Invited Review

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Apr 10, 2018 - platelet-derived growth factor receptor-alpha;. PLEKHM2, pleckstrin homology and RUN domain containing M2; RAR, retinoic acid receptor;.

J. Biochem. 2018;1—6

doi:10.1093/jb/mvy041

Invited Review RAB39A: a Rab small GTPase with a prominent role in cancer stemness Received March 20, 2018; accepted April 1, 2018; published online April 10, 2018

Tokuhiro Chano1,* and Sofia Avnet2 1 Department of Clinical Laboratory Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan and 2 Orthopaedic Pathophysiology and Regenerative Medicine Unit, Istituto Ortopedico Rizzoli IRCCS, Bologna, Italy

*Tokuhiro Chano, Department of Clinical Laboratory Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan. Tel: +81-77-548-2621, Fax: +81-77-548-2603, email: [email protected]

RAB39A is a Rab small GTPase that localizes at distinct subcellular compartments and regulates intracellular membrane trafficking pathways in vertebrate cells. RAB39A interacts with various molecules and modulates vesicular trafficking that regulates multiple biological pathways such as neuronal differentiation and/or autophagy. Among these pathways are Hippo and Notch signallings, microtubular organization and mitophagy/ autophagy. Although RAB39A has never been studied in cancer biology, it has been recently shown to promote cancer stemness and tumorigenesis. Molecular pathways regulated by RAB39A are transcriptionally maintained by the formation of molecular complex with RXRB, NCOR and HDAC that also contribute to cancer stemness. In this review, we provide current knowledge on the oncogenic function of RAB39A and summarize the effect of different microenvironments on RAB39A activity and subcellular localization in cancer cells. Keywords: cancer stemness; membrane trafficking; oncogenic mutation; RAB39A; Rab small GTPase. Abbreviations: BICD1, BICD cargo adaptor 1; CARMIL1, capping protein regulator and myosin 1 linker 1; CLEC16A, C-type lectin domain containing 16A; GAPs, GTPase-activating proteins; GEFs, guanine nucleotide exchange factors; HDAC, histone deacetylase; KIF1A, kinesin family member 1A; LPS, lipopolysaccharide; NCOR, nuclear receptor corepressor; NICD, notch intracellular domain; PDGFR-a, platelet-derived growth factor receptor-alpha; PLEKHM2, pleckstrin homology and RUN domain containing M2; RAR, retinoic acid receptor; RASSF1A, ras association domain family member 1; RXRB, retinoid X receptor beta; UACA, uveal autoantigen with coiled-coil domains and ankyrin repeats; VDR, vitamin D receptor; YAP, yes-associated protein.

RAB39A is a Rab small GTPase that localizes at distinct subcellular compartments and plays essential roles in intracellular membrane trafficking events of

vertebrate cells (1). There are two Rab39 isoforms, RAB39A and RAB39B, in vertebrates, whereas only a single Rab39 isoform is present in invertebrates such as Caenorhabditis elegans and Drosophila (2). Rab proteins are regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins and cycle between a GTP-bound active and GDP-bound inactive form, respectively. The GTP-bound active form of Rab proteins binds with the respective effector molecules to promote membrane trafficking events (2, 3); this has also been demonstrated in cancer cells (4). RAB39A is also a member of the RAS oncogene family that is understood to play a role in oncogenesis (5). As for the other Ras subfamily of small G-proteins, RAB39A has recently been suggested as a possible therapeutic target since it contributes to promote cancer stemness of sarcomas and other malignancies of the lymphoid, adrenal, glia and testicular tissues (6); cancer stemness is implicated in drug resistance, metastasis and local relapse (7). Other Rab proteins also play a role in oncogenesis. Indeed, deregulations of various Rab proteins and subsequent disruption of vesicle trafficking networks have been implicated in tumorigenesis, including loss of cell polarity, invasion and metastasis (1, 4, 8). Here, we review the current understanding of the oncogenic function of RAB39A and discuss the related pathways that are involved in the development of cancer and its stemness.

Subcellular localization of RAB39A RAB39A localizes in late endosomes and lysosomes, regulates endocytosis and acidification of maturing phagosomes and modulates the inclusion process of micro-organisms (9—11). In contrast, an important paralogue, RAB39B localizes in Golgi apparatus (11, 12) and is suggested to associate with human diseases such as Waisman syndrome and X-linked nonspecific intellectual disability (12, 13). The observations for these subcellular localizations were made through the introduction of the exogenous RAB39A or RAB39B, since no specific antibody was available to bind specifically the endogenous protein. However, a new antibody (#13355-1-AP, Proteintech, IL) that recognizes the endogenous RAB39A has recently been developed. The cancer microenvironment may develop different altered conditions, such as hypoxia and acidosis (14), which greatly impact on cancer behaviour. Using the new anti-RAB39A antibody, we have recently showed that, at low extracellular pH and low oxygen (O2) tension, in cancer cells RAB39A localized at different subcellular compartments. Under the standard culture conditions, RAB39A resides in endosomes and

