasion suppressor genes CDH1 (encoding E-cadherin) and SMAD3. Intriguingly, ectopic re-expression of SMAD3 was able to rescue Panc1 and MDA-MB-231 cells from the TGFB1 KD-induced rise in migratory activity. Together, these data suggest that RAC1B favors synthesis and secretion of autocrine TGFß1 which in a SMAD3-dependent manner blocks EMT-associated gene expression and cell motility.

The Small GTPase RAC1B: A Potent Negative Regulator of-and Useful Tool to Study-TGFß Signaling.

RAC1 and its alternatively spliced isoform, RAC1B, are members of the Rho family of GTPases. Both isoforms are involved in the regulation of actin cytoskeleton remodeling, cell motility, cell proliferation, and epithelial-mesenchymal transition (EMT). Compared to RAC1, RAC1B exhibits a number of distinctive features with respect to tissue distribution, downstream signaling and a role in disease conditions like inflammation and cancer. The subcellular locations and interaction partners of RAC1 and RAC1B vary depending on their activation state, which makes RAC1 and RAC1B ideal candidates to establish cross-talk with cancer-associated signaling pathways-for instance, interactions with signaling by transforming growth factor ß (TGFß), a known tumor promoter. Although RAC1 has been found to promote TGFß-driven tumor progression, recent observations in pancreatic carcinoma cells surprisingly revealed that RAC1B confers anti-oncogenic properties, i.e., through inhibiting TGFß-induced EMT. Since then, an unexpected array of mechanisms through which RAC1B cross-talks with TGFß signaling has been demonstrated. However, rather than being uniformly inhibitory, RAC1B interacts with TGFß signaling in a way that results in the selective blockade of tumor-promoting pathways, while concomitantly allowing tumor-suppressive pathways to proceed. In this review article, we are going to discuss the specific interactions between RAC1B and TGFß signaling, which occur at multiple levels and include various components such as ligands, receptors, cytosolic mediators, transcription factors, and extracellular inhibitors of TGFß ligands.

RAC1B Induces SMAD7 via USP26 to Suppress TGFß1-Dependent Cell Migration in Mesenchymal-Subtype Carcinoma Cells.

The small GTPase RAC1B has been shown to act as a powerful inhibitor of the transforming growth factor (TGF)ß type I receptor ALK5 and TGFß1/ALK5-induced epithelial-mesenchymal transition and cell motility. However, the precise mechanism has remained elusive. RNAi-mediated knockdown of RAC1B in the pancreatic ductal adenocarcinoma (PDAC)-derived cell line Panc1 failed to alter transcriptional activity from a transfected ALK5 promoter-reporter construct. In contrast, pharmacological inhibition of the proteasome decreased the abundance of ALK5 protein in cell lines of the mesenchymal subtype (Panc1, IMIM-PC-1, and breast cancer MDA-MB-231), but not in a PDAC cell line of the epithelial subtype (Colo357). Here, we focused on the inhibitory Smad protein, SMAD7, as a potential candidate for RAC1B-mediated inhibition of cell migration. In Panc1 cells devoid of RAC1B, SMAD7 protein was dramatically reduced and these cells were refractory to TGFß1-induced upregulation of SMAD7 protein but not mRNA expression. Intriguingly, RNAi-mediated knockdown or ectopic overexpression of SMAD7 in Panc1 cells up- or downregulated, respectively, ALK5 protein expression and mimicked the suppressive effect of RAC1B on TGFß/SMAD3-dependent transcriptional activity, target gene expression and cell migration. Transfection of SMAD7 was further able to partially rescue cells from the RAC1B knockdown-mediated increase in migratory properties. Conversely, knockdown of SMAD7 was able to partially rescue Panc1 and MDA-MB-231 cells from the antimigratory effect of ectopically expressed RAC1B. Finally, we demonstrate that RAC1B upregulation of SMAD7 protein requires intermittent transcriptional induction of the deubiquitinating enzyme USP26. Our data suggest that RAC1B induces SMAD7 by promoting its deubiquitination and establishes this Smad as one of RAC1B's downstream effectors in negative regulation of ALK5 and TGFß1-induced cell migration in mesenchymal-type carcinoma cells.

RAC1B: A Rho GTPase with Versatile Functions in Malignant Transformation and Tumor Progression.

RAC1B is an alternatively spliced isoform of the monomeric GTPase RAC1. It differs from RAC1 by a 19 amino acid in frame insertion, termed exon 3b, resulting in an accelerated GDP/GTP-exchange and an impaired GTP-hydrolysis. Although RAC1B has been ascribed several protumorigenic functions such as cell cycle progression and apoptosis resistance, its role in malignant transformation, and other functions driving tumor progression like epithelial-mesenchymal transition, migration/invasion and metastasis are less clear. Insertion of exon 3b endows RAC1B with specific biochemical properties that, when compared to RAC1, encompass both loss-of-functions and gain-of-functions with respect to the type of upstream activators, downstream targets, and binding partners. In its extreme, this may result in RAC1B and RAC1 acting in an antagonistic fashion in regulating a specific cellular response with RAC1B behaving as an endogenous inhibitor of RAC1. In this review, we strive to provide the reader with a comprehensive overview, rather than critical discussions, on various aspects of RAC1B biology in eukaryotic cells.

RAC1B Suppresses TGF-ß1-Dependent Cell Migration in Pancreatic Carcinoma Cells through Inhibition of the TGF-ß Type I Receptor ALK5.

The small GTPase Ras-related C3 botulinum toxin substrate 1B (RAC1B) has been shown previously by RNA interference-mediated knockdown (KD) to function as a powerful inhibitor of transforming growth factor (TGF)-ß1-induced cell migration and epithelial-mesenchymal transition in epithelial cells, but the underlying mechanism has remained enigmatic. Using pancreatic carcinoma cells, we show that both KD and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated knockout (KO) of RAC1B increased the expression of the TGF-ß type I receptor ALK5 (activin receptor-like kinase 5), but this effect was more pronounced in CRISPR-KO cells. Of note, in KO, but not KD cells, ALK5 upregulation was associated with resensitization of TGFBR1 to induction by TGF-ß1 stimulation. RAC1B KO also increased TGF-ß1-induced C-terminal SMAD3 phosphorylation, SMAD3 transcriptional activity, growth inhibition, and cell migration. The KD of ALK5 expression by RNA interference or inactivation of the ALK5 kinase activity by dominant-negative interference or ATP-competitive inhibition rescued the cells from the RAC1B KD/KO-mediated increase in TGF-ß1-induced cell migration, whereas the ectopic expression of kinase-active ALK5 mimicked this RAC1B KD/KO effect. We conclude that RAC1B downregulates the abundance of ALK5 and SMAD3 signaling, thereby attenuating TGF-ß/SMAD3-driven cellular responses, such as growth inhibition and cell motility.