RAB2 family comprises two isoforms (RAB2A and RAB2B) that localize to the Golgi, regulating bidirectional trafficking between the ER and the Golgi (Goud et al., 2018). High expression of RAB2A was detected in BC, compared to adjacent normal mammary tissue (Kajiho et al., 2016). Moreover, RAB2A is significantly associated with poor prognosis markers (Kajiho et al., 2016). Additionally, RAB2A was shown to mediate the exocytosis of membrane type 1-matrix metalloproteinase (MT1-MMP), an essential MMP for extracellular matrix (ECM) remodeling and BC cell invasion. Furthermore, RAB2A controls the trafficking of E-cadherin from the Golgi to the PM, ultimately regulating cell compaction, junctional stability, and tumor invasiveness (Kajiho et al., 2016; Kajiho et al., 2017).

RAB3 subfamily comprises four functionally redundant isoforms: RAB3A, RAB3B, RAB3C and RAB3D. They share a high degree of protein sequence homology (∼80%) and similar subcellular localization, namely in synaptic and secretory vesicles (Schlüter et al., 2002). RAB3A, RAB3B, RAB3C are primarily expressed in neuronal cells, while RAB3D is mostly observed in non-neuronal secretory cells, such as pancreas and mast cells (Millar et al., 2002; Riedel et al., 2002; Schlüter et al., 2002). Although the precise functions of the different RAB3 isoforms are not entirely established, they are involved in synaptic vesicle exocytosis (Schlüter et al., 2006) and secretory granule maturation (Kögel et al., 2013). Moreover, RAB3A and RAB3D were found to be required for docking and fusion of lysosomes and secretory vesicles, respectively, during regulated secretion in non-neuronal cells (Millar et al., 2002; Encarnação et al., 2016). Interestingly, RAB3A, RAB3B and RAB3D were shown to promote breast, colon, esophagus, melanoma, osteosarcoma and glioma tumor progression by increasing cell proliferation, migration, and invasion (Raffaniello, 2021). Additionally, RAB3D expression levels are higher in BC cells and positively correlate with tumor stage (Yang et al., 2015). Furthermore, RAB3D was found to regulate epithelial-to-mesenchymal transition (EMT) and promote BC tumor cell motility, invasion and metastasis through the activation of the AKT/GSK-3β/SNAIL signaling pathway (Yang et al., 2015). Finally, RAB3B expression is upregulated in aggressive luminal B BC cells in response to the amplification of the inositol-requiring enzyme 1 (IRE-1) gene, which acts as an oncogenic factor to repress a subset of tumor suppressor microRNAs (miRs) via regulated IRE1-dependent decay (Zhang et al., 2020).

RAB8 has two isoforms, RAB8A and RAB8B, which mediate the trafficking from the trans-Golgi network (TGN) to the PM, controlling the apical/basolateral transport of proteins in epithelial cells (Sato et al., 2007; Stenmark, 2009). In BC, RAB8 regulates the exocytosis of MT1-MMP, which promotes collagen degradation and cell invasion (Bravo-Cordero et al., 2007). Recently, Liu et al. showed that RAB8A is upregulated in BC (Liu et al., 2022). Moreover, RAB8A promotes the surface expression of tropomyosin-related kinase B (TRKB), which leads to the proliferation, migration and invasion of BC cells, through activation of the AKT and extracellular signal-regulated kinase (ERK) 1/2 signaling pathways (Liu et al., 2022).

The two isoforms of RAB27—RAB27A and RAB27B–are involved in regulated secretion. These have 71% of homology and recruit the same effector proteins. However, the underlying mechanisms by which they are involved in BC are different (Li et al., 2018). RAB27 isoforms are associated with lysosome-related organelles (LROs) and control exosome secretion in BC (Li et al., 2018). Indeed, RAB27A has been associated with the upregulation of exosome secretion by enhancing the secretion of insulin growth factor-II (IGF-II) in BC subtypes TNBC and HER2+ (Wang et al., 2008). RAB27B is upregulated in ER-positive BC and associated with an increased secretion of mesenchymal proteins, like vimentin and fibronectin, to the extracellular milieu (Bobrie et al., 2012; Zhang et al., 2012). Therefore, RAB27B upregulation is associated with poor prognosis in BC (Hendrix et al., 2010; Zhang et al., 2012).