ß The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

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Fig. 1 Subcellular localization of RAB39A. After 24 h of normal (pH 7.4, 20% O2), acidic (pH 6.8), or hypoxic (1% O2) culture in 143B human osteosarcoma cells, RAB39A was fluorescently labelled with anti-RAB39A antibody (#13355-AP-1, Proteintech) followed by Alexa 488. Golgi apparatus, endosome and lysosome were labelled with anti-Golgi antibody (clone 371-4, Merck) followed by Alexa 647, pHrodoTM Red dextran (P10361, Thermo) and LysoTrackerÕ Red (L7528, Thermo), respectively. Then, the subcellular localization of RAB39A was evaluated with a confocal microscopy (FV1000-D, Olympus). White square defects of right lower ends indicate 50 mm.

lysosomes in the cytoplasm and has been conformed to the previous report (11), whereas in acidic conditions, it is mainly localized at the Golgi apparatus. Interestingly, 1% O2 tension increasingly induces and rewires RAB39A into Golgi, endosomes and lysosomes (Fig. 1). Based on these findings, we speculate that, similar to the other Rab proteins, RAB39A is activated by GEFs and binds to the effector molecules, thereby promoting membrane trafficking events according to different microenvironmental conditions.

Pathways from RAB39A to cancer stemness Various molecular interactions with RAB39A have been proposed, involving multiple biological pathways. Among these, we indirectly demonstrated that RAB39A downstream drove RXRB to promote cancer stemness and tumorigenesis (6). However, the exact molecular mechanism behind this effect remains uncertain. RXRB is a member of the RXR family of nuclear receptors that mediates the effects of retinoic acid. Indeed, through the formation of heterodimers with the retinoic acid receptor (RAR), thyroid hormone and VDR, RXRB indirectly increases both DNA binding and transcriptional function on their respective response elements (15). Heterodimers, with 2

RXR and VDR, activate Ras-Raf-MAPK-ERK through the classical VDR pathway and modulate the expression of genes involved in cancer development (16). In addition, RXR, RAR and NCOR interfere with HDAC and maintain embryonic neuronal stem cells (17). Therefore, we speculate that RAB39A (containing Rab small GTPase activity) contributes to cancer development and stemness through RXR and VDR signalling thereby regulating DNA binding and transcription. Accordingly, a previous report demonstrated that RAB39A interacted with uveal autoantigen with coiled-coil domains and ankyrin repeats (UACA) and regulated retinoic acid-induced neurite differentiation in a brain neuroblastoma cell (18). RAB39A has been also suggested as a trafficking adaptor linking caspase-1 to induce the secretion of IL-1ß (19) and to functionally interact with ras association domain family member 1 (RASSF1A) (20). In this last case, RAB39A has been possibly associated with BAX and Hippo pro-apoptotic signalling pathways and with epigenetically inactivation, ultimately leading to the enhancement of Ras-driven transformation and metastasis (21, 22) (Fig. 2). To globally identify Rab effectors, Gillingham et al. (23) used a comprehensive set of Drosophila Rabs for affinity chromatography followed by mass

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RAB39A and tumorigenesis

Fig. 2 Pathways from RAB39A to cancer stemness. In the previous studies, various molecular interactions with RAB39A were predicted that could involve multiple biological pathways such as neuronal differentiation, Hippo, Notch recycling, micro-tubular organization and mitophagy/ autophagy. Downstream to these pathways, it is likely that the formation of molecular complexes with RXRB, NCOR and HDAC is important for the promotion of the transcription of genes that regulate cancer stemness.

spectrometry: various interacting effectors with Rab39 were highlighted, including BICD cargo adaptor 1 (BICD1), capping protein regulator and myosin 1 linker 1 (CARMIL1), pleckstrin homology and RUN domain containing M2 (PLEKHM2), kinesin family member 1A (KIF1A) and C-type lectin domain containing 16A (CLEC16A). The identification of these effectors suggest that RAB39A may be involved in the dynamics of Golgi, ER, endosomal and lysosomal apparatus for membrane trafficking in many different ways, and if these interactions are conserved in mammals, they might form the basis of organizing polymerization of microtubules and actin and regulating mitophagy/autophagy (Fig. 2). In agreement with this hypothesis, it has been demonstrated that RAB39A regulates lipopolysaccharide (LPS)-induced autophagosome formation and secretion of pro-inflammatory compounds through the interaction of Beclin 1 (9, 11). Similarly, in a model of rhabdomyosarcoma, we demonstrated a clear connection between lysosomal acidification and the degree of cancer stemness (24), and, in other sarcoma models, that RAB39A modulates cancer stemness and tumorigenesis (6), probably via the regulation of autophagy and lysosomal fusion, i.e. autolysosome. In the same report, the wide screening of transcriptome suggested that RAB39A deficiency reduces cancer stemness via vitamin A & D signalling and Notch signalling deregulation (6). Interestingly, Notch and Hippo pathways mutually interact through the binding between Notch intracellular domain (NICD) and yes-associated protein (YAP) (25) and through YAP1 expression, which in turn, is regulated by NICD (26). Although the direct molecular effector of RAB39A contributing to cancer stemness remains uncertain, our

