Fixing the GAP: the role of RhoGAPs in cancer

Abstract

Cancer progression and metastasis are processes that involve significant cellular changes. Many of these changes include alterations in the activity of the Rho GTPase family of proteins. Rho GTPases are signaling proteins that function as molecular switches and are involved in the regulation of most major cellular processes. Cancer development is often associated with abnormalities in Rho GTPase signaling. Rho GTPase signaling is regulated by two families of proteins, guanine nucleotide-exchange factors (RhoGEFs) and GTPase activating proteins (RhoGAPs), that function upstream of the Rho proteins to regulate their activation and inactivation, respectively. While initial work has focused on the role of RhoGEFs in cancer, the RhoGAP family members are rapidly being established as key regulators of cancer development and progression. The aim of this review is to summarize our advances in understanding the role of RhoGAPs in cancer and to discuss their significance in the development of therapeutics.

1. Introduction

Cancer progression and metastasis are processes that involve significant changes at the cellular level. One of the common changes found in developing tumors is abnormal Rho GTPase signaling (Haga and Ridley, 2016; Jansen et al., 2018; Rathinam et al., 2011; Sahai and Marshall, 2002). Rho GTPases function as molecular switches and are involved in regulating a diverse array of cellular processes including cell adhesion, motility, actin cytoskeleton organization, and cell division (Jaffe and Hall, 2005). Two families of proteins, the guanine nucleotide-exchange factors or RhoGEFs, and the GTPase activating proteins or RhoGAPs, function upstream of Rho GTPases to regulate their activation and inactivation respectively (Rossman et al., 2005; Tcherkezian and Lamarche-Vane, 2007). Beyond their diverse role in normal physiology, Rho proteins also strongly influence pathological processes such as cancer (Ridley, 2004; Sahai and Marshall, 2002). Aberrant function of Rho GTPases affects virtually all stages of cancer progression, including proliferation, invasion, and metastasis (Orgaz et al., 2014; Sahai and Marshall, 2002). Many of the best characterized Rho GTPases, including RhoA, Rac1, and Cdc42, have been shown to be involved in progression and metastasis in a variety of human tumors (for reviews see: (Haga and Ridley, 2016; Orgaz et al., 2014; Porter et al., 2016). In contrast to other small GTPases, especially Ras, which are frequently mutated in cancer, mutations within the Rho GTPase family (members of the Ras superfamily) are markedly rare. There are a few exceptions; In the last few years several studies have reported mutations in Rho proteins, particularly in Rac1, in a small number of cancers such as melanoma and brain tumors (Davis et al., 2013; De et al., 2019; Hwang et al., 2004; Krauthammer et al., 2012). The most frequently found mutation in Rac1, P29L, has been characterized as a fast-cycling mutation (Kawazu et al., 2013; Krauthammer et al., 2012). This is different from the commonly found oncogenic mutations of Ras, which are constitutively active and locked in the GTP-bound state.

What is more common than mutations is the alteration of Rho signaling resulting from the disruption of the balance between activation and inactivation of a particular Rho GTPase. There are several mechanisms by which this balance can be altered, including changes in the expression levels of the Rho GTPases or their direct regulators (RhoGEFs, RhoGAPs, and RhoGDIs), post-translational modifications of the Rho GTPases or their regulators, and alternative splicing (reviewed in (Harding and Theodorescu, 2010; Orgaz et al., 2014; Porter et al., 2016; Vigil et al., 2010)). Overall, the relationship between the Rho GTPases and cancer is complex, and a comprehensive discussion is beyond the scope of this review (see (Svensmark and Brakebusch, 2019) for more detailed commentary).

During the Rho GTPase cycle, RhoGAPs accelerate the GTP hydrolysis reaction, which is intrinsically low in Rho proteins, by several orders of magnitude, and are thus critical for the termination of Rho GTPase signal transduction (Cherfils and Zeghouf, 2013). Initially, RhoGAPs were considered simply signal terminators with a role secondary to that of RhoGEFs (Tcherkezian and Lamarche-Vane, 2007). However, this misconception has been dispelled as studies on RhoGAPs have shown their signaling is as complex as that of RhoGEFs (Tcherkezian and Lamarche-Vane, 2007). It is no surprise then, that mutations in genes encoding RhoGAPs have drastic consequences and underlie several human diseases, including cancer. Even though the early studies on Rho signaling in cancer focused predominantly on the role of RhoGEFs, we have now begun to identify functions for many of the RhoGAP family members in cancer. RhoGAPs have traditionally been considered tumor suppressors, as a loss of RhoGAP activity results in unconstrained GTPase activity, a typical characteristic of cancer (Vigil et al., 2010). However, there is a growing number of studies which show examples of RhoGAPs with oncogenic roles (Lawson and Der, 2018; Zandvakili et al., 2017). In this review, we provide an overview of RhoGAPs, their role in tumorigenesis, and the possibility of targeting them for therapeutic purposes.

2. The RhoGAP family

While there are only 20 members of the Rho GTPase family in humans, there are multiple RhoGEFs and RhoGAPs for each Rho GTPase, with 80 RhoGEFs and 66 RhoGAPs encoded in the human genome (Rossman et al., 2005; Tcherkezian and Lamarche-Vane, 2007). This redundancy of upstream Rho GTPase regulation allows for highly specific spatiotemporal control of Rho GTPase activation in the cell (Fritz and Pertz, 2016). RhoGAPs are more promiscuous than RhoGEFs, at least in vitro, with most RhoGAPs being able to inactivate multiple GTPases (Figure 1) (Amin et al., 2016; Tcherkezian and Lamarche-Vane, 2007). However, substrate selectivity is a lot narrower in vivo. This apparent discrepancy is common in the RhoGAP field and may reflect the differences between analyzing the activity in vitro using a purified RhoGAP domain versus measuring the activity of the Rho GTPases in vivo when the full-length RhoGAP is either overexpressed or silenced. This suggests that the other domains in the protein are especially important in controlling the specificity observed in vivo (Amin et al., 2016; Cherfils and Zeghouf, 2013). While there is some degree of redundancy of function in the RhoGAP family, e.g., between subfamily members such as p190A/B or RICH1/2, there is less than initially purported. Several recent RNAi screens targeting the RhoGAP and RhoGEF families, both in vitro and in vivo, suggest that the degree of redundancy is lower than expected (reviewed in (Dahmene et al., 2020)).

Figure 1. The RhoGAP family.

The 66 RhoGAPs in the human genome were aligned using CLUSTALW to produce this phylogenetic tree. The associated diagrams show the domain composition for each member (drawn at scale). The reported Rho GTPase specificity for each RhoGAP is indicated by a colored circle in each of the tree branches. Note that the specificity characterization for the RhoGAP family is never complete, as RhoGAP assays are performed primarily on RhoA, Rac1, and Cdc42, so the specificity for the other members of the 22 known human Rho GTPases is generally not known. RhoGAPs that encode an inactive RhoGAP domain or pseudo-GAPs are indicated in magenta. For descriptions of domain abbreviations and functions, the reader is referred to the SMART website (http://smart.embl-heidelberg.de/). The figure design was inspired by a similar figure describing the DH-PH RhoGEF family designed by Rossman and colleagues (Rossman et al., 2005).

The RhoGAP proteins are not only diverse in the binding specificity of their catalytic RhoGAP domain, but also in their auxiliary domains, highlighting the diverse roles of RhoGAPs which necessitates their systematic study as unique players in the development of different cancers (Figure 1). These auxiliary domains are involved in targeting, protein-protein, and protein-lipid interaction, as well as in regulating the specificity and activation state of the RhoGAPs. The accumulating evidence showing that RhoGAPs can be regulated by autoinhibitory mechanisms exemplifies the importance of RhoGAP auxiliary domains (Cherfils and Zeghouf, 2013). Most of the autoinhibitory mechanisms characterized so far in RhoGAPs involve a lipid-binding domain folding over and blocking the catalytic RhoGAP domain. Considering the abundance of membrane interaction domains in the RhoGAP family, such as BAR (Bin/Amphiphysin/Rvs), PH (pleckstrin homology), Sec14, and others, it is tempting to speculate that the association of RhoGAPs to membranes may function to coordinate activation specifically at membranes (Cherfils and Zeghouf, 2013). The best-characterized example is β2-Chimaerin, which encodes an N-terminal cysteine-rich motif (C1) that binds to diacylglycerol (DAG) and releases the autoinhibition of the RhoGAP domain (Canagarajah et al., 2004). In ARHGAP1 for example, the phospholipid binding domain Sec14 (Bankaitis et al., 2010) not only targets it to endosomes, but also binds to the RhoGAP domain and inhibits its activity (Moskwa et al., 2005). Similarly, studies on RhoGAPs that contain BAR domains, such as the Slit-Robo (Roundabout) RhoGAPs (srGAP1–3), GRAF/oligophrenin, and HMHA1 suggest that the BAR domain is also autoinhibitory (de Kreuk et al., 2013; Eberth et al., 2009; Fauchereau et al., 2003; Lucas and Hardin, 2017). A considerable number of RhoGAPs encode PH and BAR domains (Figure 1), but their role in regulating the RhoGAP activity requires further study. Nevertheless, it is clear that the diversity of RhoGAP structure is directly linked to the unique roles that the RhoGAPs play.

3. RhoGAPs in cancer

In this section, we summarize our current knowledge of the RhoGAPs that have been characterized to have a role in cancer. Here we list the RhoGAPs in phylogenetic order according to Figure 1 and starting with DLC1.

3.1. DLC1, DLC2, and DLC3

DLC1 (deleted in liver cancer 1) is the best studied RhoGAP in relation to its association with cancer. Over the years there have been several excellent reviews covering its role in cancer (Barras and Widmann, 2014; Braun and Olayioye, 2015; Popescu and Goodison, 2014; Ren and Li, 2021; Zhang and Li, 2019). Here we will summarize some of the key findings, but the reader can refer to these articles for more detail. DLC1 is part of the DLC subfamily which comprises three highly conserved genes (DLC1–3), all encoding proteins with the same domain structure: an N-terminal sterile alpha motif (SAM), a RhoGAP domain, and a StAR-related lipid-transfer (START) domain (Popescu and Goodison, 2014). The DLC proteins show strong RhoGAP activity for RhoA (as well as RhoB and RhoC), weak activity for Cdc42, and no activity for Rac1 (Ching et al., 2003; Healy et al., 2008; Kawai et al., 2007). The RhoGAP domain regulates cell migration, adhesion, and actin dynamics (Barras and Widmann, 2014), while the SAM domain is important for protein-protein interactions and DNA/RNA binding (Zhang and Li, 2019), and the START domain interacts with lipids (Alpy and Tomasetto, 2005).

DLC1 was originally identified as a tumor suppressor as it is frequently deleted in liver cancer, hence the name DLC (Yuan et al., 1998). The gene for DLC1 is localized on chromosome 8p21–22, one of the genomic regions most frequently deleted in several types of human cancer, including prostate, colon, breast, ovarian, liver, lung, bladder, and head and neck cancer (Birnbaum et al., 2003). The importance of DLC1 in cancer progression is evident in that the deletion of DLC1 is nearly as frequent as p53 deletion in common cancers such as lung, colon, pancreas, and breast (Xue et al., 2008). DLC1 expression is significantly lower in late stages of cancer compared to early stages, and in metastatic vs. non-metastatic cancers (Wang et al., 2016a). Both genomic deletions of DLC1, as well as changes in its expression levels and/or signaling have been identified in several other types of cancer as well, including liver, breast, prostate, lung, and multiple myeloma (reviewed in (Popescu and Goodison, 2014; Zhang and Li, 2019); Table 1). Restoring or overexpressing DLC1 expression suppresses cell proliferation, inhibits cell migration and invasion, and can also induce apoptosis, senescence, and autophagy (Feng et al., 2013; Hampl et al., 2013; Healy et al., 2008; Huang et al., 2015; Kim et al., 2008; Liu et al., 2012; Ng et al., 2000; Qin et al., 2014; Shi et al., 2012; Ullmannova-Benson et al., 2009; Wang et al., 2014a; Wong et al., 2005; Wu et al., 2018; Yang et al., 2016; Yuan et al., 2003; Yuan et al., 2004; Zhou et al., 2004; Zhou et al., 2008). It has also been shown that suppression of DLC1 by RNAi promotes tumorigenesis in a mouse liver cancer model (Xue et al., 2008).

Table 1.

