P-Rex in Cancer

Published OnlineFirst June 10, 2013; DOI: 10.1158/1078-0432.CCR-12-1662

Clinical Cancer Molecular Pathways Research

Molecular Pathways: P-Rex in Cancer

Atanasio Pandiella and Juan Carlos Montero

Abstract P-Rex proteins are Rho/Rac guanine nucleotide exchange factors that participate in the regulation of several cancer-related cellular functions such as proliferation, motility, and invasion. Expectedly, a significant portion of these actions of P-Rex proteins must be related to their Rac regulatory properties. In addition, P-Rex proteins control signaling by the phosphoinositide 3-kinase (PI3K) route by interacting with PTEN and mTOR. The interaction with PTEN inhibits its phosphatase activity, leading to AKT activation. The interaction with mTOR may be important in nutrient-stimulated Rac activation and migration. In humans, several studies have implicated P-Rex proteins in the pathophysiology of various neoplasias. Thus, overexpression of P-Rex proteins has been linked to poor patient outcome in breast cancer and may facilitate metastatic dissemination of prostate cancer cells. In addition, whole-genome sequencing described P-Rex2 as a significantly mutated gene in melanoma. Furthermore, expression in melanocytes of mutated forms of P-Rex2 found in patients with melanoma showed the protumorigenic role of these P-Rex mutations in melanoma genesis. These findings open interesting opportunities for P-Rex targeting in cancer. Moreover, the implication of P-Rex partner proteins such as Rac, mTOR, or PTEN in cancer has opened the possibility of acting on P-Rex to restrict protumorigenic signaling through these pathways. Clin Cancer Res; 19(17); 1–6. 2013 AACR.

Background was named P-Rex1 for PIP3-dependent Rac exchanger. Up P-Rex proteins have recently been shown to be involved to six different P-Rex proteins were derived from two genes, PREX1 PREX2 PREX1 in the pathophysiology of several neoplasias, opening the and (9). Transcription from the gene possibility of their targeting with therapeutic purposes may generate three different P-Rex1 protein isoforms of 186 (1). P-Rex proteins belong to the large family of guanine (isoform 1), 110 (isoform 2), and 175 kDa (isoform 3; Fig. PREX2 nucleotide exchange factors (GEF), which act on the Rho/ 1A). The gene generates three isoforms of 182 (also Rac family of GTPases (2, 3). Rho/Rac proteins play known as P-Rex2a), 171, and 111 kDa (also known as P- important roles in several physiologic processes, and have Rex2b; ref. 9). also been implicated in neoplastic transformation and P-Rex proteins contain an N-terminal Dbl-homology metastatic dissemination (4). Expectedly, many of the (DH) domain, the typical catalytic domain of the GEFs that roles of P-Rex proteins in cellular physiology must be act on the Rho/Rac family of GTPases (Fig. 1A). P-Rex due to their Rac regulatory properties. In addition, P-Rex proteins also include a pleckstrin homology (PH) domain, proteins may also control signaling by the phosphoinosi- as well as two pairs of DEP and PDZ domains. The C- tide 3-kinase (PI3K) route (5, 6), and carry out Rac- terminal region includes a domain homologous to inositol independent actions (7). In this article we review the polyphosphate 4-phosphatase (IP4P; 8, 10), which is functions of P-Rex proteins and the potential relevance of absent in P-Rex2b (11). their targeting in cancer. Physiologic relevance of P-Rex proteins P-Rex genes and protein isoforms P-Rex1 is mainly expressed in peripheral blood leuko- The first member of the P-Rex family, P-Rex1, was iden- cytes and in the brain and is also expressed in several other tified in a search for phosphatidylinositol-(3,4,5)-trisphos- tissues (8, 11–14). P-Rex2 is widely expressed and is strong- pahate (PIP3)-activated Rac GEFs (8). The isolated protein ly present in skeletal muscle, the small intestine, heart, lung, and placenta, being absent from peripheral blood leukocytes (10, 11, 13). Authors' Affiliation: Instituto de Biología Molecular y Celular del Cancer, P-Rex1 / mice are viable and apparently healthy but Centro de Investigacion del Cancer, CSIC-Universidad de Salamanca, Spain have mild neutrophilia (15). In the neutrophils of these mice, the G protein–coupled receptor (GPCR)-dependent Corresponding Author: Atanasio Pandiella, Instituto de Biología Molec- ular y Celular del Cancer-CIC, Campus Miguel de Unamuno, Salamanca Rac activation and recruitment to inflammatory sites were 37007, Spain. Phone: 34-923-294815; Fax: 34-923-294743; E-mail: impaired (15, 16). In leukocytes, P-Rex1 has been involved [email protected] in integrin-mediated rolling and intravascular crawling doi: 10.1158/1078-0432.CCR-12-1662 (17). In lung microvascular endothelial cells, P-Rex1 par- 2013 American Association for Cancer Research. ticipated in TNF-a–stimulated Rac activation and ROS