The RAB40 subfamily contains four isoforms (RAB40A, RAB40AL, RAB40B, and RAB40C), which are enriched in the Golgi (Stenmark, 2009; Duan et al., 2021). In BC, RAB40B was shown to interact with tyrosine kinase substrate with 5 SH3 domains (TKS5), an adaptor protein that acts as a scaffold, bringing membrane and intracellular elements close to invadopodia (Jacob et al., 2013; Jacob et al., 2016). This interaction is required to target MMP-2- and MMP-9-containing vesicles to invadopodia, promoting ECM remodeling during BC progression (Jacob et al., 2013; Jacob et al., 2016). Moreover, the same authors found RAB40B to be highly expressed in more aggressive cancers, as well as in basal BC subtype. Additionally, RAB40C was found to regulate focal adhesion number, size, and distribution in migrating BC cells (Han et al., 2022).

ARF1 localizes to the Golgi, where it regulates the function and structure of this compartment (Adarska et al., 2021). In BC, ARF1 was found to be highly expressed and fundamental for epidermal growth factor (EGF)-mediated phosphorylation of focal adhesion kinase (FAK) and Src, regulating BC cell proliferation and adhesion (Schlienger et al., 2015). The same group performed in vivo studies and showed that ARF1 promotes primary tumor growth and the formation of metastases (Schlienger et al., 2016). Indeed, in non-invasive MCF-7 BC cells, ARF1 overexpression leads to the formation of lung metastases. In the same study, the authors found a link between ARF1 and the regulation of several pathways that are involved in EMT (E-cadherin/β-catenin, RAS, ERK1/2 and PI3K/AKT), as well as the upregulation of EMT markers SLUG and SNAIL. Moreover, ARF-1-expressing cells lose their epithelial features and acquire a more mesenchymal shape. Finally, ARF1 overexpression in MCF-7 cells leads to the activation of MMP-2 via FAK, contributing to the role of ARF1 in BC invasion (Schlienger et al., 2016). Additionally, ARF1 disruption sensitizes TNBC (MDA-MB-231) cells to the anti-tumor drugs actinomycin D and vinblastine (Luchsinger et al., 2018). Furthermore, it was shown that the recruitment of ARF1 and the ARF GAP ARAP1 to circular dorsal ruffles (CDRs) promotes shear stress-induced BC cell migration (Qin et al., 2021).

ARF3 regulates the recruitment of coat complexes to the Golgi apparatus and promotes the activation of phospholipase D (PLD) and phosphatidyl-kinases (PI-kinases) (Sztul et al., 2019). In BC, it was shown that the clinical stage positively correlates with ARF3 expression, which is upregulated in 92.8% of malignant cases (Huang et al., 2019). Furthermore, ARF3 mRNA and protein expression levels were found upregulated in BC cell lines and tissues (Huang et al., 2019). It was also observed that ARF3 overexpression promotes BC cell proliferation, through the regulation of the G1/S cell cycle transition (Huang et al., 2019). A recent study, based on integrated analysis of microarray profile datasets, revealed that ARF3 is a candidate gene involved in the progression of pregnancy-associated BC (Zhang et al., 2019).

As described for ARF1 and ARF3, ARF4 also localizes to the Golgi, being required for the recruitment of coat proteins and the retrieval of ER-resident proteins (Pennauer et al., 2021). ARF4, as well as the ER-Golgi trafficking regulators COPI subunit β1 (COPB1) and USO1, were found to be upregulated in BC patient samples (Howley et al., 2018). Moreover, it was reported that ARF4, COPB1, and USO1 regulate BC cell growth and invasion by mediating the retrograde transport of proteins from the Golgi to the ER via COPI-coated vesicles (Howley et al., 2018). ARF4 was also shown to promote BC cell migration in response to phorbol-12-myristate 13-acetate (PMA), a known gene expression inducer (Jang et al., 2012).