RhoGAPs associated with cancer

RhoGAP	Gene ID and aliases	Rho GTPase specificity	Downregulated in cancer (tumor suppressor)	Upregulated in cancer (oncogene)
DLC1	DLC1, ARHGAP7, STARD12	RhoA, Cdc42 [1]	liver, lung, ovarian, renal, breast, uterine, colon, prostate, GC, NPC, esophageal carcinoma, cervical carcinoma, meningioma, multiple myeloma, oral squamous cell carcinoma, urothelial carcinoma, pancreatic, cutaneous squamous cell carcinoma, gallbladder, cutaneous melanoma, penile carcinoma, CRC [2–44]	melanoma [45]
DLC2	STARD13, ARHGAP37	RhoA, Cdc42 [46]	HCC, lung, renal, ovarian, breast, colon, uterine, gastric, CRC, rectal, astrocytoma [10, 13, 46–51]
DLC3	STARD8, ARHGAP38, STARTGAP3	RhoA, Cdc42 [52]	gastric, kidney, lung, breast, ovarian, uterine, prostate [13, 53, 54]
ARHGAP6	ARHGAP6, RhoGAP6	RhoA [55]	cervical carcinoma, lung [55–59]	colorectal [60]
ARAP1	ARAP1, Centaurin Delta 2	RhoA, Cdc42 [61]	ovarian carcinoma [62]	pediatric ALL [63]
ARAP3	ARAP3, Centaurin Delta 3	RhoA [64, 65]	GC [64–66]	metastatic breast, thyroid [67–70]
GRAF1	ARHGAP26, GRAF, OPHN1L, OPHN1L1	RhoA, Cdc42, TC10, TCL [71–73]	myeloid malignancies, highly invasive breast cancer, ovarian, metastatic brain tumors, acute myeloid leukemia [73–77]
p190A	ARHGAP35, GRF-1, GRLF1, p190RhoGAP	RhoA, Rac1, Cdc42, RhoD [72, 78–80]	HCC, glioblastoma, astrocytoma, lung, endometrial, kidney, head and neck, prostate, melanoma [81–91].	CRC, lung, osteosarcoma [92–95]
p190B	ARHGAP5, p190BRhoGAP	RhoA, Rac1, Cdc42 [80, 96]		HCC, NSCLC, breast, NPC [97–102]
FilGAP	ARHGAP24, p73, p73RhoGAP	Rac1, Cdc42 [103–105]	CRC, breast, lung, renal, astrocytoma [106–111]	B-cell lymphoma [112]
ARHGAP22	ARHGAP22, RhoGAP2, RhoGap22	Rac1 [113, 114]		RCC [115]
ARHGAP25	ARHGAP25, KIAA0053	Rac1 [116]	CRC, lung [117, 118]	ARMS [119]
ARHGAP15	ARHGAP15	Rac1 [120–122]	glioma, CRC, breast, lung [123–126]
ARHGAP9	ARHGAP9, RGL1	Cdc42, Rac1 [127]	HCC, bladder [127–129]	HNSCC, AML [130–132]
RLIP76	RLIP76, RALBP1, RIP1, RLIP1	Rac1, Cdc42 [133–135]		ovarian, bladder, CRC, adrenocortical tumors, NSCLC, glioblastomas, HCC, meningiomas, lung, breast, pancreatic ductal adenocarcinoma (PDA), gastric, oral [136–150].
ARHGAP30	ARHGAP30	RhoA, Rac1 [151]	CRC, lung, pancreatic, cervical [152–155]
CdGAP	ARHGAP31	Rac1, Cdc42 [156–159]		mouse mammary tumor explant, breast, prostate [160–162]
α-chimaerin	CHN1, ARHGAP2, N-chimaerin	Rac1 [163]		esophageal squamous cell carcinoma, cervical [164–166]
β-chimaerin	CHN2, ARHGAP3	Rac1 [167]	high-grade malignant glioma, murine mammary carcinoma, breast [168–171]	HSTL [172]
ARHGAP21	ARHGAP21	Cdc42, RhoA, RhoC [173–177]	ovarian, lung, prostate, colon, gastric [178–183]	HNSCC [184]
RacGAP1	RACGAP1, CYK-4, MgcRacGAP	Rac1, Cdc42, RhoD [72, 185–189]		HCC, meningioma, NSCLC, CRC, BLBC, uterine carcinoma, epithelial ovarian cancer, PAAD, esophageal carcinoma, HNSCC, breast, BP-NEN, GC [190–205]
ARHGAP29	ARHGAP29, PARG1	RhoA, Rac1, Cdc42 [206]	mantle cell lymphoma [207]	GC, HCC, prostate, RCC, pancreatic [208–210]
HMHA1	ARHGAP45, HA-1	RhoA, Rac1 [211, 212]	NSCLC, HCC [213]	Melanoma, leukemia lymphoma, breast, lung, renal, liver, colon, head and neck [214–216]
SrGAP1	SRGAP1, ARHGAP13	RhoA, Cdc42, Rac1 [217–220]	glioblastoma, CRC, NSCLC [221–223]	gastric [224]
SrGAP2	SRGAP2, ARHGAP34	Rac1 [225, 226]	murine osteosarcoma [227]	HCC [228]
SrGAP3	SRGAP3, ARHGAP14, MEGAP, WRP	Rac1, Cdc42 [220, 229, 230]	breast [231]
SrGAP4	ARHGAP4, p115, RhoGAP4, C1, RGC1	RhoA, Rac1, Cdc42 [232, 233]	PC [234–236]
MYO9B	MYO9B, CELIAC4, MYR5	RhoA [237, 238]		lung, prostate, esophagus [239–241]
ARHGAP18	ARHGAP18, MacGAP, Conundrum	RhoA, RhoC [242, 243]	GC, breast, melanoma [243–245]	TNBC [246, 247]
BPGAP1	ARHGAP8, PP610	RhoA, Cdc42 [248]		CRC, invasive cervical cancer [249, 250]
Cdc42GAP	ARHGAP1, RHOGAP1, p50rhoGAP	RhoA, RhoB, Cdc42, TC10, TCL [72, 185, 186, 251–253]	cervical carcinoma, PCa, ES [254–256]	breast, brain, osteosarcoma [76, 257, 258]
ARHGAP20	ARHGAP20, RA-RhoGAP, KIAA1391	RhoA [259]	HCC [260]	CLL [261, 262]
ARHGAP11A	ARHGAP11A	RhoA [195, 263]		colon, glioblastoma, lung, breast, GC, HCC, pancreas [195, 263–268]
ARHGAP19	ARHGAP19	RhoA [269]		aggressive breast and endometrial carcinoma cells [270–272]
RICH1	ARHGAP17, NADRIN	Rac1, Cdc42, TC10 [72, 273, 274]	breast, colon, cervical [275–277]
RICH2	ARHGAP44, KIAA0672, NPC-A-10	Rac1, Cdc42 [273]	HCC, lung [278, 279]
SH3BP1	ARHGAP43	Rac1, Cdc42, RhoG [280, 281]		cervical, invasive HCC [282, 283]
OPHN1	Oligophrenin-1, ARHGAP41	RhoA, Rac1, Cdc42, TC10, TCL [72, 284, 285]	CRC, prostate, GC [286–290]
GRAF2	ARHGAP10, PSGAP	RhoA, Cdc42 [291]	breast, GC [292–294]
GRAF3	ARHGAP42	RhoA, Cdc42 [295, 296]		NPC [297]
CAMGAP1	CAMGAP1 ARHGAP27	Rac1, Cdc42 [298]		ovarian, glioma [299–301]
ARHGAP12	ARHGAP12	Rac1, Cdc42 [302, 303]	melanoma [303]
ARHGAP32	ARHGAP32, GRIT, RICS, GC-GAP, p250GAP, p200RhoGAP	RhoA, Rac1, Cdc42 [304]	GC [305]
ARHGAP28	ARHGAP28, KIAA1314	RhoA [306]	colon [307]
SYDE1	SYDE1, 7h3	RhoA, Rac1, Cdc42 [308, 309]	glioma [310]
ARHGAP36	ARHGAP36	No GAP activity [72]		Medulloblastoma, pheochromocytoma, PTC [311–313]
DEPDC1B	DEPDC1B, BRCC3, XTP1	No GAP activity [72]		oral, NSCLC, prostate, soft-tissue sarcoma, malignant melanoma, neuroblastoma, chordoma, HCC [314–321]
FAM13A	FAM13A, ARHGAP48, FAM13A1	No GAP activity [72]		lung [322–324]

Most of the evidence suggests that DLC1 suppresses tumorigenesis through its RhoGAP activity for RhoA (Lahoz and Hall, 2008). However, RhoGAP independent mechanisms have also been linked to tumor cell phenotype, showing that the function of DLC1 in malignancy is complex (Healy et al., 2008; Liao et al., 2007). The activity of DLC1 for RhoA is kept in check by autoinhibition from an intramolecular interaction between the SAM and the RhoGAP domains, which can be relieved through interactions with either tensin-3 (TNS3) or PTEN (Cao et al., 2012; Kim et al., 2008). A recent report shows that peptides based in the DLC1 binding region of TNS3 and PTEN promote DLC1 activation and reduce cancer growth in some types of cancer (Joshi et al., 2020).

DLC1 does not only regulate cancer cell proliferation and migration but also regulates apoptosis, senescence, and autophagy. DLC1 localizes to both the cytoplasm and the nucleus, and its tumor suppressive function appears to be mediated by the cytosolic form, while the nuclear localized fraction plays a role during caspase 3-mediated apoptosis (Chan et al., 2011; Scholz et al., 2009; Yuan et al., 2007). The interaction between DLC1 and a caspase 3 cleavage product of p120 RasGAP (fragment N2, RasGAP158–455) may contribute to the role that the N2 fragment has in increasing sensitivity to anticancer treatments, which has been observed in various tumor cell lines, both in vitro and in vivo (Barras et al., 2014; Michod et al., 2009; Yang and Widmann, 2001). In terms of cell senescence, DLC1 has been shown to promote senescence in hepatocellular carcinoma (HCC) through suppression of RhoA activity upstream of the transcriptional coactivators Megakaryoblastic leukemia 1 and 2 (MKL1/2) (Hampl et al., 2013). DLC1 also been shown to inhibit autophagy in HCC cells (Wu et al., 2018). Autophagy inhibition appears to be regulated by the DLC1-mediated inactivation of ROCK1, which reduces the anti-autophagic interaction between the autophagy protein Beclin1 and anti-apoptotic protein Bcl2 (Gurkar et al., 2013; Wu et al., 2018). This contrasts with the DLC1-mediated regulation of cell migration which requires the RhoA effector mDia1 but not ROCK1 (Holeiter et al., 2008).

There is a wide variety of mechanisms contributing to DLC1 dysregulation in cancer beyond deletion, which include mutations, downregulation of expression through gene methylation/ubiquitination, regulation of RhoGAP activity by protein-protein interaction, and/or phosphorylation (reviewed in (Braun and Olayioye, 2015; Popescu and Goodison, 2014; Zhang and Li, 2019)). Promoter methylation is the most common mechanism of DLC1 downregulation and has been reported in several types of cancer, including lung, multiple myeloma, colorectal cancer (CRC), angiosarcoma, prostate, acute leukemia, and others (Zhang and Li, 2019). DLC1 levels are also downregulated by miR-301, a microRNA that is significantly upregulated in non-small cell lung cancer (NSCLC) tissues and cell lines (Wu et al., 2017). Inhibition of miR-301a suppresses proliferation, migration and invasion of NSCLC cells, and these effects can be partially reversed by silencing DLC1.

The literature on DLC2 (StarD13) and DLC3 (StarD8) is not as extensive as that for DLC1, but they also function as tumor suppressors. In most tissues studied, DLC1 is expressed at higher levels in tissues but is also downregulated in cancer to a greater degree than either DLC2 or DLC3 (Csepanyi-Komi et al., 2013; Wang et al., 2016a). However, downregulation of DLC2 has been documented in several types of cancers, including HCC, lung, renal, ovarian, breast, CRC, rectal tumors, and astrocytomas (Ching et al., 2003; El-Sitt et al., 2012; Gao et al., 2012; Hanna et al., 2014; Nasrallah et al., 2014; Ullmannova and Popescu, 2006; Xiaorong et al., 2008). DLC3 downregulation, on the other hand, has been reported in gastric, kidney, lung, breast, ovarian, uterine, and prostate cancer (Durkin et al., 2007; Zhang et al., 2018b). Low levels of DLC1 and DLC2, but not DLC3, have been associated with poor prognosis in lung adenocarcinoma (Du et al., 2012; Sun et al., 2019a). DLC2 has also been shown to be a target of miR-125b in breast cancer (Chang et al., 2016; Tang et al., 2012), and of miR-9–5p in prostate cancer (Chen et al., 2019a).

3.2. ARHGAP6

ARHGAP6, a RhoGAP for RhoA, is downregulated in both lung and cervical cancers (Li et al., 2016a; Prakash et al., 2000; Wu et al., 2019). Reduced ARHGAP6 in lung tumors correlates with high expression of MMP9 and vascular endothelial growth factor (VEGF), while overexpression of ARHGAP6 in lung cancer cells inhibits growth and metastasis by suppressing expression of MMP9, VEGF, and phosphorylation of STAT3, all markers of tumorigenesis (Wu et al., 2019). Decreased ARHGAP6 in lung cancers results in an increase in phosphorylated STAT3 which is associated with resistance to cisplatin chemotherapy (Li et al., 2020). The microRNA miR-96–5p has been found to target ARGHAP6 and promote cancerous phenotypes in lung adenocarcinoma (Liu et al., 2021b). In cervical cancer cells, ARHGAP6 overexpression inhibits proliferation, migration, invasion, adhesion, and induces apoptosis while, in vivo, ARHGAP6 has been found to suppress growth (Li et al., 2016a). Contrarily, one study found that ARHGAP6 may also function as an oncogene. Aberrantly growing colorectal cells and tissues have increased ARHGAP6 expression, which was associated with a low level of tumor differentiation and subsequently poor patient survival (Guo et al., 2010).

3.3. ARAP1 and ARAP3

The proteins of the ARAP (ankyrin repeat and PH domain) family are unique in that they contain both RhoGAP and ArfGAP domains and function as a link between the two signaling pathways (Miura et al., 2002). ARAP1 displays in vitro RhoGAP activity predominantly for RhoA, with lower activity for Cdc42 (Miura et al., 2002). Low expression of ARAP1 has been identified as a prognostic biomarker associated with shorter recurrence-free survival in older patients with ovarian high-grade serous carcinoma (HGSC) and receiving first-line platinum-based therapy (Nadaraja et al., 2020). ARAP1 has also been involved in the regulation of apoptosis mediated by TRAIL (TNF-related apoptosis-inducing ligand) (Simova et al., 2008). Mechanistically, ARAP1 interacts with the pro-death receptor DR4 and regulates its transport to the plasma membrane (Simova et al., 2008). ARAP1 also interacts with PTK6, a non-receptor-tyrosine kinase that is overexpressed in 60% of breast cancer tumors (Kang et al., 2010). PTK6 phosphorylates ARAP1, which inhibits EGFR internalization, resulting in longer duration of epidermal growth factor stimulation, which increases the oncogenic potential of cells overexpressing PTK6 (Kang et al., 2010).

ARAP3 displays RhoGAP activity almost exclusively for RhoA and has been found downregulated in gastric cancer (GC) tissues (I et al., 2004; Krugmann et al., 2004). Overexpression of ARAP3 in GC cells inhibits attachment to the extra cellular matrix (ECM), invasion in vitro, and peritoneal dissemination in vivo, suggesting a tumor suppressor role (Yagi et al., 2011). These effects are dependent on both the ARF (ADP ribosylation factor) and Rho-GAP activities of ARAP3, as well as tyrosine phosphorylation by Src (Yagi et al., 2011).

In other cancer types ARAP1 and ARAP3 have been shown to function as oncogenes. ARAP1 was identified as one of four signal transduction genes upregulated in drug resistance pediatric acute lymphoblastic leukemia (ALL) cells (Szczepanek et al., 2012). Bioinformatics analysis shows that ARAP3 is significantly upregulated in metastatic breast cancer, and that high ARAP3 expression is associated with increased risk of metastatic relapse in ER- breast cancer (Han et al., 2017). ARAP3 may also play an oncogenic role in thyroid cancer, as silencing ARAP3 in several thyroid cancer cell lines suppresses proliferation, migration, and invasion (Wang et al., 2016b). Whole genome next-generation sequencing identified ARAP3 mutations in both breast and thyroid cancer (Blighe et al., 2014; Trevino, 2019; Wang et al., 2016b). Some of the ARAP3 single nucleotide variants identified in breast cancer are present in both in the primary tumor and in lymph node metastasis which may suggest a role during early metastasis (Blighe et al., 2014).

3.4. GRAF1

There are three members in the GRAF (GTPase regulator associated with the focal adhesion kinase) sub-family of RhoGAPs: GRAF1 (ARHGAP26), GRAF2 (ARHGAP10, PSGAP), and GRAF3 (ARHGAP42). They all share a common domain structure consisting of an N-terminal BAR domain followed by a PH domain and an SH3 domain (Aspenstrom, 2018).

GRAF1, the first GRAF identified, was originally characterized as a Cdc42 and RhoA specific RhoGAP that targets to focal adhesions through its interaction with focal adhesion kinase (FAK) (Amin et al., 2016; Hildebrand et al., 1996; Regev et al., 2017). In vitro, ARHGAP26 can also use the Cdc42-related proteins TC10 and TCL as substrates, although this still needs to be confirmed in vivo (Amin et al., 2016). GRAF1 has been identified as a tumor suppressor, with lower expression levels associated with poor prognosis in a variety of cancers, including myeloid malignancies, invasive breast cancer, ovarian cancer, and metastatic brain tumors from primary lung adenocarcinoma (Aly and Ghazy, 2014; Bojesen et al., 2006; Chen et al., 2019b; Qian et al., 2010; Regev et al., 2017; Zohrabian et al., 2007). In acute myeloid leukemia, GRAF1 expression has been shown to be suppressed by either deletion or CpG methylation of its promoter. Even when only a single allele is deleted, the non-deleted allele is frequently mutated (Bojesen et al., 2006; Borkhardt et al., 2000). Overexpression of GRAF1 in ovarian cancer cells with low endogenous levels inhibits cell proliferation, migration, invasion, and lung metastasis in vivo (Chen et al., 2019b). These effects were associated with a decrease in β-catenin, VEGF, MMP2, and MMP7 expression. Silencing GRAF1 in ovarian cancer cells expressing higher levels of endogenous GRAF1 has the opposite effect, corroborating the results (Chen et al., 2019b). The expression of GRAF1 is regulated by the E3 ubiquitin ligase SMURF1, and is inversely correlated with β-catenin expression, which suggests migration and invasion may be regulated through the canonical Wnt pathway (Chen et al., 2019b).

Recent studies have shed some light into the mechanisms by which GRAF1 may contribute to tumor suppression. GRAF1 has been shown to play a role in the regulation of clathrin independent endocytosis (Lundmark et al., 2008). In response to a decrease in surface tension, GRAF1 is activated and recruited to the plasma membrane, where it mediates the internalization of excess membrane (Lundmark et al., 2008). Silencing GRAF1 expression stimulates cell blebbing and promotes migration and invasion in 3D (Holst et al., 2017).