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A Rac-GEF activity PIP3 binding Gβγ binding PTEN binding Interaction with PP1α

Interaction with mTOR

DH PH DEP PDZ InsPx 4-phosphatase aa 1659 P-Rex1 isoform 1

aa 956 P-Rex1 isoform 2

aa 1561 P-Rex1 isoform 3

aa 1606 P-Rex2 isoform 1 (P-Rex2a)

aa 1504 P-Rex2 isoform 2

aa 979 P-Rex2 isoform 3 (P-Rex2b)

B Ligand

GPCR RTK Extracellular

PIP3 PIP2 P P Intracellular Gα PTEN p85 γ Akt p110 β (active) P mTOR P PI3K

P-Rex Kinases Rac Rac ON Phosphatases GDP GTP Kinases (PP1α) (PKA) PP Metastasis Invasion Migration P Motility Proliferation P-Rex Adhesion Off

Figure 1. A, schematic representation of the different P-Rex protein isoforms. The different domains and the number of residues of the different proteins are shown. The properties of different regions in P-Rex, as well as proteins interacting with such regions, are described. B, regulation of P-Rex function. Underresting conditions, P-Rex proteins are located in the cytosol. In these conditions, phosphorylation at inhibitory residues (red) restricts P-Rex activity.Phosphorylationat these inhibitory residues may be mediated by kinases such as protein kinase A (PKA). Upon stimulation of GPCR or receptor tyrosine kinases (RTK), P-Rexmay translocate to the plasma membrane, an effect facilitated by the interaction between the PH domain of P-Rex and PIP3. Interaction between Gbg subunits of G proteins and the GEF domain of P-Rex may also favor membrane translocation of P-Rex. Receptor activation also causes dephosphorylation of inhibitory residues, together with phosphorylation of other activatory residues (green). This results in upregulation of P-Rex GEF function, causing release of GDP from Rac and loading of Rac with GTP. In addition, P-Rex proteins may interact and regulate the function of several key components of the PI3K pathway, such as mTOR or PTEN. The interaction of mTOR with P-Rex proteins occurs through the DEP domains of the latter, and regulates nutrient-stimulated Rac activation and cell migration. The interaction of P-Rex proteins with PTEN inhibits the phosphatase activity of the latter, increasing the amount of active AKT. The composite actions of P-Rex on various signaling pathways contribute to cancer-related functions of P-Rex, such as cell proliferation, migration, or invasion.

production (18). Removal of P-Rex1 signiﬁcantly reduced Rex1 knockout mice were also refractory to lung vascular neutrophilic transendothelial migration and leukocyte hyperpermeability and edema in a lipopolysaccharide- sequestration in TNF-a–challenged mouse lungs. The P- induced sepsis model. P-Rex2b also seems to be relevant