ARF-related protein 1 (ARFRP1) localizes to the TGN and mediates the trafficking/sorting of various cargoes (e.g., glucose transporters and E-cadherin) (Hesse et al., 2010). Furthermore, recent studies have shown that ARFRP1 regulates the recruitment of tethering factors to the TGN, upstream of ARL1 and ARL5 (Ishida and Bonifacino, 2019). In particular, ARFRP1 is involved in the recruitment of golgins, which are dependent on ARL1, and Golgi-associated retrograde protein (GARP), which is dependent on ARL5 (Ishida and Bonifacino, 2019). A recent proteome-based study proposed that ARFRP1 regulates the radioresistance of BC cells (Gao et al., 2022). Moreover, the authors found that ARFRP1 silencing leads to an increase in radiation resistance of both MCF-7 (luminal A) and MDA-MB-231 (TNBC) BC cell lines (Gao et al., 2022).

ARL4 has three paralogs (ARL4A, ARL4C and ARL4D) that localize to the PM. ARL4C was also found to localize to the cytosol and the nucleus, and interact with α-tubulin. Additionally, ARL4C regulates transferrin receptor transport from early endosomes (EEs) to recycling endosomes (REs) (Wei et al., 2009). Moreover, ARL4C was shown to be downregulated in BC samples. Furthermore, the low levels of ARL4C in BC correlate with those of the activated transcription factor 3 (ATF3), which is also downregulated in BC (Li et al., 2020). These authors also found that ATF3 promotes the transcription of ARL4C, which acts as a negative regulator of breast tumor progression. Indeed, the overexpression of ARL4C was shown to decrease BC cell proliferation, migration, and invasion, leading to cell cycle arrest (Li et al., 2020).

RAB5 has three distinct isoforms (RAB5A, RAB5B, RAB5C) that share more than 90% of sequence identity (Chen et al., 2009). RAB5 plays a key role in endocytosis regulation, mediating the transport and fusion of endocytic vesicles with EEs (Yuan and Song, 2020). In a meta-analysis of five human breast tumor gene expression datasets, RAB5A expression, but not RAB5B or RAB5C, was shown to correlate with poor prognosis (Frittoli et al., 2014). Additionally, RAB5A expression is significantly higher in matched lymph node metastases, compared to their primary tumors. RAB5A transports β3-integrin and MT1-MMP to invadopodia, through a RAB4-dependent pathway, allowing their maturation into competent ECM-degrading structures, which promotes invasion of metastatic BC cells (Frittoli et al., 2014).

RAB13 plays a role in both secretory and endocytic recycling pathways. It localizes to the TGN, REs, late endosomes and the PM (Ioannou and McPherson, 2016). Silencing of RAB13 in TNBC MDA-MB-231 cells leads to the intracellular accumulation of active β1-integrin, reducing integrin activity in focal adhesions and impairing cell migration. This suggests that RAB13 plays a role in facilitating the recycling of active β1-integrin to the PM (Sahgal et al., 2019).

RAB21 localizes mainly to EEs and the PM, being involved in the early endocytic pathway (Simpson et al., 2004). In BC, RAB21 was shown to regulate the endo/exocytic trafficking of integrins, stimulating MDA-MB-231 TNBC cell adhesion and migration (Pellinen et al., 2006).

The RAB11 subfamily encompasses three different isoforms: RAB11A, RAB11B and RAB11C, also known as RAB25. While RAB11A and RAB11B exhibit 90% amino acid identity, RAB11A/B and RAB25 share 60% homology (Kelly et al., 2012). RAB11 proteins localize to various subcellular compartments, including the endocytic recycling compartment (ERC), REs, apical recycling compartment (ARE), and the TGN (Welz et al., 2014). Moreover, they mainly regulate cargo recycling from the ERC to the cell surface, and also participate in exocytic transport from the TGN (Ullrich et al., 1996; Chen et al., 1998; Ren et al., 1998). Recently RAB11A and RAB11B were described by us to be required for Ca²⁺-dependent lysosome exocytosis (Escrevente et al., 2021).