3.5. P190A

After DLC1, the best characterized RhoGAP in cancer is p190RhoGAP. There are two p190RhoGAP paralogs in humans, p190A (also known as p190RhoGAP-A, ARHGAP35, or GRLF1 - glucocorticoid receptor DNA-binding factor 1) and p190B (or p190RhoGAP-B, ARHGAP5). Both p190A and p190B are large multi-domain proteins that include a GTP-binding domain, four FF domains, and two pseudo-GTPase domains in addition to a RhoGAP domain. The pseudo-GTPases domains are structurally like Rho GTPase domains but are catalytically inactive and regulate RhoA signaling (Stiegler and Boggon, 2017). Both p190A and B show specificity primarily for RhoA in vitro, but they also have activity for several other Rho family members including, RhoB, RhoC, RhoD, Rac1, Rac3, and Cdc42 (Amin et al., 2016; Burbelo et al., 1995; Ridley et al., 1993; Settleman et al., 1992).

In cells, both p190RhoGAPs are generally regarded as major negative regulators of RhoA, where they play key roles in integrin mediated cell adhesion, stress-fiber formation, leading edge protrusion, cell migration, and cell invasion (Arthur and Burridge, 2001; Arthur et al., 2000; Bradley et al., 2006; Nakahara et al., 1998; Vincent and Settleman, 1999). p190A has a protrusion localization sequence (PLS) that targets the protein to the leading edge where it regulates RhoA and cell migration (Biname et al., 2016). There is also evidence suggesting that some of the cellular processes regulated by the p190RhoGAPs are not mediated by RhoA. For example, p190A has been shown to regulate lamellipodia and invadopodia dynamics through local control of RhoC activity, a process that also involves the RhoC exchange factor p190RhoGEF (Bravo-Cordero et al., 2011; Bravo-Cordero et al., 2013). Additionally, both p190A and p190B have the fascinating ability to switch specificity from RhoA to Rac1 upon interacting with phospholipids (Lévay et al., 2013; Lévay et al., 2009; Ligeti et al., 2004).

Most of the p190RhoGAP mediated cellular processes are important during cancer progression, so it is unsurprising that both proteins have been associated with several types of cancer (reviewed in (Heraud et al., 2019)). Unlike DLC1, which is often deleted in cancer, p190A is instead typically mutated, with many of the mutations being nonsense or frameshift deletions/insertions (Heraud et al., 2019). Large genome-wide studies have identified several mutations spanning the whole protein in a wide range of cancer types (Campbell et al., 2016; Kandoth et al., 2013; Lawrence et al., 2014). A recent study has characterized several of these high frequency occurring mutants, many of them in the PLS region, and shows the mutations affect both the localization and GAP activity of p190A (Biname et al., 2016).

Both p190A and p190B promote contact inhibition in epithelial cells in a RhoGAP-dependent manner, and thus may act as tumor suppressors (Frank et al., 2018). p190RhoGAPs regulate contact inhibition by repressing the activity of Yes-associated protein (YAP) activity, and the loss of either p190A or p190B is sufficient to perturb contact inhibition through YAP signaling (Frank et al., 2018). p190A (but not p190B) also interacts with multiple translation preinitiation complex subunits, including all the subunits of eIF3, and may function in the assembly of translation pre-initiation complexes (Parasuraman et al., 2017).

Consistent with a tumor suppressor role, p190A expression is downregulated in HCC and endometrial samples (Kurokawa et al., 2003; Wen et al., 2020). In addition, the gene for p190A maps to a chromosomal region that is frequently deleted in a variety of human solid tumors including glioblastomas, astrocytomas, lung, endometrial, kidney, and head and neck tumors (Lawrence et al., 2014; Tikoo et al., 2000; Wen et al., 2020; Wolf et al., 2003b; Zack et al., 2013). p190A activation state has been found to be misregulated in different types of cancer. In prostate cancer, a microRNA (miRNA), miR-20A, that targets the p190A kinase ABL2 is overexpressed and is associated with poor prognosis (Qiang et al., 2014). Since phosphorylation by ABL2 promotes p190 activation, downregulation of ABL2 by miR-20A results in a decrease in p190A activation. Inhibiting miR-20A decreases migration and invasion through the activation of p190A (Qiang et al., 2014). Similarly, the regulation of p190A activity plays a key role during regulation of melanoma invasion and metastasis (Bartolome et al., 2014; Bartolome et al., 2008; Molina-Ortiz et al., 2009). In melanoma cells, p190A is phosphorylated and activated by the Src family kinase Blk, resulting in a decrease in RhoA activity and impaired migration, invasion, and metastasis (Bartolome et al., 2014; Bartolome et al., 2008; Molina-Ortiz et al., 2009). p190A also correlates with the expression levels of E-cadherin, which is downregulated at the initial stages of melanoma when the cells are more invasive (Molina-Ortiz et al., 2009). Interestingly, overexpression of RhoA with the RhoGAP domain of p190A fused to the C-terminus has been tested successfully in mice as a therapeutic approach to reduce RhoA-mediated invasion and metastasis in pancreatic cancer cells (Kusama et al., 2006).

p190 deletion/downregulation may promote tumor progression in oligodenrogliomas by preventing cells from reaching a further differentiated phenotype. Deletion or downregulation of p190RhoGAP alters the proper differentiation of oligodendrocytes, a cell type that is presumed to be the progenitor for oligodendrogliomas. p190A phosphorylation by the tyrosine kinase Fyn, leads to an in increase in its RhoGAP activity and a subsequent decrease in RhoA activity, promoting the differentiation of oligodendrocytes (Wolf et al., 2001). Coincidingly, overexpression of the RhoGAP domain of p190A suppresses proliferation and reduces the incidence of oligodendrogliomas, with cells adopting a more differentiated phenotype (Wolf et al., 2003b). Another study found that p190A levels are downregulated by platelet derived growth factor (PDGF), which is upregulated in oligodendrogliomas and is known to block the progression of oligodendroglial precursors to oligodendrocytes (Bögler et al., 1990; Guha et al., 1995; Wolf et al., 2003b). Similarly, there is some evidence that p190A inactivation in endometrial cancer leads to aberrant activation of Hippo-YAP pathway outputs resulting in malignant transformation. It may therefore be possible to treat endometrial cancers with YAP small-molecule inhibitors when p190A is suppressed (Wen et al., 2020).

Even though most of the documented cases describe p190A as a tumor suppressor, there are some examples that suggest it can also function as an oncogene (Heraud et al., 2019). In CRC, increased expression of p190A is associated with poor prognosis and shorter disease-free survival (Li et al., 2016b). Expression of specific polymorphic variants at the 3'-UTR of p190A in osteosarcoma has also been correlated with poor outcome, larger tumor size, tumor dedifferentiation, and increasing metastatic risk (Zhao et al., 2014). In breast cancer, the expression levels of p190A were associated with the incidence of bone metastasis (Chiu et al., 2017). p190A is also expressed at high levels and associated with poor prognosis in lung adenocarcinoma (Notsuda et al., 2013). When the expression of p190A is silenced in lung adenocarcinoma cell lines, cell proliferation and cell migration/invasion are significantly impaired. Mechanistically, p190A phosphorylation downstream of EGFR promotes the formation of a p190A/p120RasGAP complex formation, which reduces the RhoGAP activity of p120RasGAP. In the absence of p190A, or when EGFR is inhibited, the p190A/p120RasGAP complex is disrupted and Ras is inactivated (Notsuda et al., 2013).

3.6. P190B

Unlike p190A, the evidence suggests that p190B acts predominantly as an oncogene despite having similar specificity and functions (see section on p190A) (Frank et al., 2018). The expression of p190B is upregulated in HCC by CD147, a transmembrane glycoprotein that has been shown to regulate adhesion, migration, invasion, and metastasis (Chen et al., 2016b). Elevated levels of p190B inhibit RhoA activity in HCC, which promotes cell migration (Chen et al., 2016b). p190B levels are also regulated by microRNAs. Wang and colleagues have found that miR-486–5p, one of the most downregulated microRNAs in lung cancer, targets p190B in NSCLC (Wang et al., 2014c). Reduced expression miR-486–5p in NSCLC correlates with increased p190B expression, enhanced migration and invasion, advanced stage, and lymph node metastasis (Wang et al., 2014c). Silencing p190B inhibits migration and invasion in NSCLC, which supports the role of p190B as a functional target of miR-486–5p (Wang et al., 2014c). In contrast, in nasopharyngeal cancer (NPC) the microRNA miR-744 is upregulated and targets the promoter region of p190B, which stimulates p190B transcription, resulting in an increase in expression. Unsurprisingly, miR-744 expression was found to stimulate migration and invasion in vitro and promotes tumor growth and metastasis in vivo (Fang et al., 2015). p190B upregulation in clinical specimens of NPC correlates with advanced clinical stage, lymph node metastasis, and miR-744 expression (Fang et al., 2015). Finally, p190B activity is also upregulated in breast cancer, where high expression levels of the oncogene MCT-1 (multiple copies in T-cell malignancy) correlate with p190B stimulation (Wu et al., 2014). MCT-1 interacts with p190B and enhances its activity through Src phosphorylation of p190B, which results in asymmetrical cell division, chromosomal miss-segregation, and cytokinesis failure (Wu et al., 2014). This study proposes that abrogating MCT-1 function may be a viable therapeutic in breast cancer, as targeting MCT-1 suppresses tumor growth. Similarly, p190B overexpression has been shown to alter normal mammary gland development, and induces hyperplastic lesions, while p190B haploinsufficiency has been shown to inhibit mouse mammary tumor virus (MMTV)-Neu breast tumor initiation (Heckman-Stoddard et al., 2009; Vargo-Gogola et al., 2006). There is also a report that shows copy number variants identified for p190B in colorectal adenoma patients that may be predictive of tumor predisposition (Horpaopan et al., 2015).

3.7. FIlGAP (ARHGAP24), ARHGAP22, and ARHGAP25

The FilGAP subfamily of RhoGAPs comprise three members, FilGAP, ARHGAP22, and ARHGAP25, all sharing a similar domain structure and specificity for Rac1 (Akilesh et al., 2011; Csepanyi-Komi et al., 2012; Itoh et al., 2002; Lavelin and Geiger, 2005; Ohta et al., 2006; Sanz-Moreno et al., 2008). While FilGAP has been shown to have activity for Cdc42 in vitro, in vivo studies support FilGAP as a Rac1 specific RhoGAP (Ohta et al., 2006). In terms of expression, both FilGAP and ARHGAP22 are generally ubiquitous, while ARHGAP25 is mostly in myeloid tissues (Csepanyi-Komi et al., 2012). FilGAP has been studied extensively, and it represents one of the best characterized examples of how crosstalk between RhoA and Rac1 is regulated (Guilluy et al., 2011). In brief, FilGAP functions downstream of RhoA and its effector ROCK, which phosphorylates and activates FilGAP to specifically inactivate Rac1 at the leading edge (Ohta et al., 2006; Saito et al., 2012).

All the RhoGAPs in the FilGAP family appear to have similar functions, although there is some discrepancy in whether they have tumor suppressive or oncogenic roles in cancer. Expression of FilGAP is decreased in several cancers, including CRC, breast cancer, lung cancer, and renal cancer, suggesting a tumor suppressor role (Dai et al., 2018b; Wang et al., 2019a; Wang et al., 2019b; Xu et al., 2016; Zhang et al., 2018c). In NSCLC and breast cancer cells, silencing FILGAP expression promotes cell migration and invasion, while overexpression has the opposite effect, with an increased expression of E-cadherin and decreased expression of MMP9, VEGF, Vimentin, and β-catenin (Dai et al., 2018b; Wang et al., 2019a). Similarly, ectopic expression of FilGAP in triple-negative breast cancer (TNBC) cells attenuates phosphorylation of STAT3 and expression of MMP-2 and MMP-9 (Dai et al., 2018b). FilGAP overexpression has been also shown to promote cell cycle arrest and apoptosis in NSCLC cells, in a process that involves signaling through STAT6 (Wang et al., 2019b). In addition, overexpression of FilGAP inhibited the formation of lung and renal tumors in vivo (Wang et al., 2019b; Xu et al., 2016). Interestingly, FilGAP expression was found to be elevated in GII astrocytomas as compared to normal astrocytes. However, FilGAP levels decreased stepwise from GII through GIII to GIV tumors, and the patients who displayed high FilGAP expression had more favorable overall survival and progression free survival as compared to the low FilGAP score patients, which suggests FilGAP has a tumor suppressive role in astrocytomas despite the elevated expression observed (Hara et al., 2016). There is at least one example where FilGAP may display oncogenic properties. In B-cell lymphomas FilGAP expression is higher when compared to normal lymph nodes and this high expression levels correlate with poor prognosis (Nishi et al., 2015).

Cancer cells utilize different strategies for migration and invasion. For example, tumor cells can switch between two motility modes: mesenchymal, which is characterized by an elongated morphology and depends on extracellular proteases, and amoeboid, characterized by a rounded, high actomyosin contractility and independence from extracellular proteases (Friedl and Wolf, 2003). These two modes of movement are interconvertible, and tumor cells may undergo amoeboid-mesenchymal and mesenchymal-amoeboid transitions (Friedl and Wolf, 2003). This plasticity may allow cancer cells to adapt to different environments and can also limit the potential of some therapies that target a particular migration mode, e.g., protease dependent (Wolf et al., 2003a). Interestingly, FilGAP has been shown to play a role in regulating the switch between mesenchymal and amoeboid motility. In breast, prostate, lung, and CRC cancer cells, high levels of FilGAP correlate with low Rac1 activity and round/amoeboid morphology, while low levels of FilGAP correlate with high Rac1 activity and mesenchymal morphology (Saito et al., 2012). This correlation between cell morphology and FilGAP expression has also been observed in grade IV astrocytomas, with elongated cells enriched in tumor lesions displaying low FilGAP expression, and rounded cells with high FilGAP levels in the peripheral areas adjacent to non-neoplastic brain tissues (Hara et al., 2016). Silencing endogenous FilGAP expression increases the proportion of mesenchymal elongated cells in astrocytomas, probably through increased Rac1 activity (Hara et al., 2016). FilGAP suppression of mesenchymal movement is regulated by the Ras-GAP RASAL2, which is overexpressed and promotes tumorigenesis in TNBC in a RAS-independent manner (Feng et al., 2014). RASAL2 binds to and inhibits FilGAP, which results in increased Rac1 activation and mesenchymal invasion (Feng et al., 2014).

In melanoma and TNBC cells, the alternative splicing regulator RBM10 binds to FilGAP and targets it to the cell periphery in a process that requires the activity of Fyn (Yamada et al., 2016). Association of RBM10 with FilGAP may stimulate the RacGAP activity of FilGAP and suppress cell spreading. In MDA-MB-231 TNBC cells, FilGAP functions downstream of the small GTPase ARF6 to negatively regulates pseudopod formation (Uehara et al., 2017).

Both ARHGAP22 and ARHGAP25 seem to be regulated in a similar manner to FilGAP to modulate amoeboid/mesenchymal motility (Ladhani et al., 2011; Sanz-Moreno et al., 2008; Thuault et al., 2016). In melanoma cells, the RhoGEF DOCK3 activates Rac1 and promotes mesenchymal movement, while during amoeboid movement, the RhoA effector ROCK activates ARHGAP22, which then downregulates Rac1 activity (Ladhani et al., 2011; Sanz-Moreno et al., 2008). ARHGAP22 and DOCK3 can also be regulated at the level of expression during these processes (Ladhani et al., 2011; Sanz-Moreno et al., 2008). For example, pigment epithelium–derived factor (PEDF) promotes the upregulation of DOCK3 and downregulation of ARHGAP22 (Ladhani et al., 2011), and the promotor region of ARHGAP22 is hypermethylated in invasive melanoma cells (Koroknai et al., 2019).

A recent article has characterized the role of ARHGAP25 as a tumor suppressor in pancreatic adenocarcinoma (PAAD) (Huang et al., 2021). Both PAAD tissues and cell lines express lower levels of ARHGAP25 compared to their normal controls, and the expression levels of ARHGAP25 were inversely correlated with proliferation and tumor growth in mouse xenografts (Huang et al., 2021). Mechanistically, ARHGAP25 appears to control proliferation through the Akt/mTOR pathway, and at low levels of ARHGAP25, there is an increase of mTOR/HIF-1α/PKM2 mediated glycolysis, which is inhibited when ARHGAP25 is overexpressed (Huang et al., 2021).

