THE RAC GTPase IN CANCER: FROM OLD CONCEPTS TO NEW PARADIGMS

Cancer associated mutations in Rho GTPases

Unlike Ras mutations, Rho GTPases were only recently recognized to be mutated in human cancer, as a result of large genomic sequencing studies. Nonetheless, the causal association between these mutations and disease progression remains to be fully determined in most cases. One of the most prominent driver mutations in Rac has been found in melanoma, specifically a hot spot in the RAC1 gene leading to missense mutations in P29, occurring with frequency of ~5% and up to 9% in chronically sun-exposed melanomas (4,19). Rac1^P29S, the most common mutation, leads to gain-of-function and consequently enhances downstream Rac signaling. This mutation confers resistance to Raf and MEK inhibitors, thus having significant clinical therapeutic significance (20). Interestingly, expression of Rac1^P29S in melanoma patients correlates with PD-L1 up-regulation, thus potentially contributing to suppression of an anti-tumor immune response (21). These findings could also be extended to other cancers since the Rac1^P29S hotspot mutation has been identified in head and neck, and endometrial cancers (22). A 5% incidence in Rac1 mutations (Rac1^G12V, Rac1^G12R, Rac1^P34R, Rac1^Q61R, and Rac1^Q61K) has been found in germ cell testicular tumors, which makes these tumors, along with melanoma, the cancer types with the highest incidence of Rac1 mutations reported to date (23). Gain-of-function mutants Rac1^A159V (paralogous to A146 mutants in KRas) and Rac1^Q61R (paralogous to Q61 mutants in KRas) have been also identified in head and neck, and prostate cancer, respectively (22). It should not be assumed that the same scenario is true for other Rho GTPases, since RhoA mutations found in gastric adenocarcinomas, head and neck cancer, and lymphomas could be either gain-of function (for example RhoA^Y42C) or inactivating (for example RhoA^G17V), further emphasizing the complex behavior of this GTPase in cancer, as described above (3,4). Discerning the functional significance of these mutations in the context of additional oncogenic and tumor suppressing alterations is vital to establish their utility as biomarkers, as well as their role in therapy responsiveness.

Anomalous Rac degradation and localization

Rac degradation is partially modulated by post-translational modifications. Ubiquitination by FBXL19 and HACE1 E3 ligases influences Rac expression levels (24,25). Consistent with the role of Rac in the control of NADPH oxidase complexes, deficiency in the tumor suppressor HACE1 increases reactive oxygen species (ROS) production, and this in turn leads to an addiction to Gln, a major nutrient source for tumor cells (26). Moreover, HACE1 down-regulation cooperates with ErbB2/HER2 overexpression in mammary cells to induce Rac1 hyperactivation, migration, malignant transformation, and tumorigenesis in vivo, effects that are sensitive to pharmacological Rac inhibition (27). A very recent report that identified cancer-associated missense mutations in HACE1 leading to defective Rac1 ubiquitination highlights the relevance of the control of Rac1 expression in cancer cell proliferation (28).

Spatial localization of Rho GTPases is key to fine-tune signaling outputs, and is tightly regulated by post-translational modifications and protein-interactions. In the case of Rac, prenylation of the C-terminal CAAX motif contributes to membrane redistribution from a cytosolic compartment (3). Abnormal localization of Rac, particularly in the nucleus, has been shown to occur in cancer cells. Increased nuclear Rac1 in tumor cells may be the result of an imbalance in the nucleocytoplasmic shuttling of this protein, causing a reduction in active cytoplasmic Rac1 and a concomitant elevation of active RhoA that favors actomyosin contractility required for cell invasion. In addition, nuclear Rac1 regulates actin polymerization at this cellular location, a function important for tumor cells as it confers the nuclear plasticity necessary for invasion (29). Adding more complexity to the role of nuclear Rac in cancer, a recent study demonstrated that deregulated Rac-GEF activity in tumor cells accumulates Rac in the nucleolus, which supports protein biosynthesis required for cancer cell growth (see below) (30).