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P-Rex in Cancer

in the regulation of Rac1 activation and cell migration of Mutagenesis experiments showed that phosphorylation of endothelial cells (19). Ser1169 facilitated P-Rex1 GEF function. Therefore, stimu- P-Rex proteins are widely expressed in the nervous system lation of ErbB receptors activates a phosphorylation/ (13) and have been involved in neuronal migration (14), dephosphorylation cycle of P-Rex1, which results in differentiation (7), and memory-related functions (20). dephosphorylation of inhibitory residues (Ser313, Ser319) Transfection of P-Rex1 inhibited neurite sprouting in PC12 and phosphorylation of stimulatory residues (Ser605, cells (7). Moreover, expression of an N-terminal truncated Ser1169) to control the GEF activity of P-Rex1. form of P-Rex had the same action as full-length P-Rex1, suggesting that the neurite-regulating functions of P-Rex1 The PI3K/mTOR route and P-Rex proteins may be partially independent of its Rac regulatory proper- Of particular relevance is the interplay between the ties (7). On the other hand, expression of a mutant lacking PI3K route and P-Rex proteins. P-Rex1 was initially the DH domain, which acts as a dominant-negative form described as a Rac GEF upmodulated by the bg subunits with respect to the GEF activity of P-Rex1, decreased neu- of heterotrimeric G proteins and by PIP3, which is gen- ronal migration induced by neurotrophic factors (14). With erated by PI3K (8). P-Rex proteins interact with mTOR respect to P-Rex2, mice lacking the PREX2 gene are viable and PTEN, two important players in PI3K signal trans- and fertile but develop a mild motor coordination defect duction (5, 6). The interaction of P-Rex1 and P-Rex2 with and have alterations of the Purkinje cell dendrite morphol- mTOR occurs through the DEP domains of the P-Rex ogy (13). proteins and regulates nutrient-stimulated Rac activation Together, these studies show the nonredundant functions and cell migration (6). of P-Rex proteins in animal physiology. This is relevant if P-Rex may participate in the upregulation of the activity one considers the large complement of Rac GEF factors of the PI3K pathway by antagonizing PTEN (5). This latter reported that are also widely expressed (21). protein dephosphorylates PIP3, an important lipid second messenger implicated in the regulation of cellular prolifer- Regulation of P-Rex proteins ation and survival (26). Inactivation of PTEN leads to The activation of P-Rex is directly and synergistically increased levels of PIP3 and therefore promotes cellular produced by the binding of PIP3 to the PH domain and responses, such as augmented cell proliferation or the Gbg subunits to the DH domain (8, 22, 23; Fig. 1B). decreased cell death, which are hallmarks of oncogenic These bindings favor translocation of P-Rex from the cytosol progression. In breast cancer cells, Fine and colleagues to the plasma membrane, where P-Rex GEF activity is higher (5) found that PTEN was able to bind P-Rex2a. Such (8, 