ARHGAP25 was also found to be a negative regulator in both CRC and lung cancer migration and invasion, and its expression is significantly lower in patients with CRC and lung cancer (Tao et al., 2019a; Tao et al., 2019b; Xu et al., 2019a). Overexpression of ARHGAP25 inhibits CRC and lung cancer cell growth, suppresses cell migration and invasion, and reduces the expression of MMPs, EMT-associated factors, and β-catenin (Tao et al., 2019b; Xu et al., 2019a). The anti-metastatic role of ARHGAP25 can be attributed, at least in part, to its role as a negative regulator of the Wnt/β-catenin pathway (Tao et al., 2019a; Tao et al., 2019b; Xu et al., 2019a). ARHGAP25 was found to be upregulated in alveolar rhabdomyosarcoma (ARMS) (Thuault et al., 2016). ARMS-derived cell lines are highly invasive and adopt a rounded morphology during invasion. ARHGAP25 is activated by ROCKII and inhibits Rac1 activity, favoring the rounded morphology. Surprisingly, the activation of ROCKII is independent of RhoA. Instead, ROCKI activity increase is due to the downregulation of RhoE, which functions as a ROCK inhibitor (Thuault et al., 2016).

There is one report showing ARHGAP22 is upregulated in renal cell carcinoma (RCC) cells treated with JQ1, an inhibitor of BRD4, a member of the bromodomain family proteins which have been shown to regulate oncogene expression in different cancer types (Sakaguchi et al., 2018).

3.8. ARHGAP15

ARHGAP15 is one of the smallest RhoGAPs, consisting of a RhoGAP domain and an N-terminal PH domain, and shows specificity for Rac1 in vitro (Seoh et al., 2003). The expression of ARHGAP15 is suppressed in malignant gliomas, CRC, lung cancer, and breast cancer tumors, and this downregulation correlates with shorter disease-free survival in lung, CRC, pancreatic and breast tissues and with tumor grade in gliomas (Liao et al., 2017; Liu et al., 2019c; Pan et al., 2018a; Sun et al., 2017; Takagi et al., 2018).

In lung cancer cells, overexpression of ARHGAP15 suppresses cell proliferation, migration, and invasion, and reduces the expression of MMP2, MMP9, VEGF, as well as the phosphorylation of STAT3 (Liu et al., 2019c). ARHGAP15 also suppresses migration, cell growth, and invasion (Pan et al., 2018a). In glioma cells, ARHGAP15 expression is regulated by FOXP3 (forkhead box protein P3) (Sun et al., 2017), a transcription factor that functions as a tumor suppressor in a wide variety of cancers (Martin et al., 2010). Overexpression of FOXP3 increases the expression of ARHGAP15, which in turn inhibits the activity of Rac1 (Sun et al., 2017). Interestingly, ARHGAP15 was detected in 47% of breast carcinoma samples, while it was almost negligible in morphologically normal tissues (Takagi et al., 2018). However, patients that presented higher ARHGAP15 immunoreactivity had improved prognosis and decreased risk of recurrence, suggesting ARHGAP15 is still acting as a tumor suppressor when expressed in breast tumors (Takagi et al., 2018).

3.9. ARHGAP9

ARHGAP9 is a Cdc42 and Rac1-specific RhoGAP that has both tumor suppressive and oncogenic roles. The domain structure of ARHGAP9 is comprised of an SH3 domain, a WW domain and a PH domain followed by the RhoGAP domain. ARHGAP9 is associated with poor prognosis and disease-free survival in HCC tissue, as well as in both non-muscle invasive and muscle invasive bladder cancer (BCa) (Furukawa et al., 2001; Piao et al., 2020; Zhang et al., 2018a). In HCC cells, ARHGAP9 overexpression suppresses migration, invasion, and in vivo lung metastasis by upregulating the expression of E-cadherin through the transcription factor FOXJ2 (Forkhead box protein J2) (Zhang et al., 2018a). There is no conclusive evidence on whether the catalytic activity of ARHGAP9 is required for its tumor suppressor activity. However, a clue on the potential mechanism comes from a report showing that ARHGAP9 interacts with the MAP kinases Erk2 and p38α, which are frequently activated during metastasis, and inhibits their activity through a RhoGAP domain independent mechanism (Ang et al., 2007). A study by Sun et al. found that ARHGAP9 could be upregulated in liver cancer cells through treatment with Rg3 (Ginsenoside), a naturally occurring phytochemical with anti‑metastasis effects in many types of cancer (Sun et al., 2019b). The increased ARHGAP9 expression via Rg3 treatment resulted in suppressed migration and invasion of liver cancer while the effects were significantly reduced when ARHGAP9 expression was silenced (Sun et al., 2019b).

ARHGAP9 has also been characterized as a potential oncogene. An analysis of The Cancer Genome Atlas (TCGA) shows that ARHGAP9 forms part of a hub of 16 genes that are highly upregulated in head and neck squamous cell carcinoma (HNSCC) and have a negative impact on survival (Song et al., 2019). In acute myeloid leukemia (AML), ARHGAP9 overexpression was associated with decreased overall survival and resistance to chemotherapies (Han et al., 2021a while silencing the expression of ARHGAP9 was associate with a reduced metastatic phenotype {He, 2021 #2708). However, patients with high ARHGAP9 expression were more responsive to autologous or allogeneic hematopoietic stem cell transplantation suggesting ARHGAP9 expression could be a useful prognostic tool with AML patients (Han et al., 2021a).

3.10. RLIP76

RLIP76 (RALBP1, RIP1, or RLIP1) is a well-studied RhoGAP domain containing protein that also has a polybasic region (PBR) and a Ral binding domain (RALBD). While predominantly known as an effector for Ral, RLIP76 has been shown to have RhoGAP activity for Cdc42 and, to a lesser extent, Rac1 (Cantor et al., 1995; Jullien-Flores et al., 1995; Park and Weinberg, 1995). RLIP76 is a frequent target for novel therapeutics as it is overexpressed in many cancers, including ovarian cancer, bladder cancer, CRC, adrenocortical tumors, NSCLC, glioblastomas, HCC, meningiomas, lung cancer, breast cancer, pancreatic ductal adenocarcinoma (PDA), gastric cancer, and oral cancer (Durand et al., 2011; Ezzeldin et al., 2014; Fan et al., 2015; Haixia et al., 2015; Hudson et al., 2007; Ieong et al., 2019; Male et al., 2012; Mollberg et al., 2012; Seifert et al., 2016; Smith et al., 2007; Wang et al., 2015; Wang et al., 2013; Wang et al., 2016c; Yoshida et al., 2010; Zhang and Li, 2016). Additionally, several single nucleotide polymorphisms (SNPs) in RLIP76 have been identified to be associated with metastatic colorectal cancer and increased recurrence-free survival (Stremitzer et al., 2015; Volz et al., 2015). Even though the role of RLIP76 in cancer is well established, little is known about its RhoGAP activity in cancer. It may be that the tumor promoting role of RLIP76 is largely independent of its activity for Cdc42 and Rac1 although this would need to be verified.

RLIP76 has been linked to several pathways that can explain its role in tumorigenesis. For example, RLIP76 is an effector for Ral and RalA promotion of anchorage-independent growth in CRC is dependent on binding to RLIP76 (Martin et al., 2011). Although, RLIP76 can also promote tumorigenesis through other mechanisms as expression of RLIP76 was found to be necessary for metastasis in prostate and bladder cancer cell lines in a RalA independent manner (Wu et al., 2010). While still poorly understood, the RhoGAP activity of RLIP76 may also play a role in cancer progression as RhoGAP activity is activated by Aurora-A which is frequently overexpressed in cancers (Lim et al., 2010). RLIP76 also interacts with Epsin, a clathrin-mediated endocytic adaptor protein that is frequently upregulated in cancer, to enhance invasion and migration in fibrosarcoma cells (Coon et al., 2010).

RLIP76 promotes the development of drug resistance in many cancers (Awasthi et al., 2007). It is a trans-membrane protein that functions as a high-capacity mercapturic acid pathway (MAP) transporter (Cornish et al., 2021) that is responsible for the excretion of many chemotherapeutics such as doxorubicin (Adriamycin) (Awasthi et al., 2002; Singhal et al., 2021). Furthermore, high expression levels are associated with reduced sensitivity to radiation and therefore provides resistance to radiation therapy (Awasthi et al., 2005). This radiation resistance has been mapped to RLIP76 modification by SUMO-1 and ubiquitin in response to DNA damage which are required for the activation of NF-κB and tumor cell survival (Yang et al., 2011).

One of the key functions of RLIP76 is in regulating apoptosis. The high expression of RLIP76 in many cancers protects against apoptosis as depletion results in enhanced apoptosis in several models (Singhal et al., 2018; Singhal et al., 2006; Singhal et al., 2009; Singhal et al., 2007; Singhal et al., 2005; Stuckler et al., 2005; Zhang et al., 2015). This has also been shown in mice. RLIP76 and p53 depleted mice are highly resistant to carcinogenesis when compared to p53(-/-) mice (Awasthi et al., 2018). Depletion of RLIP76 is a promising means for cancer treatment as the presence of RLIP76 on the outside of the cell makes it an accessible target for therapeutics (Singhal et al., 2017).

3.11. ARHGAP30

ARHGAP30 is a RhoGAP with specificity for Rac1 and RhoA and no known auxiliary domains (Naji et al., 2011). ARHGAP30 also binds to the atypical Rho GTPase Wrch-1 (RhoU) in a RhoGAP-independent manner and seems to function downstream of Wrch-1 to regulate cell adhesion and stress fibers (Naji et al., 2011). Expression of ARHGAP30 is downregulated in lung cancer tissues and cell lines, where it functions as a tumor suppressor by via reducing migration, invasion, and the Wnt/β-catenin pathway (Mao and Tong, 2018). ARHGAP30 is also downregulated in CRC patients, with low levels being associated with poor survival (Wang et al., 2014b). In CRC, ARHGAP30 binds to p53 and its transcriptional co-activator p300, which promotes the activation of p53 by facilitating its acetylation by p300 (Wang et al., 2014b). These effects are independent of ARHGAP30 RhoGAP activity, as a catalytic dead mutant can still mediate the transactivation of p53 (Wang et al., 2014b). In pancreatic cancer, ARHGAP30 is significantly increased in tumors, and patients with higher expression have better prognosis (Zhou et al., 2020). Overexpression and knock down experiments show that ARHGAP30 reduces pancreatic cancer cell proliferation and metastasis and promotes apoptosis. ARHGAP30 expression is accompanied by reduced β-catenin, MMP2, and MMP9 and with increased levels of Bcl-2-associated X protein (Bax) and cleaved caspase-3 (Zhou et al., 2020). Similarly, ARHGAP30 overexpression significantly inhibited lung metastasis in vivo in nude mice and increased the survival of mice with lung metastases (Zhou et al., 2020) ARHGAP30 also has low expression in cervical cancer and overexpressing ARHGAP30 results in reduced proliferation and migration while promoting apoptosis. Furthermore, ARHGAP30 was found to interact with and suppress nucleolin, and subsequently, global protein synthesis (Wu et al., 2021).

3.12. CDGAP

CdGAP (ARHGAP31) is closely related to ARHGAP30 (see previous section). However, despite having some overlapping roles, CdGAP promotes tumor invasiveness while ARHGAP30 is a tumor suppressor (He et al., 2017; Naji et al., 2011). This may be due to their differences in specificity, with ARHGAP30 showing RhoGAP activity for RhoA and Rac1, and CdGAP for Rac1 and Cdc42 (Karimzadeh et al., 2012; LaLonde et al., 2006; Lamarche-Vane and Hall, 1998; Naji et al., 2011; Wormer et al., 2012) or possibly because the oncogenic role of CdGAP may not require RhoGAP activity. CdGAP localizes to cell-matrix adhesions where it regulates focal adhesion dynamics, lamellipodia formation, and cell migration in a Rac1-dependent manner (LaLonde et al., 2006; Wormer et al., 2012; Wormer et al., 2014).

Despite its role inhibiting migration in vitro, CdGAP is expressed at high levels in a mouse mammary tumor explants and basal-type breast cancer cells, and silencing its expression inhibits cell migration and invasion downstream of TGFβ (He et al., 2017; He et al., 2011). This was confirmed by silencing the expression of CdGAP in ErbB2-transformed breast cancer cells which inhibited tumor growth and metastasis to the lungs in mice (He et al., 2017). Surprisingly, the regulation of TGFβ-mediated cell migration and invasion by CdGAP does not require its catalytic domain, suggesting that the activities of Rac1 and Cdc42 are not essential in this pathway (He et al., 2011)

Beyond regulating cell migration, CdGAP was also found to be important for EMT (He et al., 2017; McCormack et al., 2017). CdGAP forms a complex with the transcriptional repressor Zeb2 to suppress the expression of E-cadherin and disrupt cell-cell junctions (He et al., 2017). Interestingly, CdGAP-Zeb2 mediated repression of E-cadherin is also RhoGAP-independent, suggesting some of the key functions of CdGAP may be as an adaptor or scaffold (He et al., 2017). This process is regulated by the actin binding and bundling protein Ajuba, which binds to and inhibits CdGAP and prevents CdGAP-mediated downregulation of E-Cadherin at cell-cell junctions and subsequent EMT (McCormack et al., 2017). CdGAP also regulates EMT, apoptosis, and cell cycle progression in prostate cancer where expression was found to be associated with early biochemical recurrence and bone metastasis (Mehra et al., 2021).

3.13. α-CHIMAERIN and β-CHIMAERIN

The chimaerin sub-family of RhoGAPs comprises two genes, α- and β-chimaerin (CHN1 and CHN2), which encode at least four alternative spliced isoforms: α1-, α2-, β1-, and β2-chimaerin (Yang and Kazanietz, 2007). Besides the RhoGAP domain, which is specific for Rac1 (Diekmann et al., 1991; Leung et al., 1993); chimaerins also contain a C1 domain, which allows them to function as receptors for the lipid second messenger DAG and the phorbol ester tumor promoters. α2- and β2-chimaerins represent the longer isoforms and encode an additional SH2 domain (Yang and Kazanietz, 2007).

Most reports show the β-chimaerin isoform playing a tumor suppressor role, with decreased expression in high-grade malignant gliomas and breast cancer (Menna et al., 2003; Yang et al., 2005; Yuan et al., 1995). Overexpression of β-chimaerin in MCF-7 breast cancer cells inhibits cell proliferation in a Rac-GAP dependent manner (Yang et al., 2005). Additionally, expression of the RhoGAP domain of β-chimaerin significantly reduced the growth rate and invasive ability of mammary carcinoma tumors in mice (Menna et al., 2003). In vivo, deletion of β-chimaerin in the MMTV-Neu/ErbB2 mouse breast cancer model increases their susceptibility to develop breast tumors, with more tumors initiated in β-chimaerin-KO mice during the time of primary tumor growth (Casado-Medrano et al., 2016). However, even though more tumors are formed in the absence of β-chimaerin, tumor progression seems to be delayed and most tumors formed are of lower grade and less aggressive (Casado-Medrano et al., 2016). These discrepancies are not uncommon and may reflect differences in the cell types and mouse models utilized for these experiments.

A recent report has shown that the activity of β-chimaerin is repressed downstream of VEGF and NRP2 (neuropilin 2) signaling (Elaimy et al., 2018). NRP2 is highly expressed in breast cancer-stem cells (CSCs) and VEGF−/NRP2 signaling contributes to breast tumor initiation (Goel et al., 2013). Mechanistically, VEGF/NRP2 signaling promotes the activation of Rac1, which inhibits LATS, allowing TAZ to target to the nucleus where it associates with TEAD and represses β-chimaerin, which helps maintaining high Rac1 activity in a positive feedback loop (Elaimy et al., 2018).