observation for subcellular localization of RAB39A has suggested that the redistribution process such as molecular recycling may be stimulated in acidic extracellular condition, whereas the involving molecules are degradative under the standard condition. In addition, RAB39A is fully employed to rewire the relevant signallings under hypoxic condition. Similarly to RAB35, another Rab small GTPase, promoting the ligand-independent activation of platelet-derived growth factor receptor-alpha (PDGFR-a) and the subsequent oncogenic role (27); RAB39A functions in endomembrane trafficking, and its deregulation (probably via its constitutive activation) will enable to enhance the stemness-relevant pathway such as Notch signalling in a ligand-independent manner. RAB39A possibly attributes cancer stemness to constitutive activation of Notch signalling with or without involving Hippo signalling deregulation (Fig. 2).

RAB39A mutations and their oncogenic roles Several Rab small GTPases, their deregulations and the subsequent disruption of vesicle trafficking networks have been often involved in tumorigenesis. For example, aberrantly increased expression of RAB1A, RAB5A, RAB7, RAB14, RAB23 and RAB40B promote the lack of cell polarity, cell migration, invasion and metastasis in several types of cancers (1, 4, 8). In addition, similar to cases of KRAS mutations, somatic mutations of RAB35 promote the constitutive activation of PI3K/AKT signalling and RAB35-dependent recycling and ligand-independent activation of PDGFR- a have caused an oncogenic role in cancer development (27—29). Conversely, RAB25 promotes

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Fig. 3 Compared alignments of RAB39A/B, RAB35 and KRAS; and presumable pathogenic mutations from TCGA database. (A) Alignment of amino acid sequences among RAB39A, Rab39a (mouse), RAB39B, RAB35 and KRAS was performed using ClustalW v2.1 software. Square circled boxes indicating GXXXXGK(S/T) and DXXGQ are switching motifs for GTP and GDP, respectively. Mutated sites speculatively holding highly pathogenic effects are indicated by arrows. (B) Somatic mutations of RAB39A in cancers are found in the TCGA database, and the mutations corresponding to arrows of schematic A are demonstrated in table B.

or suppresses tumorigenesis depending on the type of cancer cells due to its interplay with cell-type specific effectors (1, 4, 8). Through the alignment and comparison of RAB39A with RAB39B, RAB35 and KRAS, we identified somatic mutations of RAB39A that could have pathogenic effects during cancer development (Fig. 3). However, the precise annotations of these mutations have never been described in The Cancer Genome Atlas (TCGA) database: http://www.cbioportal.org/index.do? session_id=5a850e7a498eb8b3d564c7c6. Here, RAB39A mutations are sometimes amplified or deleted, suggesting that, like RAB25, RAB39A deregulation plays a role in tumorigenesis depending on the types of cancers. Furthermore, similar to the reported mutations of RAB35, and as suggested by Wheeler et al. (27), we speculate that the identified mutations in RAB39A induce GTP-bound, constitutively active PI3K/AKT signalling.

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Concluding remarks and perspectives In this article, we have briefly reviewed the current knowledge on the functions of RAB39A, a small Rab GTPase that contributes to cancer development, paying particular attention to cancer stemness. The crosstalk between extracellular hostile environments and the rewired localizations of RAB39A in cancer cells suggests that RAB39A functions as a member of Rab proteins in endomembrane trafficking. RAB39A was predicted to interact with various molecules and could play roles in vesicular trafficking in multiple biological pathways such as neuronal differentiation, Hippo and Notch signallings, microtubular organization and mitophagy/autophagy. However, other systems of transcriptional maintenance that involve RAB39A and include molecular complexes with RXRB, NCOR and HDAC, may also play a role in cancer stemness and deserve further investigation.

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RAB39A and tumorigenesis

Acknowledgements The authors thank Takefumi Yamamoto (T.Y.) and Hiroko Kita (H.K.), Shiga University of Medical Science, for experimental assistance in analyzing the subcellular localization of RAB39A.

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Funding This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan [Grant-in-Aid for Scientific Research (C) 18K09192 to T.C.].

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Conflict of Interest None declared.

Patent RAB39A is submitted as a targeting molecule for cancer treatments into Japanese patents (#NP151029: JP2015-123952), by the applicant, Shiga University of Medical Science.

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