23). Deletion studies indicated that the DH/PH domains interaction decreased the phosphatase activity of PTEN and are sufﬁcient to activate P-Rex by PIP3 (22). The other resulted in augmented phosphorylation of AKT at activating domains (2 DEP, 2 PDZ, and IP4P) keep the catalytic residues. Interestingly, this action on PTEN was indepen- activity of full-length P-Rex1 low (22), suggesting that dent of the GEF activity of P-Rex2a. In MCF10A cells that interactions between the DH/PH domains and the other express endogenous active PTEN, forced expression of P- domains may autoinhibit P-Rex activity. Rex2a augmented AKT phosphorylation at Ser473 and Phosphorylation of P-Rex represents an important favored the formation of multiacinar structures (5), a fea- mechanism of regulation of its function. Several reports ture that in these cells has been associated to a pretumori- have indicated that P-Rex1 is phosphorylated at multiple genic status (27). In ovarian cancer cells, expression of P- sites (9, 24, 25), and the phosphorylation of these sites Rex1 has been shown to promote activation of AKT1 and P- regulates P-Rex1 activity in an opposite fashion. In breast Rex1 knockdown inhibited AKT1 (28). cancer cells, phosphorylation of Ser313 and Ser319,located in the PH domain, inhibits P-Rex1 activity under resting Clinical–Translational Advances conditions, and mutation to nonphosphorylatable resi- Several circumstances indicate that P-Rex proteins may dues augments Rac activity (9). Stimulation of ErbB play a role in the pathophysiology of various neoplastic receptors, which increases Rac activity in those cells, was diseases. On the one hand, P-Rex expression levels have accompanied by dephosphorylation of Ser313 and Ser319 been shown to correlate with patient outcomes in several (9). Interestingly, b-adrenergic receptor activation neoplasias. Moreover, molecular alterations of P-Rex have increases the phosphorylation of P-Rex1, likely through been described in melanoma, and some of these altera- PKA, and such a phosphorylation diminishes P-Rex1 GEF tions promote tumorogenesis. On the other hand, P-Rex activity (24). Moreover, dephosphorylation of P-Rex1 by proteins have been shown to participate in the regulation l-phosphatase (24) or protein phosphatase 1a (PP1a; of several prooncogenic properties, such as migration, ref. 25) increases the activity of P-Rex1. In the aggregate, cell proliferation, or invasion. These clinicopathologic these reports support the concept that phosphorylation of and biologic ﬁndings open the possibility of targeting P-Rex1 at certain residues inhibits its GEF activity toward P-Rex proteins with therapeutic purposes. Moreover, Rac. the implication of P-Rex partnerproteinssuchasRac, Phosphorylation of P-Rex1 at other sites activates its Rac mTOR, or PTEN in cancer offers the possibility of acting GEF function. In breast cancer cells, stimulation of ErbB on P-Rex to restrict protumorigenic signaling through receptors caused phosphorylation of Ser605 and Ser1169 (9). these pathways.