There is at least one report suggesting a potential oncogenic role for β-chimaerin. β-chimaerin was identified as one of the genes upregulated in Hepatosplenic T-cell lymphoma (HSTL), an aggressive lymphoma cytogenetically characterized by isochromosome 7q [i(7)(q10)] (Finalet Ferreiro et al., 2014). The authors propose that the enhanced expression of β-chimaerin leads to downregulation of the NFAT pathway and a proliferative response (Finalet Ferreiro et al., 2014).

Like β-chimaerin, α-chimaerin (originally named n-chimaerin) is also a receptor for phorbol esters, but most of the studies on α-chimaerin are related to brain function, as it is expressed almost exclusively in the brain and has been implicated in the regulation of the dynamics of the neurocytoskeleton organization (Ahmed et al., 1990; Ahmed et al., 1993; Hall et al., 1990; Yang and Kazanietz, 2007). However, a small number of studies suggests that α-chimaerin plays a role in cancer but, unlike β2-chimaerin, it may function as an oncogene. α-chimaerin is expressed at high levels in esophageal squamous cell carcinoma and cervical cancer tissues, where the expression levels in cervical cancer were associated with increased risk of metastasis and shorter survival. Overexpression of CHN1 in cervical cancer cells promoted cell proliferation, migration, invasion, and EMT via the Akt/GSK-3β/Snail pathway whereas silencing α-chimaerin had the opposite effect. Overexpression of α-chimaerin in vivo promoted tumor formation (Zhao et al., 2021). In prostate cancer, α-chimaerin is target of the tumor suppressive miR-205, which is downregulated in prostate cancer (Couch et al., 2016; Gandellini et al., 2009). Surprisingly, a recent study showed that in cervical cancer miR-205 may act as an oncogene, by positively regulating α-chimaerin expression to mediate cell growth, apoptosis, migration, and invasion (Liu et al., 2020).

3.14. ARHGAP21

ARHGAP21 is a large RhoGAP (1958 aa) that bridges the ARF and Rho families of small GTPases by functioning as an effector for Arf1 at the Golgi complex and as a RhoGAP for Cdc42 or RhoA/C (Bigarella et al., 2009; Dubois et al., 2005; Javadi et al., 2017; Lazarini et al., 2013; Sousa et al., 2005). There are discrepancies in the reported specificity of ARHGAP21, which may be in part attributed to a historic misunderstanding, as ARHGAP21 is frequently misnamed as ARHGAP10, and several reports use both gene names interchangeably. However, ARHGAP21 and ARHGAP10 are different genes located on chromosomes 10 and 4 respectively and have quite different domain structures. It is therefore necessary to take care when reading the literature for either of the proteins so as not to make errors.

The catalytic activity of ARHGAP21 can be regulated through protein-protein interaction. ARHGAP21 forms a complex with β-arrestin that suppresses its RhoGAP activity for RhoA (Anthony et al., 2011). This interaction is stimulated by PTEN, one of the most frequently mutated tumor suppressor genes in human cancer (Lee et al., 2018), which is both recruited to the membrane and activated by β-arrestin (Lima-Fernandes et al., 2011). In the absence of PTEN, the interaction between ARHGAP21 and β-arrestin is disrupted, which results in higher ARHGAP21 activity, decreased Cdc42, and defects in 3D glandular morphogenesis (Javadi et al., 2017).

ARHGAP21 has been identified in multiple studies as a negative regulator of cancer cell migration, invasion, EMT, and tumor growth (Barcellos et al., 2013; Bigarella et al., 2009; Luo et al., 2016). ARHGAP21 expression is decreased in ovarian, lung, prostate, and colon cancer with low expression levels generally correlating with worse prognosis (Gong et al., 2019; Liu et al., 2019a; Luo et al., 2016; Teng et al., 2017). In ovarian cancer cells, CXCL12 stimulation results in a significant decrease in ARHGAP21 expression levels, a process that is mediated by VEGF/VEGFR signaling and results in enhanced cell invasion (Luo et al., 2019). ARHGAP21 mRNA and protein levels are also downregulated in GC by miR-337–3p, which targets the 3’-untranslated region of ARHGAP21 (Wang et al., 2020). While a transcriptome analysis has shown increased levels of ARHGAP21 in HNSCC (Carles et al., 2006), more work needs to be done to determine if ARHGAP21 expression is positively associated with any other cancers.

ARHGAP21 function has been linked to several key pathways, including the endothelin-1 pathway, the Wnt signaling pathway, and the PI3K/AKT pathway, and while the details of ARHGAP21 involvement have not been fully characterized, some studies have shed light on potential mechanisms (Bigarella et al., 2009; Gong et al., 2019; Lazarini et al., 2013; Liu et al., 2019a; Teng et al., 2017). For example, in glioblastoma cells, ARHGAP21 interacts with FAK and prevents its phosphorylation (Bigarella et al., 2009). Silencing ARHGAP21 expression increases FAK phosphorylation, Cdc42 activity, MMP-2 production, and subsequently cell migration. Similarly, in lung cancer ARHGAP21 overexpression inhibits MMP-2, MMP-9, VEGF, and the Wnt pathway proteins β-catenin and c-Myc (Teng et al., 2017). Finally, ARHGAP21 also interacts with α-tubulin and may contribute to the regulation of α-tubulin acetylation and EMT (Barcellos et al., 2013).

3.15. RACGAP1

RacGAP1 (also known as MgcRACGAP or Cyk4) is one of the best characterized RhoGAPs and plays a key role in cytokinesis during the formation of the cleavage furrow. The formation of the cleavage furrow requires RhoA activation by the RhoGEF Ect2, which is regulated by autoinhibition and is activated by the centralspindlin complex, a tetramer comprised of a dimer of the kinesin motor KIF23 and a dimer of RacGAP1. Centralspindlin localizes to the equatorial plasma membrane in anaphase where it recruits and activates Ect2, which in turn activates RhoA to promote the formation of the contractile ring (Basant and Glotzer, 2018; Chen et al., 2015). The catalytic specificity of RacGAP1 in the centralspindlin complex has been a matter of active debate for several years (for a review see (Basant and Glotzer, 2017). In vitro, RacGAP1 shows specificity for Rac1, Cdc42, and RhoD, with only minimal activity towards RhoA. However, the evidence in vivo is not conclusive and the RhoGAP activity of RacGAP1 during cytokinesis has been found to be required for RhoA activation, for Rac1 inactivation, or totally dispensable, depending on the experimental system used (Amin et al., 2016; Barfod et al., 1993; Bastos et al., 2012; Jantsch-Plunger et al., 2000; Toure et al., 1998; Verma et al., 2019).

RacGAP1 has been identified as an oncogene in many types of cancer, with high levels of expression observed in HCC, meningioma, NSCLC, CRC, basal-like breast cancer, uterine carcinoma, epithelial ovarian cancer, pancreatic ductal adenocarcinoma, and esophageal carcinoma (Imaoka et al., 2015; Ke et al., 2013; Khalid et al., 2019; Lawson et al., 2016; Liang et al., 2013; Mi et al., 2016; Wang et al., 2018a; Wang et al., 2011; Yang et al., 2018b; Yin et al., 2019). RacGAP1 is associated with poor patient survival and resistance to the chemotherapy drug doxorubicin in HNSCCs, early recurrence and poor prognosis in breast cancer (especially in luminal (ER+) tumors), and poor survival in bronchopulmonary neuroendocrine neoplasms (BP-NEN) (Hazar-Rethinam et al., 2015; Milde-Langosch et al., 2013; Neubauer et al., 2016; Pliarchopoulou et al., 2013; Sahin et al., 2016). Similarly, expression of RacGAP1 at the invasive front in GC correlates with tumor size, lymph node metastasis, lymphatic invasion, vascular invasion, advanced stage, and poor prognosis (Saigusa et al., 2015).

RacGAP1 has also been associated with the regulation of migration and invasion. Phosphorylation of RacGAP1 promotes its recruitment to invasive pseudopods through its interaction with IQGAP1, where it suppresses the activity of Rac1 while promoting the activity of RhoA (Jacquemet et al., 2013). Interestingly, RacGAP1 is also enriched 10-fold in exosome vesicles secreted from colon carcinoma cells which, when isolated from metastatic cells, transfer amoeboid phenotype to non-metastatic cells (Schillaci et al., 2017).

The localization of RacGAP1 in cells may be key in understanding its function as an oncogenic RhoGAP. Early work found that RacGAP1 localizes to the nucleus in interphase, the mitotic spindle in metaphase, and the midbody during cytokinesis (Hirose et al., 2001). In HCC, RacGAP1 promotes tumor growth by inhibiting the Hippo and YAP pathways and promoting cytokinesis (Yang et al., 2018b). In NSCLC and basal-like breast cancer (BLBC), silencing the expression of RacGAP1 causes cytokinesis defects (Lawson et al., 2016; Liang et al., 2013). Without RacGAP1 expression to regulate RhoA, BLBC cells grow slower due to a combination of cytokinesis failure, CDKN1A/p21-mediated RB1 inhibition, and the onset of senescence (Lawson et al., 2016). Nuclear expression of RacGAP1 was associated with worse survival in primary CRC patients when compared to cytoplasmic expression of RacGAP1 (Imaoka et al., 2015; Yeh et al., 2016). Also, RacGAP1 is largely nuclear in both ovarian and uterine cancer and, in NSCLC, RacGAP1 was found to localize to the nucleus in non-dividing cells and in the cytoplasm in dividing cells (Liang et al., 2013; Mi et al., 2016; Wang et al., 2018a).

RacGAP1 expression is also regulated by miRNA. A recent study has shown that miR-15a-5p, which functions as a tumor suppressor in HCC, targets both RacGAP1 and its pseudogene RacGAP1P (Wang et al., 2019c). The RacGAP1P pseudogene was identified to be upregulated in HCC and leads to larger tumors and reduced survival (Wang et al., 2019c). RacGAP1P acts as an oncogene by serving as a sponge for miR-15–5p and subsequently increasing RacGAP1 expression (Wang et al., 2019c). RacGAP1 is also targeted by miR-4324, a tumor suppressor miRNA which has been found to be significantly downregulated in BCa (Ge et al., 2019). Consequently, RacGAP1 expression levels are significantly upregulated in BCa. Restoring miR-4324 expression in BCa cells suppresses cell proliferation and metastasis in vivo and enhances chemotherapy sensitivity to doxorubicin in vitro by repressing RacGAP1 expression (Ge et al., 2019). Similarly, silencing RacGAP1 expression significantly inhibited BCa malignant biological behavior in vitro (Ge et al., 2019). Furthermore, rescuing RacGAP1 expression in miR-4324 expressing cells inhibited miR-4324-mediated suppression of BCa cell proliferation and migration (Ge et al., 2019). The report by Ge and colleagues also dissects a feedback loop controlling miR-4324 downregulation through RacGAP1. RacGAP1 induces the phosphorylation of STAT3, which mediates the methylation of the estrogen receptor 1 (ESR1) promoter resulting in downregulation of ESR1. Since the expression of miR-4324 is regulated by the estrogen receptor 1, high levels of Rac1GAP correlate with a decrease in miR-4324 levels and promotes the oncogenic behavior of BCa cells (Ge et al., 2019).

Because of its oncogenic role, RacGAP1 is a potential target for therapeutics. Chang and colleagues found that FIP-fve, a protein isolated from Flammulina velutipes that possesses anti-inflammatory and immunomodulatory activities, has potential anti-cancer effects as it downregulates RacGAP1 mRNA and protein expression and therefore cell migration in lung cancer cells (Chang et al., 2013). Also, of interest are BET inhibitors (BETi), which are a potential therapeutic agent being tested in clinical trials. BETi target the transcription of several oncogenic genes, including RacGAP1 (Pham et al., 2019).

3.16. ARHGAP29 and HMHA1

ARHGAP29 (PTPL1 associated RhoGAP1 or PARG1), and minor histocompatibility antigen 1 (HMHA1 or ARHGAP45) are two closely related RhoGAP proteins that also encode an F-BAR domain and a C1 domain. HMHA1 was Initially characterized as an autosomal minor H antigen, and only later identified as a RhoGAP (de Kreuk et al., 2013; Goulmy et al., 1983). HMHA1 is a RhoGAP for RhoA, Rac1, and Cdc42 in vitro, but there is evidence suggesting it may prefer Rac1 in vivo. ARHGAP29 prefers RhoA as a substrate, but also shows reduced activity in vitro towards Rac1 and Cdc42 (Amado-Azevedo et al., 2018; de Kreuk et al., 2013; Saras et al., 1997). The F-BAR domain of HMHA1 plays an autoinhibitory role in its RhoGAP function, whereas the C1 domain suggests that these proteins may be regulated by DAG like chimaerins, although this has yet to be tested (de Kreuk et al., 2013).

Like other RhoGAPs, ARHGAP29 seems to have both oncogenic and tumor suppressing roles. For example, expression of ARHGAP29 is increased in GC, HCC, prostate cancer, and RCC and correlates with poor prognosis, but it is decreased in mantle cell lymphoma (Miyazaki et al., 2017; Qiao et al., 2017; Ripperger et al., 2007; Shimizu et al., 2020). Circulating tumor cells (CTCs) also show increased ARHGAP29 expression in a pancreatic cancer mouse model, and the elevated expression of ARHGAP29 is positively correlated with metastatic potential of CTCs (Qiao et al., 2017). Similarly, in an orthotopic spontaneous HCC metastasis assay, silencing ARHGAP29 significantly reduced metastasis to the lung in vivo (Qiao et al., 2017). ARHGAP29 likely controls metastasis by regulating cancer cell migration, which increases when ARHGAP29 is transcriptionally activated by YAP (Qiao et al., 2017). This leads to the suppression of the RhoA-LIMK-cofilin pathway and inhibits actin polymerization (Qiao et al., 2017). In RCC, ARHGAP29-mediated increase in proliferation and invasion through the inhibition of the RhoA/ROCK (Miyazaki et al., 2017).

The expression of HMHA1 is dramatically increased in melanoma and aberrantly expressed in several leukemia/lymphoma cells lines, as well as in different solid tumor cell lines (Fujii et al., 2002; Klein et al., 2002; Miyazaki et al., 2003; Xu et al., 2017b). Since minor histocompatibility H antigens are important targets for immunotherapy, HMHA1 could potentially be used as a therapeutic target (Spierings et al., 2004).

3.17. SRGAP subfamily (SRGAP1–4)

The Slit-Robo GTPase-activating protein family of RhoGAPs (srGAP1–4) were first identified as potential Slit-Robo effectors because their C-terminal SH3 domain binds the intercellular domain of the Robo family receptors, whose canonical ligands are members of the Slit family of secreted proteins (Lucas and Hardin, 2017). In the last decade, the Slit/Robo pathway, which was originally associated with axon guidance in the nervous system, has been shown to play a role in a wide variety of cancer types, where it can act to either promote or suppress tumorigenesis (Ballard and Hinck, 2012). All srGAPs encode a F-BAR domain at the N-terminus, which allows for interactions with membranes, followed by the RhoGAP catalytic domain, and the above mentioned SH3 domain. SrGAPs show varying specificities for Rho GTPases, although that may be attributed to the assays utilized and whether the activity was analyzed in vitro or in vivo. srGAP2, srGAP3, and srGAP4 have preferential activity for Rac1, but srGAP3 and srGAP4 can also inactivate Cdc42 (Foletta et al., 2002; Guerrier et al., 2009; Ma et al., 2013; Mason et al., 2011; Soderling et al., 2002; Yang et al., 2006). SrGAP1 on the other hand, seems to preferably inactivate RhoA, although there are reports showing activity on Cdc42 and Rac1, (Liang et al., 2017; Liang et al., 2018; Ma et al., 2013; Wong et al., 2001). SrGAP4 has also been reported to inactivate RhoA (Christerson et al., 2002; Foletta et al., 2002).