Deregulation in Rac-GEF function

One of the best characterized mechanisms of Rho protein signaling deregulation involves the hyperactivation of GEFs, either by excessive upstream oncogenic activation or deregulated GEF expression/activity. The multiplicity of cellular outcomes regulated by the more than 70 identified Rho GEFs is dictated by their differential pattern of expression and selectivity for Rho proteins, as well as by the intricate mechanisms governing their activation. Various studies have linked enhanced Rac exchange activity, resulting from GEF overexpression, to an invasive and metastatic phenotype (5).

Among the many Rho family GEFs regulating Rac activity and implicated in cancer progression, Ect2, Tiam1, P-Rex and Vav family members stand as the most prominent ones associated with tumorigenesis and metastasis. Their anomalous expression and/or activation may impact patient outcome prediction and therapeutic management. For example, Vav3 overexpression in ovarian cancer associates with poor prognosis and confers resistance to established therapeutic regimes (31). Genetic alterations in the VAV1 gene, including activating mutations and fusions, have been found in peripheral T-cell lymphomas, establishing an oncogenic role for this GEF in the development of this disease (32).

The PI3K/Gβγ-regulated Rac-specific GEFs P-Rex1 and P-Rex2 have received significant attention in recent years as they are either overexpressed or mutated in cancer. P-Rex1 up-regulation occurs through enhanced gene expression, epigenetic deregulation, or increased protein stability (12,33–35). P-Rex1 overexpression in luminal breast cancer plays a fundamental role in integrating upstream signals emanating from tyrosine-kinase and G-protein-coupled receptors, and has been associated with metastasis in breast cancer patients. Indeed, lymph node metastasis and metastatic breast tumors display elevated P-Rex1-positive immunostaining (12). A particular role for ErbB3/HER3 signaling in CXCR4-mediated P-Rex1/Rac1 activation via hypoxia-inducible factor 1α (HIF-1α) has been recently described in luminal breast cancer models (36). Deletion of the PRex1 gene in a melanoma mouse model prevents metastatic dissemination (13). A potential role for P-Rex1 beyond solid tumors has been recently highlighted in acute myeloid leukemia (AML), particularly in AML cells harboring mutant Ras proteins (37). Whereas P-Rex1 mutations are infrequent, a high incidence of P-Rex2 point mutations has been described in melanoma (14%, third in prevalence after BRaf and NRas) (38). Although the biological consequences of the multiple P-Rex2 mutations, observed in melanoma and pancreatic cancer, have yet to be fully determined, a recent study has demonstrated enhanced Rac1-GEF activity and a pro-tumorigenic function of a truncated P-Rex2 mutant (39).

Ect2, a GEF that displays exchange activity on Rac, Rho, and Cdc42, plays an essential role in cell division. ECT2 gene co-amplification with PRKCI and SOX2 genes has been described in human tumors (40,41), although other mechanisms may also account for deregulated Ect2 expression. Targeted shRNA depletion of Ect2 from cancer cells impairs tumorigenic growth in a manner that is independent of its role in cytokinesis (40). Ect2 is required for the growth of stem-like tumor-initiating cells and thereby contributes to KRas-driven lung cancer (30). In normal cells, Ect2 is sequestered within the nucleus. Upon breakdown of the nuclear envelope, Ect2 diffuses throughout the cytoplasm and associates with the mitotic spindle, promoting cytokinesis via RhoA activation. In cancer cells, however, a significant fraction of Ect2 mislocalizes to the cytoplasm, where it promotes Rac activation and transformed growth via the MEK/Erk pathway (5,30,40,42,43). Furthermore, a recent model described the activation of Rac1 by Ect2 in the nucleolus of cancer cells, where it promotes the synthesis of ribosomal RNA (rRNA), a major component of the ribosomal machinery. rRNA and protein biosynthesis are key requirements for abnormal cancer cell proliferation, and rRNA biogenesis inhibitors are currently in clinical trials as anti-cancer agents (30,42). The recent intriguing finding that nuclear Tiam1 is a negative regulator of colorectal cancer cell proliferation and invasion via suppression of a TAZ/YAP genetic program, and that it serves as a good prognostic factor for colorectal cancer patients (44) highlights the complexities of Rac-GEF signaling in different cancer types. Overall, these new findings indicate that different subcellular pools of Rac controlled by specific Rac-GEFs play distinctive roles in cancer progression.