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Expression and molecular alterations of P-Rex proteins facilitated cellular migration in a P-Rex–dependent manner in cancer (9, 29). Studies in breast (9, 29) and prostate cancer (30) have In the skin, Lindsay and colleagues showed that P-Rex1 shown a link between P-Rex expression and patient out- was required for efficient melanoblast migration and mel- come. Sosa and colleagues found that more than 50% of anoma dissemination (37). Using an animal model of breast tumors stained for P-Rex1 (29). Normal breast tissue melanoma, which included mutant N-Ras as well as dele- and the stroma did not stain for P-Rex1. P-Rex1 staining was tion of the INK4a tumor suppressor, the authors described higher in patients with metastatic disease, suggesting a role decreased melanoma metastases when these mice were of P-Rex1 in metastatic dissemination. Montero and col- crossed with P-Rex1 / mice (37). Interestingly, no differ- leagues analyzed P-Rex expression in breast tumors by ences in tumor latency or burden in the primary melanomas þ þ quantitative Western blotting (9). Expression of P-Rex was were observed when P-Rex / mice and P-Rex / mice were found to be variable among different patient samples. compared. In line with these data, Campbell and colleagues Kaplan–Meier analyses confirmed that patients whose (38) reported that P-Rex1 favored the invasiveness of breast tumors expressed high levels of P-Rex1 had a shorter WM852 melanoma cells. Therefore, targeting of P-Rex1 in disease-free survival. prostate, ovarian, breast, or skin cancer may be beneficial in PREX1 is located on chromosome 20q13.13, a region fighting disease dissemination. commonly amplified in breast tumors (31) and some breast cancer cell lines (32). The PREX2 gene is located on chro- Role of P-Rex proteins in proliferation mosome 8q13, a region frequently amplified in breast, Although the above reports support a prometastatic role prostate, or colorectal cancer (33–35). Analysis of the of P-Rex proteins, their intermediate action in cell prolif- COSMIC database indicates that mutations in PREX2 are eration is less clear. In breast cancer cells, knockdown of present in 3% of tumors, a frequency slightly higher than P-Rex1 reduced cell proliferation both under resting con- that of mutations in PREX1 (2% of tumors). Mutations in ditions and after ligand-induced stimulation of ErbB recep- PREX1 and PREX2 have a tendency to cluster in the GEF tors (9, 29). However, the lack of effect of P-Rex knockdown exchange region. Additional mutations in PREX1 accumu- on the proliferation of prostate cancer cells (30) or tumor late in the IP4P region. Skin and lung cancers represent the burden in melanoma (37) contrasts with the data obtained tumors that most frequently bear PREX mutations. Around in breast cancer and indicates intertumoral differences in 10% of lung cancers contain PREX1 or PREX2 mutations. the protumorigenic actions of P-Rex proteins. Mutations of PREX1 (0.99%) or PREX2 (1.17%) in breast cancer are scarce and are absent in hematopoietic cancers. P-Rex proteins in pro-oncogenic signaling In skin cancer, 23% of tumors carry PREX2 mutations, The role of P-Rex proteins as mediators of signaling by the and 3.6% of tumors bear PREX1 mutations. Massive cell surface receptors, especially those involved in the path- sequencing of melanoma genomes identified PREX2 as a ophysiology of several neoplasias, merits consideration. significantly mutated gene, accounting for a 14% frequency Initial work suggested a potential role of P-Rex1 in signaling in a cohort of 107 human melanomas (36). These muta- by GPCRs. This idea was confirmed by showing the inter- tions were distributed along the entire length of P-Rex2 and mediate action of P-Rex1 in migration upon activation of included nonsynonymous PREX2 mutations as well as the chemokine receptor CXCR4 (29). truncations (36). Biologic experiments in which some of In addition to the intermediate role of P-Rex in GPCR the P-Rex2 mutations found in patients were reproduced signaling, evidence is accumulating suggesting that P-Rex in TERT-immortalized human melanocytes showed the proteins may play a relevant role in signaling by different role of this GEF in melanoma genesis and showed that classes of cell surface receptors. This idea is supported by the PREX2 mutations generate altered proteins with oncogenic fact that the receptor tyrosine kinases (RTK) ErbB (9), activity (36). platelet-derived growth factor receptor b (38), TrkA (7), and IGF-I receptor (IGF-IR; ref. 39) use P-Rex. That P-Rex Prometastatic properties of P-Rex proteins may act as general intermediates in signaling by In prostate cancer cell lines and human samples, expres- RTKs is a relevant concept, especially because of the impor- sion of P-Rex1 strongly correlated with the metastatic phe- tant roles of these receptors in oncology and physiology. It notype (30). Qin and colleagues (30) reported that P-Rex1 will be necessary to analyze whether RTKs other than those acted through Rac to facilitate migration and invasion of already reported use P-Rex as part of their signal transduc- prostate cancer cells upon activation of the EGF receptor. tion machinery. These studies may uncover novel roles of P- Moreover, overexpression of P-Rex1 in a nonmetastatic cell Rex proteins in mediating physiologic actions of RTKs that line increased its motility and invasion properties. In their may be relevant in tumor growth. An example of the latter is prostate cancer models, P-Rex1 did not affect prostate the role of P-Rex proteins in glucose metabolism. P-Rex1 has tumor growth but caused spontaneous lymph node metas- been reported to regulate the membrane exposure of the tases. Similarly, in ovarian cancer cells, knockdown of P- glucose transporter GLUT4 in adipocytes in response to IGF- Rex1 inhibited insulin-like growth factor-I (IGF-I)–induced IR activation (39). This action indicates that P-Rex proteins activation of cell migration or invasion (28). In breast may control supply of metabolic energy to cells by upre- cancer cells, activation of ErbB or chemokine receptors gulating glucose transport into their cytosol. Obviously, this