SrGAP1 expression is downregulated in several cancer types, including CRC, glioblastoma, and NSCLC, suggesting it functions a tumor suppressor (Feng et al., 2016; Koo et al., 2015; Tang et al., 2010). In glioblastoma multiform cells, downregulation of srGAP1 appears to be mediated by miR-145, which is overexpressed in highly invasive glioblastoma cells (Koo et al., 2015; Koo et al., 2012). In CRC, decreased srGAP1 expression is associated with lymphatic invasion, poor tumor differentiation, high tumor, node, metastasis (TNM) stage, and poor survival (Feng et al., 2016). SrGAP1 interacts with Robo1 upon stimulation by Slit2 in CRC and suppresses Cdc42 activity and cell motility (Feng et al., 2016).

Depending on the cancer type, srGAP1 may also have an oncogenic role. For example, srGAP1 was found to be a target of two tumor suppressive miRNAs, miR-340 and miR-124, which are downregulated in primary gastric tumors (Huang et al., 2018). The downregulation of miR-340 and miR-124 observed in GC is, at least in part, responsible for srGAP1 overexpression, and silencing srGAP1 expression in GC cells reduces cell proliferation, colony formation, cell invasion, and migration (Huang et al., 2018). This is supported by data which shows that srGAP is upregulated in 75% of GC cell lines tested, and the high srGAP1 levels predict poor survival. The oncogenic role of srGAP1 seems to also be mediated by the activation of the Wnt/β-catenin pathway (Huang et al., 2018) although further study is required.

Compared to srGAP1, there is not as much information regarding the role of the other srGAP family members in cancer. SrGAP2 expression is reduced or absent in primary osteosarcoma samples, and overexpression of srGAP2 reduces cell migration in osteosarcoma cell lines, suggesting it may function as a tumor suppressor (Marko et al., 2016). However, in HCC srGAP2 has increased expression and is associated with poor patient outcomes, likely by promoting migration and invasion (Li et al., 2021). SrGAP3 functions as a tumor suppressor in human mammary epithelial cells (HMECs), where its expression is absent in 70 percent of the breast cancer cell lines tested (Lahoz and Hall, 2013). Silencing srGAP3 expression in HMEC cells promotes Rac dependent, anchorage-independent growth, a process that requires Rac1 activity (Lahoz and Hall, 2013). In contrast, srGAP3 expression inhibits HMEC cell invasion and anchorage dependent cell growth in a RhoGAP dependent fashion and correlates with an increase in RhoA/ROCK signaling, suggesting srGAP1 may contribute to the regulation of the known crosstalk between Rac1 and RhoA (Lahoz and Hall, 2013).

SrGAP4 (ARHGAP4) protein expression is markedly downregulated in pancreatic cancer tissues, and low ARHGAP4 levels correlate with reduced survival in patients (Shen et al., 2020; Shen et al., 2019a; Shen et al., 2019b). Recent studies show that srGAP4 is a target of miR-939–5p, which is upregulated in pancreatic cancer tissues and contributes to the malignant phenotype (Shen et al., 2020; Shen et al., 2019a). Overexpression of srGAP4 inhibits the increased viability, migration, and invasion induced by miR-939–5p upregulation in pancreatic cancer cells (Shen et al., 2020). A recent report has shed some light on the role of srGAP4 in pancreatic cancer and suggests may play a role in the regulation of the Warburg effect, an increase in glucose uptake and lactate production observed in tumors, which occurs even in the presence of oxygen and mitochondria (Shen et al., 2019b). The study shows that overexpression of srGAP4 inhibits cell viability, glucose uptake, lactate release, mTOR phosphorylation, and expression of HIF-1α and PKM2, all key players in the regulation of metabolic adaptations in tumors, whereas srGAP4 silencing has the opposite effect. Finally, srGAP4 silencing has been shown to promote EMT and increased migration in MCF10a cells (Kang et al., 2020).

3.18. MYO9B

Myo9B belongs to the Mammalian class IX myosin, which includes both myosin IXa (Myo9A) and myosin IXb (Myo9B) and encode for a single-headed motor with a RhoA-specific RhoGAP domain in the tail region (Muller et al., 1997; Reinhard et al., 1995; Wirth et al., 1996). Myo9B is also known by the name CELIAC4 because polymorphisms in this gene have been suggested to be a marker for celiac disease (Chen et al., 2016c). Polymorphisms have been identified in Myo9B in association with other disorders such as multiple sclerosis, Crohn’s, and ulcerative colitis (Kemppinen et al., 2009; Li et al., 2016c).

Myo9B is part of a Slit2/Robo/Myo9B/RhoA signaling pathway that restricts lung cancer growth and metastasis (Kong et al., 2015). Increased Slit2 expression or Slit2 treatment leads to RhoA activation and inhibits cancer cell migration. Upon Slit2 stimulation, the transmembrane receptor Robo binds to and inhibit the RhoGAP activity of Myo9B, which results in an increase in GTP-bound RhoA and a subsequent suppression of cell invasion and migration (Kong et al., 2015). Similarly, in prostate cancer cells, high levels of Myo9b inhibit RhoA activity resulting in reduced number stress fibers and increased migration, which is inhibited when Myo9B is silenced (Makowska et al., 2015). In lung and prostate cancer, Myo9B was found expressed at high levels and associated with poor prognosis (Kong et al., 2015; Makowska et al., 2015) and in esophageal adenocarcinoma, Myo9B is also associated with increased risk (Menke et al., 2012).

3.19. ARHGAP18

ARHGAP18, also referred to by the name MacGAP, is a RhoA/C-specific RhoGAP that encodes a RhoGAP domain but no other known domains (Chang et al., 2014; Maeda et al., 2011). ARHGAP18 appears to have both tumor suppressing and oncogenic roles in cancer. In GC, ARHGAP18 functions as a tumor suppressor, with lower expression levels in GC tumors when compared to normal tissue (Li et al., 2018). Overexpression of ARHGAP18 in GC cells suppresses cell viability, migration, invasion, as well as in vivo tumor formation (Li et al., 2018). In neuroendocrine tumors, treatment with the synthetic somatostatin analogue octreotide triggers a progressive increase in ARHGAP18 expression (along with other genes), also suggesting a tumor suppressor role (Li et al., 2012). ARHGAP18 is downregulated during lymphovascular invasion in breast cancer, an indicator of metastatic potential and poor outcome, and increased expression of ARHGAP18 mRNA and protein levels is associated with a better outcome (Aleskandarany et al., 2017). Similarly, loss of ARHGAP18 was found to promote tumor vascularization and subsequent tumor growth in melanoma tumors in ARHGAP18 negative mice (Chang et al., 2014). ARHGAP18 appears to function as a negative regulator of angiogenesis by promoting vascular stability and limiting pro-angiogenic signaling through RhoC, which is a contributing factor to cancer development (Chang et al., 2014).

The ARHGAP18 gene locus is a common integration site for the cancer-causing MMTV (Kim et al., 2011). MMTV promotes transformation by integrating near cellular oncogenes in mammary epithelial cells (Callahan and Smith, 2000). The insertional mutation caused by MMTV identifies ARHGAP18 as a potential oncogene in breast cancer (Kim et al., 2011). ARHGAP18 has also been shown to be oncogenic by acting as a RhoGAP for RhoA in TNBC and its expression levels correlate with shorter patient survival (Aguilar-Rojas et al., 2018; Humphries et al., 2017). Downregulation of ARHGAP18 increases RhoA activation but impairs growth, migration, and metastatic capacity as part of a miR-200b and ROCK pathway (Humphries et al., 2017).

3.20. BPGAP1 and CDC42GAP

BPGAP1 (ARHGAP8) and Cdc42GAP (also known as ARHGAP1, RhoGAP1, or p50RhoGAP) are two very similar RhoGAPs, both encoding a BNIP-2 and Cdc42GAP/p50RhoGAP Homology (BCH) domain at the N-terminus, followed by a proline rich region and the RhoGAP domain. Cdc42GAP was the first identified RhoGAP and displays activity on a wide range of Rho proteins, including Cdc42, which appears to be the preferred substrate, but also RhoA, RhoB, Rac1, TC10, and TCL (Amin et al., 2016; Barfod et al., 1993; Lancaster et al., 1994; Neudauer et al., 1998; Ridley et al., 1993; Zhang and Zheng, 1998). BPGAP1 on the other hand, shows specificity for Cdc42 and RhoA in vitro, and no significant activation toward Rac1 GTPase activity (Shang et al., 2003). However, in vivo it seems to preferentially modulate RhoA activity (Shang et al., 2003).

The BCH domain was initially identified as a region of high protein sequence homology between BNIP-2 (BCL2/adenovirus E1B 19kDa interacting protein-2) and Cdc42GAP/p50RhoGAP (Low et al., 2000). BCH domains belong to a larger family of lipid binding domains named Sec14, also named CRAL_TRIO (Bankaitis et al., 2010). However, they are considered ‘Sec14-like’ domains, with only 14% sequence identity with the CRAL_TRIO domain of the Saccharomyces cerevisiae Sec14p protein (Gupta et al., 2012). Unlike other CRAL_TRIO domains of the Sec14 superfamily, BCH domains are not known to interact with lipid molecules and their postulated non-protein ligands are currently unknown (Gupta et al., 2012). Instead, BCH domains have the unique ability to interact with and control the activation/inactivation of Rho GTPases and their regulators (including RhoGEFs and RhoGAPs) (Chichili et al., 2021; Soh and Low, 2008; Zhou et al., 2010; Zhou et al., 2005, 2006). For example, the BCH domain of Cdc42GAP serves as a local modulator by binding to RhoA and preventing its inactivation by the adjacent RhoGAP domain (Chichili et al., 2021; Zhou et al., 2010).

The gene for BPGAP1 is in a region of the genome that suffers frequent loss of heterozygosity in CRC and breast tumors (Johnstone et al., 2004). However, mutational analysis revealed no somatic mutations in CRC and breast tumors, which suggests that BPGAP1 is unlikely to be a tumor suppressor (Johnstone et al., 2004). Instead, BPGAP1 has been found to be upregulated in primary CRC tumors and in invasive cervical cancer (Johnstone et al., 2004; Song et al., 2008). While little is known about the mechanisms by which BPGAP1 may promote cancer, it is important in the regulation of cell motility. BPGAP1 has been shown to promote protrusion and enhance cell migration through binding to cortactin (Lua and Low, 2004; Shang et al., 2003).

BPGAP1 also regulates the internalization of EGFR, through its interaction with EEN/endophilin I and the phosphorylation of Erk1/2 in both a RhoGAP-dependent and -independent manner (Lua and Low, 2005; Pan et al., 2010). BPGAP1 activity and Erk1 phosphorylation are controlled by a feedback loop that includes Mek1 and the peptidyl-prolyl cis/trans isomerase (PPI) Pin1, which associates with BPGAP1 and inhibits its RhoGAP activity, the magnitude of the Erk1 response, and subsequently cell motility (Pan et al., 2010). Similarly, in breast cancer cells, BPGAP1 promotes Erk1 activation on late endosomes through its interaction with MP1, a specific endosomal scaffold for Mek1 and Erk1. This regulatory function requires phosphorylation of BPGAP1 by JNK, which releases its autoinhibitory conformation and promotes cell proliferation and transformation (Jiang et al., 2017).

Unlike BPGAP1, Cdc42GAP does not have an obvious oncogenic or tumor suppressive role as it has been found both up and downregulated in different cancers. For example, Cdc42GAP is upregulated in metastatic brain tumors from primary lung adenocarcinoma (Zohrabian et al., 2007), whereas in cervical carcinoma cells and in prostate cancer, the expression of Cdc42GAP is lower in cancerous tissue when compared to normal tissues (Davalieva et al., 2015; Li et al., 2017b).

Several reports show that Cdc42GAP expression can be regulated by miRNAs in different cancer types although there are discrepancies on whether Cdc42GAP functions as an oncogene or tumor suppressor. An oncogenic role is supported by reports showing that Cdc42GAP is targeted by the tumor suppressor miRNAs miR-509–3p, which is downregulated in osteosarcoma and ovarian cancer, and miR-34b-5p, which is downregulated in breast cancer (Dong et al., 2020; Pan et al., 2016; Patil et al., 2019; Zhang et al., 2018d). However, downregulation of the Cdc42GAP-specific miRNAs miR-34b-5p and miR-509–3p, in breast cancer and osteosarcoma respectively, were found to be associated with worse prognosis and metastatic phenotype (Dong et al., 2020; Patil et al., 2019). Also, hsa-miR-940, which is highly enriched in exosomes, is secreted by metastatic prostate cancer cells and targets Cdc42GAP (Hashimoto et al., 2018). hsa-miR-940 secreted from prostate cancer cells promotes the osteogenic differentiation of mesenchymal stem cells (hMSCs) in vitro and induces osteoblastic lesions in the bone metastatic microenvironment in vivo, which is accompanied by a reduction of Cdc42GAP levels (Hashimoto et al., 2018). miR-130b, which is upregulated in Ewing sarcoma (ES), also targets Cdc42GAP (Satterfield et al., 2017). The miR-130b-mediated downregulation of Cdc42GAP in ES is correlated with poor prognosis. miR-130b promotes invasion, migration, and proliferation in vitro, and increased metastatic potential in vivo (Satterfield et al., 2017). By targeting Cdc42GAP, miR-130b promotes the activation of Cdc42, which in activates its downstream effector PAK1, a well characterized contributor to oncogenic signaling cascades in different cancer types (Radu et al., 2014). Activation of PAK1 stimulates the JNK and AP-1 cascades and their downstream transcriptional targets, which include IL-8, MMP1, and CCND1. AP-1 also promotes the expression of miR-130b, thus completing an oncogenic positive feedback loop. Interestingly, the oncogenic properties of miR-130b can be blocked by a PAK inhibitor, highlighting its therapeutic potential (Satterfield et al., 2017).

3.21. ARHGAP20

ARHGAP20 (also known as RA-RhoGAP) is a PH and Ras-Associating domain-containing RhoGAP. ARHGAP20 was initially identified by Katoh and Katoh, who characterized the KIAA1391 (ARHGAP20) gene within the 11q23.1 commonly deleted region in a variety of human tumors, including breast cancer, lung cancer, colorectal cancer, ovarian cancer, endometrial cancer, cervical cancer, malignant melanoma, and neuroblastoma (Katoh and Katoh, 2003). ARHGAP20 was also identified as one of the genes disrupted in a translocation detected in two B-cell chronic lymphocytic leukemia patients (B-CLL) along with BRWD3, which codes for a putative novel transcription factor located at Xq13 (incorrectly identified as Xq21 in the paper). Both genes are inactivated because of the translocation however the remaining copy was significantly upregulated (Kalla et al., 2005). A second study found that 11q22–23 deletions were common in CLL, associated with worse prognosis, and were associated with upregulated ARHGAP20 mRNA despite the deletion (Herold et al., 2011). In HCC however, ARHGAP20 was found to be downregulated in tumors compared with normal controls, with downregulation being correlated with larger tumor size and vascular invasion. Exogenous expression of ARHGAP20 in HCC cell lines significantly inhibited wound healing, cell migration, and cell invasion, as well as lung metastasis in nude mice (Liu et al., 2021a).

3.22. ARHGAP11A

ARHGAP11A is an oncogenic RhoGAP with no defined domains other than the RhoGAP domain, which is specific for RhoA (Kagawa et al., 2013; Lawson et al., 2016). It has been found to be upregulated in many cancers including colon cancer, glioblastoma, lung cancer, breast cancer, GC, HCC, and pancreatic cancer (Chen et al., 2021; Dai et al., 2018a; Fan et al., 2021; Fu et al., 2015; Kagawa et al., 2013; Lawson et al., 2016; Lu et al., 2017; Yan et al., 2021a). The upregulation of ARHGAP11A in cancer inhibits RhoA dependent processes, including the formation of stress fibers and focal adhesions (Kagawa et al., 2013). ARHGAP11A-mediated RhoA suppression promotes Rac1 activation, which results in a more migratory and invasive phenotype (Kagawa et al., 2013). Interestingly, ARHGAP11A also binds to Rac1b, an alternatively spliced isoform of Rac1 associated with cancer progression, although independently of its RhoGAP activity (Dai et al., 2018a; Melzer et al., 2019). This interaction has been shown to facilitate HCC malignant progression (Dai et al., 2018a).