In BC, RAB11 controls the transport of α6β4-integrin from REs and the TGN to the PM, enabling cell invasion under hypoxic conditions (Yoon et al., 2005). The RAB11 isoforms have also been associated with breast carcinogenesis. A miR that targets RAB11A (miR-320a) was found to inhibit proliferation, migration, and invasion of MDA-MB-231 cells in vitro, as well as tumor growth in a mouse xenograft model (Wang et al., 2015). Additionally, the authors found that RAB11A mRNA is overexpressed in BC samples, compared to normal adjacent tissues (Wang et al., 2015). In a subsequent study, miR-452 was found to be a tumor suppressor gene that also inhibits BC cell migration and invasion by targeting RAB11A (Li et al., 2017b). Notably, RAB11A is upregulated in DCIS, a non-obligatory precursor of invasive BC, when compared to adjacent normal tissues (Palmieri et al., 2006). In the same study, the overexpression of a dominant-negative RAB11A mutant (S25N) was observed to lead to decreased EGF receptor (EGFR) recycling and cell proliferation in MCF10A human breast epithelial cell line (Palmieri et al., 2006).

The role of RAB11B in BC remains relatively unexplored. However, a recent study highlighted the importance of RAB11B in the adaptation of BC metastases to the brain microenvironment. Specifically, RAB11B regulates the recycling of β1-integrin, enabling effective interaction between BC cells and the brain ECM (Howe et al., 2020).

Interestingly, RAB25 plays a dual role in BC, functioning as a tumor promoter in luminal cancers, and as a tumor inhibitor in TNBC. Indeed, expression of RAB25 positively correlates with ER- and PR-positive BCs and lymphatic metastasis (Yin et al., 2012). Moreover, it was shown that RAB25 increases proliferation and migration of luminal B BC cell lines (Mitra et al., 2016). On the other hand, RAB25 expression is lost in hormone receptor-negative BC compared to matched normal tissues and overexpression of this isoform reduces the proliferation of MDA-MB-231 cells (Cheng et al., 2010).

RAB34 regulates endosomal trafficking, autophagy and lysosome maturation, by mediating the distribution of lysosomes to the perinuclear region (Starling et al., 2016). Moreover, it facilitates the internalization of receptors, transport of endocytic vesicles to peri-Golgi regions, and regulates the fusion of phagosomes with lysosomes (Kasmapour et al., 2012). In BC, RAB34 overexpression was associated with increased tumor invasiveness, migration and metastatic potential, as well as the recycling of β3-integrin (Wang and Hong, 2005; Sun et al., 2018).

RAB35 localizes to REs and the PM of different cell types, and plays a role in several cellular functions, including endocytic recycling, cytokinesis, cell polarity, exosome release, immunity, lipid homeostasis, and phagocytosis (Klinkert and Echard, 2016). Therefore, it is not surprising that RAB35 can exert an important role in different types of cancer by controlling several aspects of cancer progression, such as cell migration, proliferation and survival (Shaughnessy and Echard, 2018). In MCF-7 luminal A BC cells, RAB35 activation by Wnt5a promotes cell migration via the DVL2/RAB35/RAC1 signaling pathway (Zhu et al., 2013). EGF-activated RAB35 can also lead to a more invasive phenotype in BC cells. In its active form, RAB35 binds to microtubule-associated monooxygenase, calponin and LIM domain containing-1 (MICAL-1) and promotes its activation. MICAL-1 activation increases reactive oxygen species (ROS) generation and AKT phosphorylation, leading to a more invasive phenotype (Deng et al., 2016). Additionally, RAB35 expression was found to be downregulated in highly invasive BC tumors, where ARF6 is hyperactivated (Allaire et al., 2013). Enhanced ARF6 activation leads to integrin and EGFR recycling to the cell surface, promoting cell migration (Allaire et al., 2013). Finally, it was shown that some RAB35 pathogenic somatic mutations (G18V, A29V and F45L) in BC can activate this protein and confer it oncogenic properties (Shaughnessy and Echard, 2018).