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effect of P-Rex could be of relevance for the fitting of the or PTEN. Inhibition of the Rac-GEF activity of P-Rex pro- tumoral tissues, but such physiologic action may even teins could be possible by using compounds that prevent outweigh its oncological relevance in case P-Rex proteins interaction of the PH domain of P-Rex proteins with PIP3, are shown to play important roles in insulin receptor– or that directly inhibit the GEF activity of the DH domain. mediated glucose transport. Interference with the GEF activity should reduce the Rac- Mechanistically, it will be important to define whether mediated actions of P-Rex proteins, especially those related RTKs and other cell surface receptors activate P-Rex by to cellular motility and invasion. Reduction of the interac- the same mechanism used by the ErbB receptors, i.e., by tion of P-Rex proteins with mTOR and PTEN could be used activating the P-Rex phosphorylation/dephosphorylation to restrict P-Rex–dependent PI3K signaling. Compounds cycle. It will also be relevant to identify the kinases and in development that may inhibit interaction of the DEP phosphatases involved in controlling that cycle, and the domain of P-Rex proteins with mTOR are expected to reduce signaling cascades that mediate activation of those kinases P-Rex/mTOR-dependent Rac activation and migration. and phosphatases. Finally, elucidation of additional P-Rex– Similarly, preventing the binding of the PH domain of P- interacting proteins could help in understanding the pro- Rex proteins to PTEN may allow the latter to inhibit PI3K tein networks in which P-Rex proteins participate. signaling. In addition to the above-mentioned strategies that direct- Targeting P-Rex in cancer ly target P-Rex proteins, indirect ways to reduce P-Rex Given the ample number of Rac GEFs (21), we may signaling may be contemplated. These strategies rely on the consider it unreasonable to target P-Rex proteins. However, use of drugs that act on signaling proteins upstream or clinicopathologic as well as preclinical data support roles of downstream of P-Rex, such as inhibitors of RTKs, GPCRs, P-Rex proteins in human cancer, opening therapeutic Rac, or dual PI3K/mTOR inhibitors. It is expected that options for the targeting of P-Rex proteins in cancer. In increasing the knowledge about the regulatory mechanisms fact, P-Rex expression has been correlated with patient responsible for controlling P-Rex functioning may allow the outcome in breast and prostate cancers, and reduction of development of additional strategies. As an example, aug- P-Rex expression has been shown to restrict proliferation menting the phosphorylation of P-Rex1 at inhibitory resi- and/or metastatic dissemination (9, 29, 30). Therefore, dues may also be exploited to reduce its functionality. This targeting P-Rex proteins may be therapeutically relevant in would require unveiling the kinases and phosphatases neoplasias in which those proteins play an important role in responsible for maintaining P-Rex in its "off" state (Fig. 1B). regulating their growth or dissemination. In conclusion, the ample range of physiologic cancer- Of particular relevance is the role of P-Rex proteins in related functions of P-Rex, together with the accumulating melanoma pathophysiology. The fact that mutated forms of evidence that links P-Rex proteins to several types of neo- P-Rex2 have been detected in melanoma and are tumorigenic plasias, opens possibilities for the targeting of P-Rex pro- (36) raises the possibility of targeting P-Rex2 in melanomas teins with therapeutic purposes. bearing such mutations. Moreover, Lindsay and colleagues showed that P-Rex1 was required for efficient melanoblast Disclosure of Potential Conflicts of Interest migration and melanoma dissemination (37). Interestingly, No potential conflicts of interest were disclosed. expression of P-Rex1 in melanoma cell lines has been reported to be controlled by the extracellular signal-regulated Authors' Contributions kinase (ERK) route (40). This finding is relevant given the Conception and design: A. Pandiella, J.C. Montero Writing, review, and/or revision of the manuscript: A. Pandiella, J.C. high frequency of activation of the ERK pathway in mela- Montero nomas due to upstream mutations in BRAF (36). It is possible Study supervision: A. Pandiella that BRAF inhibitors used in the clinic, such as vemurafenib (41) or other BRAF/ERK pathway inhibitors, may reduce Grant Support P-Rex1 levels and therefore contribute to preventing P-Rex1– This work was supported by grants from the Ministry of Science and facilitated melanoma dissemination. Innovation of Spain (BFU2009-07728/BMC) to A. Pandiella; and Instituto de Salud Carlos III (CP12/03073 to J.C. Montero). Our Cancer Research Besides controlling P-Rex proteins by downregulating Institute and the work carried out at our laboratory received support from the their cellular levels, another way of acting on these proteins European community through the regional development funding program may be the use of strategies that affect their functionality. (FEDER) and from the Fundacion Ramon Areces. Such strategies may include inhibition of the Rac-GEF Received February 4, 2013; revised May 13, 2013; accepted May 20, 2013; activity of P-Rex proteins or their interaction with mTOR published OnlineFirst June 10, 2013.

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OF6 Clin Cancer Res; 19(17) September 1, 2013 Clinical Cancer Research

Molecular Pathways: P-Rex in Cancer

Atanasio Pandiella and Juan Carlos Montero

Clin Cancer Res Published OnlineFirst June 10, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-12-1662

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