ARHGAP11A expression is regulated throughout the cell cycle and increases in S, G2, and M phases (Kagawa et al., 2013). Knockdown of ARHGAP11A arrests breast cancer cells in G1 phase, suppresses migration, and increases cell spreading (Lawson et al., 2016). However, the specific role of ARHGAP11A in the cell cycle may be cell type specific. In a study by Xu and colleagues, ARHGAP11A was characterized as a regulator of cell cycle but in a tumor suppressive capacity (Xu et al., 2013). They show that ARHGAP11A induces cell cycle arrest and apoptosis by binding to the tumor suppressor p53 through a mechanism independent of the RhoGAP activity (Xu et al., 2013). The RhoGAP domain of ARHGAP11A binds to the tetramerization domain of p53 and stabilizes the tetrameric conformation of p53, enhancing its DNA-binding activity (Xu et al., 2013). Upon DNA damage stress, ARHGAP11A expression is upregulated and translocated to the nucleus, where it binds to p53 and induces cell-cycle arrest and apoptosis (Xu et al., 2013).

RNA can also regulate ARHGAP11A expression in cancer. Expression of ARHGAP11A is decreased in breast cancer by the PIWI-interacting RNA (piRNA) piR-021285, which promotes methylation of the ARHGAP11A gene (Fu et al., 2015). A SNP in piR-021285 has been associated with attenuated methylation of the ARHGAP11A gene along with increased mRNA expression of ARHGAP11A and increased invasiveness in breast cancer cells (Fu et al., 2015).

3.23. ARHGAP19

ARHGAP19 is a small RhoA-specific RhoGAP that is mostly expressed in hematopoietic cells (David et al., 2014). While not much is known about this RhoGAP, it plays a role in lymphocyte mitosis, where it controls two key processes: the timing of morphological changes in early mitosis and chromosome segregation in anaphase (David et al., 2014; Marceaux et al., 2018). Phosphorylation by ROCK and CDK1 controls ARHGAP19 localization and prevents its translocation to the cell cortex, which is essential for normal mitosis progression by preserving RhoA activity in the cortex, which is needed for cortical contractility (David et al., 2014; Marceaux et al., 2018).

There is only one report showing ARHGAP19 as an oncogene with increased expression in carcinoma cells (aggressive breast and endometrial). This is in response to downregulation of miR-200c, which targets ARHGAP19 and is frequently observed to have reduced levels in carcinoma samples that have undergone EMT (Howe et al., 2011). Restoring miR-200c expression inhibits aberrant expression of ARHGAP19 and suppresses cell migration in both aggressive breast and endometrial cancer cells (Gregory et al., 2008; Howe et al., 2011; Park et al., 2008). ARHGAP19 has also been identified as a target of the tumor suppressor miR-193b in ovarian cancer cells but its physiological relevance has not been characterized (Nakano et al., 2013).

3.24. RICH1 and RICH2

RICH1 (ARHGAP17 or Nadrin) and RICH2 (ARHGAP44) are two closely related RhoGAP proteins that contain N-BAR domains and display RhoGAP activity for Rac1 and Cdc42 (Amin et al., 2016; Richnau and Aspenstrom, 2001; Wells et al., 2006). Both proteins appear to be negative regulators of cancer development.

The expression of RICH1 is reduced in colon cancer, cervical cancer, and breast cancer, suggesting a potential tumor suppressor role (Guo et al., 2019; Kiso et al., 2018; Pan et al., 2018b). In TNBC, RICH1 expression is downregulated through VEGF/NRP1 signaling, which results in increased Cdc42 activity, filopodia formation, and cell migration (Kiso et al., 2018). In colon cancer, RICH1 overexpression inhibits cell growth and invasion and restricts metastasis to the lung in vivo (Pan et al., 2018b). This is likely associated with the Wnt signaling pathway and β-catenin expression as they are negatively correlated with RICH1 expression in colon cancer (Pan et al., 2018b). In cervical cancer, RICH1 inhibits PI3K/AKT signaling, which increases P21 and P27 expression to suppresses cell proliferation in vitro and tumor growth in vivo (Guo et al., 2019).

RICH1 has also been shown to play a role in neurofibromatosis Type 2 (NF2), an autosomal dominant disorder characterized by development of schwannomas, a tumor that develops from the Schwann cells in the peripheral nervous system (Yi et al., 2011). NF2 is caused by mutations and loss of heterozygosity of the NF2/Merlin tumor suppressor gene (Hanemann, 2008). Interestingly, both Merlin and RICH1 bind to Angiomotin and are recruited to cell-cell junctions where they are incorporated to the PatJ/Pals1/Angiomotin complex (Yi et al., 2011). Merlin and RICH1 compete for binding to Angiomotin, and this competition regulates the equilibrium between contact inhibition and proliferation (Yi et al., 2011). Under growth inhibition conditions such as the maturation of cell junctions, Merlin is recruited to cell junctions and releases RICH1 from the Angiomotin complex, allowing it to inhibit Rac1 activity. In response to growth stimuli, or when Merlin is mutated or inactivated, Merlin dissociates (or cannot associate) from Angiomotin (Yi et al., 2011). Binding of free Angiomotin to RICH1 inhibits its RhoGAP activity, leading to an increase in Rac1 activity and the downstream Ras-MAPK mitogenic pathways (Yi et al., 2011).

RICH2 has also been found to be downregulated in cancer, specifically in HCC and lung cancer (Xu et al., 2017a; Zhang et al., 2019). In HCC, RICH2 expression correlates negatively with tumor size, TNM stage, and metastasis, and, when overexpressed in vitro, it inhibits proliferation and invasion (Zhang et al., 2019). In lung cancer, RICH2 is a target of p53, which suppresses RICH2 transcription (Xu et al., 2017a). Increased expression of mutant p53 correlates with reduced RICH2 expression and higher levels of active Cdc42 (Xu et al., 2017a). In addition, RICH2 is upregulated in breast cancer cells when expression of the breast cancer susceptibility gene BRIP1 is silenced (Daino et al., 2013)

3.25. SH3BP1

SH3BP1 (ARHGAP43) is closely related to RICH1 and RICH2 (see section 3.24). However, unlike RICH1/2, SH3BP1 appears to function as an oncogene. SH3BP1 has RhoGAP activity for both Rac1, Cdc42, and RhoG, and is overexpressed in cervical cancer tissues, HCC, and prostate cancer (Cicchetti et al., 1995; Liu et al., 2017; Parrini et al., 2011; Tao et al., 2016; Wang et al., 2018b). In HCC and cervical cancer, overexpression of SH3BP1 promotes cell motility and invasion by regulating Rac1 activity and its effector WAVE2 (Tao et al., 2016; Wang et al., 2018b). Surprisingly, SH3BP1 expression promotes Rac1 activity, and silencing SH3BP1 reduces Rac1 activity. This suggests that SH3BP1 is somehow activating Rac1 despite having RhoGAP activity for Rac1 (Tao et al., 2016; Wang et al., 2018b). The mechanism by which SH3BP1 promotes Rac1 activation is not clear, but it probably involves crosstalk with a Rac-GEF. SH3BP1 has been proposed to regulate the local GDP/GTP cycling of Rac1 at the leading edge of migrating cells, and maybe overexpressing SH3BP1 perturbs this balance and results in increased Rac1 activity (Parrini et al., 2011). SH3BP1-mediated increase in Rac1-WAVE2 signaling promotes VEGF secretion to stimulate cell invasion and microvessel formation (Tao et al., 2016). SH3BP1 was also found to promote migration and invasion in prostate cancer as a direct downstream target of the Hippo pathway protein TAZ, which is known to be overexpressed in many cancers (Chen et al., 2016a; Liu et al., 2017; Skibinski et al., 2014; Yuen et al., 2013).

4. Lesser studied RhoGAPs in cancer

There are a handful of RhoGAPs that have little to no evidence for a role in cancer. The RhoGAPs ABR, TCGAP, ARHGAP23, GMIP, MYO9A, SYDE2, TAGAP, ARHGAP11B, ARHGAP39 currently have no known role. Here, we describe the RhoGAPs for which there is limited evidence to substantiate a role in cancer. However, further study is required to verify their role in tumorigenesis and the pathways in which they are involved.

4.1. OPHN1

Oligophrenin-1 (OPHN1) is a RhoGAP protein that, while ubiquitously expressed, is predominantly expressed in the neural tube during development and has been associated with X-linked intellectual disability (Billuart et al., 1998). OPHN1 shows specificity to a wide range of Rho GTPases, including RhoA, Rac1, Cdc42, TC10, and TCL (Amin et al., 2016; Billuart et al., 1998; Fauchereau et al., 2003). There is limited information on the role of OPHN1 in cancer, with reports showing increased levels in CRC tumors, glial tumors, and prostate cancer (Goto et al., 2014; Liu et al., 2022; Ljubimova et al., 2001; Pinheiro et al., 2001), as well as differential expression in GC exhibiting lymphovascular invasion (Dicken et al., 2006; Kim et al., 2012). OPHN1 expression increases in response to androgen deprivation therapy, a common method of treating prostate cancer, possibly facilitating therapy resistance. Increased OPHN1 in prostate cancer contributes to cancer progression by promoting cell survival and migration (Liu et al., 2022)

4.2. GRAF2 and GRAF3

GRAF2 was initially characterized as a RhoGAP with activity for Cdc42 and RhoA and is also a tumor suppressor (Ren et al., 2001). Sometimes GRAF2 (ARHGAP10) has been incorrectly referred to as ARHGAP21, a different RhoGAP gene that encodes PDZ domain and a PH domain (see section 3.14). GRAF2 is directly targeted and downregulated by miR-3174, which is significantly upregulated in GC and is associated with poor prognosis (Li et al., 2017a). miR-3174 prevents GC cell-death by inhibiting both apoptosis and autophagic cell death, and these effects can be rescued by re-expressing GRAF2 (Li et al., 2017a).

GRAF3 functions as a RhoGAP for RhoA primarily, but also shows activity for Cdc42 (Bai et al., 2013; Luo et al., 2017). Not much is currently known about GRAF3, although it has been identified to have an oncogenic role in NPC, where its expression is increased and it is associated with shorter metastasis-free survival (Hu et al., 2018). Silencing the expression of GRAF3 reduces NPC migration and invasion in vitro, and the opposite effect is observed when GRAF3 is overexpressed (Hu et al., 2018).

4.3. CAMGAP1

CAMGAP1 (ARHGAP27) is closely related to ARHGAP12 (see section 4.4), and shares the same domain structure, except for an additional WW domain. The RhoGAP activity of ARHGAP27 is specific for Rac1 and Cdc42 and shows no significant activity towards RhoA (Sakakibara et al., 2004). There is not much evidence supporting the role of ARHGAP27 in cancer, although it has been identified as a potential oncogene. ARHGAP27 is expressed at high levels in epithelial ovarian cancer, where it was identified as a candidate gene that affects tumor initiation and development (Permuth-Wey et al., 2013). Two methylation sites in the promoter of ARHGAP27 were found to be associated with decreased expression of ARHGAP27 and lower risk of epithelial ovarian cancer (Yang et al., 2019). Fang and colleagues found that rat glioma cells overexpressing CD133, a cell surface marker associated with CSCs, displayed enhanced stem cell properties, increased cell proliferation and migration, and higher ARHGAP27 expression (Fang et al., 2013). Silencing ARHGAP27 in these cells reduced invasion, which suggested a potential role for ARHGAP27 in cancer cell migration (Fang et al., 2013).

4.4. ARHGAP12

ARHGAP12 is a multidomain protein with an SH3 domain, two WW domains, and a PH domain, in addition to the RhoGAP domain, which is specific for Rac1 and, to a lesser extent, Cdc42 (Diring et al., 2019; Gentile et al., 2008). ARHGAP12 has been found downregulated by hepatocyte growth factor in epithelial cells, where it negatively regulates cell adhesion and invasion (Gentile et al., 2008). In melanoma cells, ARHGAP12 inhibits Rac1 and Cdc42 activity, F-actin assembly, invadopodia formation, and lung metastasis in mouse (Diring et al., 2019). Interestingly, ARHGAP12 interacts with G-actin, and since the interaction appears to inhibit its RhoGAP activity, it has been proposed that the binding to G-actin allows ARHGAP12 to function as an actin sensor to regulate cellular process that depend on local changes of F-actin concentration (Diring et al., 2019).

4.5. ARHGAP32

ARHGAP32 (also known as GRIT, RICS, GC-GAP, p250GAP, or p200RhoGAP) is a large RhoGAP containing only an SH3 domain besides the RhoGAP domain. ARHGAP32 is expressed mostly in the brain and has RhoGAP specificity for Cdc42, RhoA, and to a lesser extent Rac1 (Nakamura et al., 2002).

A recent article though, reported that in GC cells, the expression ARHGAP32 is regulated by CD73, (ecto-5′-nucleotidase; NT5E), a protein that catalyzes the conversion of extracellular AMP to membrane-permeable nucleosides (Xu et al., 2020). CD73 is overexpressed in several cancers and plays a role in cancer progression and survival via conversion of immunostimulatory ATP into immunosuppressive adenosine. In gastric cancer, overexpression of CD73, promotes the upregulation of ARHGAP32. CD73-mediated upregulation of ARHGAP32, inhibits RhoA signaling and promotes EMT, migration, and invasion (Xu et al., 2020).

4.6. ARHGAP28

ARHGAP28 is a negative regulator of RhoA and actin stress fiber formation and may also play a role during focal adhesion turnover, in a process controlled at the transcription level by YAP and TAZ (Mason et al., 2019; Yeung et al., 2014). There is virtually no information on ARHGAP28 in cancer, except for a report showing that in the highly metastatic colon cancer cell line KM12SM, the ARHGAP28 promoter region is highly methylated when compared to the parental cell line KM12C (Kasuya et al., 2016). This hypermethylation is correlated with an increase in RhoA activation suggesting downregulation of ARHGAP28 (Kasuya et al., 2016).

4.7. SYDE1

SYDE1 is a poorly characterized RhoGAP that has specificity for RhoA (Lo et al., 2017) and Rac1 and Cdc42 but (Amado-Azevedo et al., 2017). In terms of cancer, the expression of SYDE1 was found to be higher in gliomas compared to healthy tissues and the expression promotes invasion and metastasis but not growth. SYDE1 may have value as a marker for predicting prognosis of gliomas (Han et al., 2021b).

5. Pseudo-RhoGAPs

There are several RhoGAP domain containing proteins which have mutations that prevent them from stimulating the intrinsic Rho GTPase GTP-to-GDP hydrolysis (Amin et al., 2016). These RhoGAPs, often referred to as ‘pseudo-RhoGAPs,’ lack the arginine finger residue that is critical for catalytic activity. The function for these pseudo-RhoGAP domains is not known, but RhoGAP mutants at this site are still able to interact with their target Rho protein, so they may be able to influence Rho signaling by binding and sequestering their cognate Rho GTPase (Amin et al., 2016). Despite no known catalytic activity, these proteins still have important roles in the cell as scaffolding platforms, and their misregulation can still promote tumorigenesis. Here, we highlight three pseudo-RhoGAPs that have known roles in cancer.

5.1. ARHGAP36

There is not much known about the small RhoGAP ARHGAP36. The expression of ARHGAP36 is upregulated in human pheochromocytomas but not in other cancers tested such as colon, lung, bladder, breast, kidney, and prostate (Croise et al., 2016). High ARHGAP36 expression has been observed in medulloblastomas, the most common malignant childhood brain tumor, where it is correlated with poor prognosis, both in patients and in a mouse model (Beckmann et al., 2019; Rack et al., 2014). ARHGAP36 has been shown to activate the Hedgehog (Hh) signaling pathway, which is often misregulated in medulloblastoma (Kool et al., 2012; Rack et al., 2014). ARHGAP36 is also highly expressed in papillary thyroid carcinoma (PTC) where it promotes proliferation and migration (Yan et al., 2021b).