ARF6 is mainly localized at the cell periphery, where it regulates endocytic recycling and cortical actin dynamics. Moreover, this small GTPase is involved in the regulation of cell division (Sztul et al., 2019). ARF6 has been extensively studied in cancer and it is known to regulate cancer cell growth, angiogenesis, invasion, and the formation of metastases (Li R. et al., 2017). Moreover, high ARF6 expression and the activation of downstream signaling pathways were associated with poor overall survival of BC patients (Hashimoto et al., 2004). It was also shown that ARF6 has a role in BC cell invasion, as it was found to localize to invadopodia and regulate the activity of these actin-rich structures, which promote ECM degradation in tumors (Hashimoto et al., 2004). Indeed, a study using BC cell lines with different invasive capacities showed a correlation between ARF6 protein levels and BC cell invasiveness (Hashimoto et al., 2004). The same authors also observed that ARF6 silencing decreases the invasion capacity of BC cells and regulates EGFR signaling (Morishige et al., 2008). A recent study showed that ARF6 targets palmitoylated EGFR to promote its trafficking from the Golgi to the PM, through the recruitment of the exocyst tethering complex (Guo et al., 2022). As mentioned, matrix remodeling/degradation is a key feature of BC cell invasion. ARF6 activity and its effectors JIP3 and JIP4 have been associated with MDA-MB-231 cell invasion through invadopodia-mediated mechanisms (Marchesin et al., 2015). In particular, the authors found that the binding of active ARF6 to JIP3/JIP4 regulates the trafficking and exocytosis of MT1-MMP, a key regulator of invadopodia function (Tanaka and Sakamoto, 2023). Furthermore, this study showed that the regulation of MT1-MMP trafficking occurs via the recruitment of motor proteins (dynactin-dynein and kinesin-1) by JIP3/JIP4 (Marchesin et al., 2015). Recently, the role of ARF6 was associated with the tumor microenvironment. Specifically, the chemokine (C-C motif) ligand 18 (CCL18), which is mainly produced by tumor-associated macrophages and has been linked to the formation of metastases in BC, was found to increase the expression of ARF6 and the phosphorylated form of its downstream effector AMAP1, an ARF GAP, in MCF-7 cells (Huang et al., 2022). In the same study, the authors observed that CCL18 increases the levels of miR-760 in exosomes, which activates an ARF6/Src/PI3K/AKT signaling cascade and induces BC cell proliferation, migration, invasion, and drug resistance.

RAB26 is involved in several processes, including exocrine granule maturation, amylase release from parotid acinar cells and lysosome clustering in the perinuclear region (Nashida et al., 2006; Tian et al., 2010; Li et al., 2012). Moreover, RAB26 increases the integrity of adherens junctions in acute lung injury and regulates the trafficking of cell surface receptors such as α2-adrenergic receptor (Li et al., 2012; Dong et al., 2018). In BC, RAB26 acts as a tumor suppressor gene by reducing focal adhesion association of Src kinase and inducing autophagic degradation of phosphorylated Src, which results in the inhibition of migration and invasion of BC cells (Liu et al., 2021). Furthermore, BC datasets showed that higher RAB26 expression is associated with a significantly higher overall survival (Liu et al., 2021).