Deregulation of Rac-GAPs: opposite roles in cancer

Based on their roles as catalyzers of Rac mediated GTP hydrolysis, the general assumption has been that Rac-GAPs would act as tumor suppressors. Although cellular-based analyses consistently demonstrated anti-growth and anti-migratory properties for most Rac-GAPs, only a few studies identified tumor suppressor activities for these proteins in vivo. Furthermore, this view has been recently challenged by the identification of Rac-GAPs with unexpected oncogenic properties in certain cancers.

The Rac-specific GAP β2-chimaerin was originally identified as a potential tumor suppressor protein down-regulated in human glioblastoma (45). This GAP negatively regulates Rac-mediated processes such as cell cycle progression and migration, although the effects vary depending on whether cancer cells are in epithelial or mesenchymal stages (46–48). A recent study demonstrated that genetic ablation of the β2-chimaerin gene (Chn2) in the MMTV-Neu/ErbB2 mouse model of breast cancer accelerates tumor onset and increases the number of preneoplastic lesions. However, tumor progression is substantially delayed, and tumors in a β2-chimaerin-null background are less aggressive, unveiling a dual role for β2-chimaerin as a suppressor of tumor initiation and a promoter of tumor progression (48).

Reduced expression of SrGAP2 and SrGAP3, two members of the Slit-Robo GTPase-activating proteins (srGAPs), has been observed in human osteosarcomas and invasive ductal breast carcinomas, suggesting a tumor suppression function for these proteins. Notably, while down-regulation of SrGAP2 contributes to a more aggressive phenotype, most likely by enhancing cell migration, down-regulation of srGAP3 promotes Rac1-dependent, anchorage-independent cell growth (49,50).

The Rac-GAP ArhGAP24 (also known as FilGAP) is down-regulated in renal tumors, and this reduced expression correlates with poor survival. Conversely, ectopic overexpression of ArhGAP24 in renal cancer cells reverts their tumorigenic potential by inhibition of G1/S cell cycle transition, induction of apoptosis, and reduction of invasion (51). ArhGAP24 also has an important role in breast cancer metastasis, although contrasting results reported both pro- and anti-metastatic effects of this protein in triple negative breast cancer (TNBC). Most notably, inhibition of ArhGAP24 by RASAL2 (a Ras-GAP) enhances mesenchymal invasion by increasing Rac activity, a mechanism that is clinically relevant since high RASAL2 expression is predictive of poor outcome in TNBC patients (52).

The remarkable complexity of Rac-GAP function is also well illustrated by studies showing a pro-tumorigenic function for RacGAP1 (MgcRacGAP), whose elevated expression has been linked with aggressiveness in several human cancers and has been recently identified as a metastatic driver in uterine carcinosarcoma (53). Similarly, high expression of the Rac/Cdc42 GAP CdGAP correlates with poor prognosis in breast cancer patients, and its expression is particularly elevated in the basal breast cancer subtype. Interestingly, CdGAP is required for TGFβ and Neu/ErbB2-induced cell motility and invasion independently of its GAP activity. Through its proline-rich domain, CdGAP forms a functional complex with the transcription factor Zeb2, leading to repression of E-cadherin, and thus favoring epithelial-to-mesenchymal transition (EMT) (54). Another intriguing example is p190B RhoGAP, whose overexpression in the mammary gland results in Rac1 activation and enhanced ErbB2-driven tumorigenesis and metastasis (55). Thus, most probably the oncogenic activity of these GAP proteins involves functions independent of their ability to accelerate GTP hydrolysis from Rac.