The role of ARHGAP36 during cancer progression may be related to its ability to suppress PKA signaling, a signaling pathway frequently altered in cancer and a negative regulator of the Hh pathway, which suggests that ARHGAP36 may activates Hh signaling by inhibiting PKA (Briscoe and Therond, 2013; Eccles et al., 2016). ARHGAP36 inhibits PKA through two distinct mechanisms: it binds to and inhibits PKAC (the catalytic subunits of PKA) and it also promotes its degradation (Eccles et al., 2016). While ARHGAP36 is a strong regulator of PKA, further study is required to determine of aberrant ARHGAP36 expression in cancer is related to its PKA activity.

5.2. DEPDC1B

DEPDC1B, named after its DEP (Dishevelled, Egl-1, Pleckstrin) domain, is overexpressed in oral cancer, NSCLC, prostate cancer, soft-tissue sarcomas, malignant melanoma, and neuroblastoma (Bai et al., 2017; Liu et al., 2019b; Pollino et al., 2018; Su et al., 2014; Xu et al., 2019c; Yang et al., 2014). In some cancer types, high DEPDC1B levels have been associated with shorter survival and metastatic progression (Pollino et al., 2018; Yang et al., 2014).

DEPDC1B is an effector of the proto-oncogene kinase Raf-1 and promotes a variety of tumorigenic process including cell survival, proliferation, anchorage-independent growth, migration, and invasion (Boudreau et al., 2007; Su et al., 2014; Yang et al., 2014). In NSCLC, the regulation of migration and invasion by DEPDC1B is mediated by the Wnt/β-catenin pathway (Yang et al., 2014). Despite being catalytically inactive, DEPDC1B has been shown to indirectly suppress RhoA activity, although the mechanism is not completely understood (Garcia-Mata, 2014; Marchesi et al., 2014). In neuroblastoma cells overexpressing N-Myc protein, the expression of DEPDC1B is regulated by the long non-coding RNA lncNB1, which is one of highest overexpressed transcripts in these tumors (Liu et al., 2019b). LncNB1 promotes DEPDC1B expression, which in turn induces ERK protein phosphorylation and N-Myc protein stabilization (Liu et al., 2019b). Silencing expression of IncNB1 in mice reduces the expression of DEPDC1B, which results in tumor regression (Liu et al., 2019b). DEPDC1B expression was found to promote metastatic phenotypes in chordoma through UBE2T-mediated ubiquitination of BIRC5 (Wang et al., 2021) and in HCC through interactions with CDK1 (Dang et al., 2021).

5.3. FAM13A

The RhoGAP FAM13A (ARHGAP48) is an important RhoGAP in the development of lung cancer. GWA studies have consistently identified FAM13A to be associated with pulmonary function in several lung diseases, including chronic obstructive pulmonary disease (COPD), asthma, pulmonary, fibrosis, and lung cancer (Cho et al., 2010; Fingerlin et al., 2013; Hancock et al., 2010; Pillai et al., 2010; Young et al., 2011). Interestingly, some FAM13A variants identified are associated with reduced risk, both in lung cancer (and COPD) (rs7671167) and breast cancer (rs1059122) (Cho et al., 2010; Wei et al., 2019; Young et al., 2011), while a different variant (rs9224), is associated with an increased risk in lung cancer (Yu et al., 2019). In NSCLC cell lines, the expression of FAM13A is upregulated by hypoxia, a major negative prognostic factor in cancers, and cells expressing FAM13A were found at higher frequency in the tumoral regions of the tissues in NSCLC patients (Eisenhut et al., 2017; Ziolkowska-Suchanek et al., 2017; Ziółkowska-Suchanek et al., 2021). Silencing FAM13A expression inhibits proliferation and promotes migration of NSCLC cells (Eisenhut et al., 2017). A recent study has shown that higher expression of FAM13A is associated with increased tumor shrinkage in response to neoadjuvant endocrine therapy (NAET) in estrogen receptor-positive primary breast cancer (Goto-Yamaguchi et al., 2018). This suggests that the FAM13A gene and other genes identified in this study may be used as marker for the choice of treatment in breast cancer patients (Goto-Yamaguchi et al., 2018).

FAM13A regulates RhoA activity indirectly in the lungs, as well as Wnt activity in both lung cancer cells and tissues which is associated with more aggressive tumor growth (Corvol et al., 2018; Jin et al., 2015). FAM13A shuttles between the nucleus and the cytosol depending on the phosphorylation status of Ser-322, which is regulated by the antagonistic action of the kinase Akt and B56-containing PP2As (protein phosphatase 2A) (Jin et al., 2015). When FAM13 is dephosphorylated, it moves to the nucleus and indirectly activates the Wnt pathway although the exact mechanism by which it does this is still poorly understood (Jin et al., 2015).

6. RhoGAP fusions

Genomic rearrangements can dramatically alter gene functions by amplification, deletion, or fusion. Since the discovery of the Philadelphia chromosome in 1960, gene fusions have been identified in a variety of cancer types (Dai et al., 2018c). Coincidentally, the Philadelphia chromosome (9–22 translocation), which is found in over 95% chronic myelogenous leukemia (CML), results in the fusion of the tyrosine kinase ABL with BCR, a protein encoding both a RhoGEF and a RhoGAP domain (Laurent et al., 2001). The BCR-ABL fusion has been extensively used as a genetic test for CML. The fusion cuts off the RhoGAP domain of BCR and results in constitutive tyrosine kinase activity through the Abl region, increasing cell migration and proliferation (Laurent et al., 2001; Sattler and Griffin, 2003). The ubiquity of BCR-ABL in CML has made it a promising target for a variety of therapeutics (see (Lin et al., 2016) for review). With advances in sequencing technologies, several other fusions involving RhoGAP genes have been observed in cancers. In many of these cases, these fusions promote tumorigenesis and are potential markers for cancer severity.

A fusion between GRAF1 and the mixed-lineage leukemia (MLL) gene was identified in an infant with juvenile myelomonocytic leukemia (Borkhardt et al., 2000). The predicted fusion protein consists of the N-terminal part of MLL and the C-terminal part of GRAF1. The RhoGAP domain of the fused allele is lost, and in several cases, the second allele presents mutations within the RhoGAP domain, suggesting that the RhoGAP domain of GRAF1 functions as a tumor suppressor (Borkhardt et al., 2000).

In some GCs, ARHGAP26 is found fused with the tight junction gene CLDN18 (claudin 18), which results in the loss of epithelial phenotype and with cells forming long protrusions suggestive of EMT (Yao et al., 2015). The CLDN18-ARHGAP26 gene product encodes a fusion protein containing all four transmembrane domains of CLDN18 and the RhoGAP domain of ARHGAP26 but lacking the C-terminal PDZ-binding motif of CLDN18 that mediates its interaction with TJ proteins. Cell lines expressing the CLDN18-ARHGAP26 fusion protein show reduced cell-cell adhesion, impaired barrier function, and are more invasive (Yang et al., 2018a; Yao et al., 2015). The CLDN18-ARHGAP26 fusion gene was also found in 25% of Signet-ring cell carcinoma in GC patients, who subsequently had worse survival outcomes and increased chemotherapy resistance (Shu et al., 2018). Interestingly, the CLDN18-ARHGAP26 fusion and two other fusions involving RhoGAPs, CTNND1 (p120 catenin)-ARHGAP26 and ANXA2 (Annexin A2)-MYO9A have been found to be common in diffuse-type gastric cancers (DGC) (Yang et al., 2018a). All these RhoGAP fusions have a similar genomic rearrangement with the RhoGAP domain at the C-terminus and have been associated with a significant worse prognosis (Tanaka et al., 2018; Yang et al., 2018a).

In gastric cardia adenocarcinomas (GCA), a fusion protein of bone marrow X kinase (BMX) and ARHGAP12 (BMX-ARHGAP) was identified by using paired-end RNA-Seq technology (Xu et al., 2014). Expression of BMX-ARHGAP in GC is associated with poor prognosis of patients with GC tumors and silencing its expression increased survival in a mouse xenograft model (Xu et al., 2019b). Mechanistically, when BMX-ARHGAP expression was silenced in GC, it reduced drug resistance, migration, invasion, EMT protein expression, and lymph node metastasis via downregulation of RhoA activity and the JAK/STAT3 pathway (Xu et al., 2019b).

In uterine perivascular epithelioid cell tumors, the fusion gene RAD51B-OPHN1 has been identified, although its role and prevalence in cancer is largely unknown (Agaram et al., 2015; Bennett et al., 2018). However, in the four instances where it has been identified, the RAD51B-OPHN1 fusion has been associated with an increased mitotic index as well as with recurrence and/or mortality (Agaram et al., 2015; Bennett et al., 2018).

Another RhoGAP protein that forms a gene fusion in cancer is srGAP3, which was found fused to RAF1 in astrocytoma (Forshew et al., 2009; Jain et al., 2017; Jones et al., 2009). SRGAP3-RAF1 is an oncogenic fusion that activate the MAPK and PI3K/mTOR signaling pathways, confers resistance to some RAF inhibitors, and is associated with poor survival (Jain et al., 2017). The RhoGAP domain of srGAP3 is missing in the fusion so it does not contribute to the phenotype. The fusion protein is also missing the autoinhibitory domain of RAF1, resulting in an increase in the kinase activity of RAF1, which is proposed to stimulate tumorigenesis in astrocytomas and pediatric low-grade gliomas (PLGGs) by activating both the MAPK and PI3K/mTOR signaling pathways (Jain et al., 2017; Jones et al., 2009).

Finally, in recent years, transcriptome-profiling studies have identified a different type of gene fusion, which originate from read-through transcription and are frequently overexpressed in tumors (Sultan et al., 2008; Wang et al., 2008). These read-through fusions have been also found to include RhoGAPs, including PRR5-ARHGAP8, which has been identified in colon, renal, and GCs, where it expresses at high levels, and TIMM23-ARHGAP32 in ER+ breast cancer cells (Choi et al., 2016; Fukamachi et al., 2014; Schulte et al., 2012).

7. RhoGAPs as drug targets

RhoGAPs are generally considered tumor suppressors, and the loss of RhoGAP activity allows for uncontrolled GTPase activity, which can promote transformation and cancer progression (Sahai and Marshall, 2002; Vigil et al., 2010; Zandvakili et al., 2017). However, an increasing number of oncogenic RhoGAPs have also been described recently. For the tumor suppressor RhoGAPs, a logical therapeutic approach would then be to develop small molecule agonists to increase the activity of a specific RhoGAP. Since it is generally easier to develop small molecule antagonists than agonists, this can represent a challenge (Vigil et al., 2010). For that reason, RhoGAPs have been considered less attractive as drug targets than RhoGEFs. Oncogenic RhoGAPs are more promising targets for small molecules, but so far, there are no reported inhibitors targeting RhoGAPs. There is, however, some encouraging evidence suggesting it may be possible to develop strategies to restore or enhance the activity of tumor suppressor RhoGAPs that are inactivated or downregulated in cancers. The best example is β2-chimaerin, which has a C1 domain that binds to DAG and phorbol esters, which function to relieve intramolecular inhibition, and thus activate the enzyme (Canagarajah et al., 2004). This suggests it may be possible to develop drugs targeting the C1 domain to selectively inactivate Rac1 in cancer cells. It would still be challenging, as other proteins encode C1 domains. However, it is still considered possible to achieve specific C1 activation (Blumberg et al., 2008).

An alternative strategy could be to restore the expression of the tumor suppressor RhoGAPs that are downregulated in cancer. For example, silencing FBXW5, which is part of the ubiquitin ligase complex that degrades DLC1, restores DLC1 expression (Kim et al., 2013). There are also many reports showing that RhoGAP expression is regulated by miRNAs in different cancer types (see above sections for examples). Therapies that target those miRNAs may help rescue the normal expression of specific RhoGAPs and restore their tumor suppressor function. Finally, it could also be possible to target other proteins in the RhoGAP pathways, such as those which regulate the expression or activity of the RhoGAP of interest.

8. Conclusion

In the last two decades, it has become increasingly clear that RhoGAPs play key roles in almost every aspect of cancer progression, including proliferation, migration, invasion, and metastasis. Many members of the RhoGAP family are directly involved in cancer progression. For the others, interfering with the homeostasis of the Rho GTPases network, which is intimately interconnected, may result in transformation indirectly. To understand the mechanisms of action of RhoGAPs in cancer transformation, it is essential to identify their interacting partners and define the signaling pathways that regulate their activation and function. This will allow for the identification of novel therapeutic strategies to target them.

There are several challenges ahead. The RhoGAP family is large and there is significant crosstalk among its members, making it difficult to isolate the signaling contribution of each individual RhoGAP. There are also considerable technical shortcomings in the study of RhoGAPs. First, most of the studies on RhoGAP function, especially the early ones, have relied on overexpressing the RhoGAP of interest (or its RhoGAP catalytic domain) in cells. The problem is that introducing large amounts of RhoGAP can result in a global, unregulated inhibition of its cognate Rho GTPase, which could muddle the interpretation of the results. Novel technologies, including CRISPR-mediated KO, knock-in or mutagenesis will allow the study of RhoGAP function at endogenous levels of expression and may help to sort out these discrepancies. Second, there are significant discrepancies in the studies that characterize the specificity of each RhoGAP. In vitro RhoGAP specificity assays are the gold standard in determining the substrate preference and have been used extensively. However, they have the disadvantage of using, most of the time, only the RhoGAP domain. Since the other domains are essential for the regulation of the activity in vivo, there are several examples in which the substrate preference differs between in vitro and in vivo data. Interpreting the results from different types of experimental approaches can be difficult and time consuming but it is essential to continue moving the field forward. Third, the RhoGAP family encodes a wide variety of domains other than the catalytic RhoGAP domain. The function of these non-catalytic domains has been chronically understudied. Understanding their function may prove essential to properly targeting the RhoGAP family. Finally, the models used to evaluate the role of RhoGAPs in cancer should be considered. It is necessary to study the mechanisms by which RhoGAPs regulate the development of cancer and not simply correlative studies. These mechanisms should be studied in models that are representative of more physiologically relevant environments, such as 3D spheroids, as well as in vivo studies using patient derived xenografts or other animal models. This will accelerate the process of the development of therapeutics and provide more solid evidence for the role of a given RhoGAP in cancer.

Table 2.

RhoGAP fusions

RhoGAP fusion	Fused gene	Cancer	Function	References
BCR-ABL	ABL proto-oncogene 1, non-receptor tyrosine kinase	Chronic myelogenous leukemia	Promotes tumorigenesis through a variety of mechanisms, particularly through constitutive kinase activity of ABL	Reviews: [325–327]
MLL-GRAF1	mixed-lineage leukemia	Juvenile myelomonocytic leukemia	No known function, although the GAP domain is either deleted or mutated suggesting a tumor suppressing role of the GAP	[328]
CLDN18-ARHGAP26	Claudin 18	Gastric cancer, Signet-ring cell carcinoma, diffuse-type gastric cancers	Loss of epithelial phenotype reminiscent of EMT	[329, 330]
BMX-ARHGAP12	Bone marrow X kinase	Gastric cardia adenocarcinoma	BMX-ARHGAP promotes proliferation and invasion likely via RhoA and JAK/STAT3 pathways	[331, 332]
PRR5-ARHGAP8	Proline-rich 5	Colon, renal, and gastric cancer	Frequently overexpressed in cancers	[333, 334]
CTNND1-ARHGAP26	Catenin Delta 1	Diffuse-type gastric cancers	Associated with worse prognosis	[330]
ANXA2-MYO9A	Annexin A2	Diffuse-type gastric cancers	Associated with worse prognosis	[330]
SrGAP3-RAF1	Raf-1 Proto-Oncogene, Serine/Threonine Kinase	Astrocytoma and pediatric low-grade gliomas	Activates MAPK and PI3K/mTOR signaling, confers resistance to some RAF inhibitors, and is associated with poor survival	[335–337]
RAD51B-OPHN1	RAD51 paralog B	Uterine perivascular epithelioid cell tumors	Associated with an increased mitotic index as well as with recurrence and/or mortality	[338, 339]
TIMM23-ARHGAP32	Mitochondrial import inner membrane translocase Tim23	Breast cancer (ER+)	Not known	[340]