ARL8 has two paralogs–ARL8A and ARL8B–both localized to lysosomes. Whereas the function of ARL8A is still poorly understood, ARL8B is well studied and known to regulate the kinesin-dependent anterograde movement (towards the cell periphery) of lysosomes (Rosa-Ferreira and Munro, 2011; Khatter et al., 2015). Interestingly, two recent reports showed evidence that ARL8 also regulates long-range endolysosomal retrograde movement (towards the perinuclear region) (Keren-Kaplan et al., 2022; Kumar et al., 2022). ARL8A and ARL8B were found to be upregulated in both luminal A and TNBC cell lines, upon silencing of the Ca²⁺-binding protein TBC1 domain family member 9 (TBC1D9, a RAB GAP), which is associated with an impairment of TNBC progression (Kothari et al., 2021). A recent study showed evidence that the movement of lysosomes towards the cell periphery promotes the invasion of radiation-surviving BC cells in vitro, as well as tumor metastasis in vivo (Wu et al., 2020). In particular, the recruitment by the biogenesis of lysosome-related organelles complex 1 (BLOC-1)-related complex (BORC) and activation of ARL8B leads to binding to the effector Sif-A and kinesin-interacting protein (SKIP), which promotes lysosome anterograde movement (Wu et al., 2020). At the cell periphery, lysosomes can be exocytosed and release ECM-degrading proteases, leading to BC cell invasion (Machado et al., 2015; Wu et al., 2020).

ARL2 is highly conserved, and it was found to localize to the cytosol and mitochondria (Sztul et al., 2019). Moreover, this small GTPase was shown to regulate the α/β-tubulin biogenesis and microtubule dynamics, as well as mitochondria motility, fusion, and ATP levels (Bhamidipati et al., 2000; Newman et al., 2014; Francis et al., 2017; Newman et al., 2017). ARL2 was also shown to regulate α/β-tubulin polymerization in MCF-7 cells. Furthermore, it was also observed that a lower expression of ARL2 leads to enhanced resistance to cytotoxic agents (Beghin et al., 2007; Beghin et al., 2008). This mechanism is regulated by protein phosphatase 2A (PP2A), which fails to dephosphorylate p53 when ARL2 expression is low (Beghin et al., 2008). The same group also used orthotopic mouse models to show that ARL2-depleted BC cells have an enhanced clonogenic potential, less contact inhibition, and proliferation, as well as impaired tumor growth (Beghin et al., 2009).

ARL13 presents two paralogs (ARL13A and ARL13B) that only share around 43% sequence homology (Marwaha et al., 2019). ARL13B is a well-established regulator of cilia structure and function (Seixas et al., 2016; Revenkova et al., 2018). Moreover, our group described that the interaction of ARL13B with the actin cytoskeleton, mediated by its effector non-muscle myosin IIA (NMIIA), promotes the formation of CDRs and, consequently, cell migration (Casalou et al., 2014). Furthermore, we showed that ARL13B plays a role in BC progression, through a mechanism likely independent of cilia (Casalou et al., 2019). Specifically, we found that the silencing of ARL13B leads to an impairment in BC cell migration and invasion in vitro, as well as tumor growth and metastasis in vivo. Moreover, we gathered evidence to support that ARL13B promotes BC cell migration and invasion through the regulation of integrin-dependent signaling and cell-ECM adhesion (Casalou et al., 2019). The results obatined in this study suggest that ARL13B interacts with β3-integrin, regulates the formation of stress fibers and the size of focal adhesions, which results in the modulation of the cell-ECM adhesion and cell motility (Casalou et al., 2019).

ARL11, which is also named ADP-ribosylation factor-like tumor suppressor gene-1 (ARLTS1), was initially described as a tumor suppressor (Calin et al., 2005). Yet, little is known about its function(s) and localization. Nevertheless, in a recent study, it was observed that ARL11 localizes to the nucleus, cytosol, and cortical actin (Arya et al., 2018). These authors also found that ARL11 is essential for liposaccharide (LPS)-induced macrophage activation by interacting with phosphorylated ERK1/2 on actin structures (Arya et al., 2018). Different variants of ARL11 have been linked to familial and sporadic cases of cancer. In this regard, the mutations W149Stop and C148R are the best characterized (Yendamuri et al., 2008). The former is a nonsense mutation that results in the production of a truncated protein that is unable to bind GTP, which leads to a decrease in apoptosis (Petrocca et al., 2006). Furthermore, both mutations were associated with familial cases of BC (Frank et al., 2006; Yendamuri et al., 2008).