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Melanoma goes on a diet to get moving: toning down phagocytic Rab7 expression helps melanoma to metastasise Alistair N. Hume and Miguel C. Seabra e-mail: [email protected]

A grand challenge in oncology is the identification of genetic drivers of tumourigenesis as targets for future therapeutic interventions. In the case of cutaneous melanoma, this is particularly challenging, due to the high degree of genetic variation between tumours. To address this problem, some recent studies have focused on identifying lineage-specific drivers of tumour progression, rather than genes and pathways common to the multiple tumour types. In melanoma, the subversion of the master transcriptional regulator of melanocyte development microphthalmiaassociated transcription factor (MITF) and its targets by cancer cells to promote tumour development has been a recent focus. These studies have highlighted the role of membrane trafficking pathways regulated by Rab GTPases in tumour progression (Goldenring, 2013). Rabs are p21-Ras-related small GTPases that fulfil highly conserved functions in regulating vesicular transport pathways throughout eukaryotes. Most evidence suggests that Rabs function as compartment-specific molecular switches directing membrane remodelling and transport in the secretory and endocytic pathways. They do this by regulating the specificity of cytosol-tomembrane recruitment of a diverse group of effector proteins such as coats, motors, tethers and fusion factors that transduce Rab function in vesicle formation, transport and fusion. Recent studies have linked overexpression of the MITF target RAB27A, a regulator of the transport and secretion of lysosome-related organelles including melanosomes in melanocytes, with

Coverage on: Alonso-Curbelo, D., Riveirorez-Guijarro E et al. Falkenbach, E., Pe (2014) ‘RAB7 controls melanoma progression by exploiting a lineage-specific wiring of the endolysosomal pathway’. Cancer Cell 26(1), 61–76. doi: 10.1016/ j.ccr.2014.04.030. doi: 10.1111/pcmr.12310


melanoma progression. In this context, this Rab is thought to act via the secretion of growth factor receptor-rich exosomes that promote proliferation and metastasis (Peinado et al., 2012). However, this does not appear to be a lineage specificity of this effect as RAB27A/B products are linked with exosome secretion in tumour lines of diverse origin including breast cancer (Recchi and Seabra, 2012). In line with an important role for Rabs and vesicular transport in melanoma, a recent report from Maria Soengas group in Madrid published in Cancer Cell presents evidence that another Rab, RAB7A, which regulates autophagy, plays an important tumour-type specific role in driving melanoma. In this study, Alonso-Curbelo et al. sought to identify new melanoma-specific oncogenes. To do this, they used Gene Set Enrichment Analysis (GSEA) to analyse transcriptional data sets the NCI-60 panel and the Cancer Cell Line Encyclopedia, from 35 different tumour types. GSEA is a powerful bio-informatic tool for interpretation of gene expression that focuses on sets of genes linked by function, regulation or chromosomal location. Strikingly, this analysis revealed that in melanoma, the highest enrichment was for gene sets associated with the function of the lysosome. As lysosome-related organelles melanosomes share some characteristics with lysosomes; however, GSEA highlighted melanoma-specific enrichment in ‘lysosome-only’ genes encoding hydrolytic proteins rather than those common to melanosomes. Critically, Alonso-Curbelo et al. found that survival of melanomaderived cell lines, but not those derived from other tumours, was significantly reduced by attenuation of the hydrolytic function of lysosomes using chloroquine. This suggests that enhanced lysosomal function specifically promotes melanoma cell survival. Alonso-Curbelo et al. then validated the role of one of the identified genes, namely the small GTPase RAB7A, in melanoma survival. In melanocytes,

Rab7 protein is reported to function in regulating the transport of melanogenic proteins tyrosinase-related protein 1 (Tyrp-1) and gp100/Pmel/Silv. RAB7A was selected for further analysis based on several observations; firstly, it is an established regulator of endo/lysosome function; secondly, it was among the most highly expressed lysosomal genes identified; and thirdly, it is found in a region of the genome that is often amplified in melanoma. Using Western blotting and tissue microarray analysis, they confirmed that Rab7 protein is more highly expressed in melanoma compared with other cancer cell lines and normal human skin. They then used shRNA knockdown and dominant negative mutants to test the role of RAB7A in melanoma proliferation and migration, key processes in melanoma pathogenesis. These studies revealed surprisingly that while proliferation correlated positively with RAB7A expression, the opposite was true for motility. These observations raise the possibility that different levels of RAB7A expression might correlate with different behaviour of cells in the proliferating primary and motile metastatic melanoma, respectively. Alonso-Curbelo et al. then investigated the mechanism by which RAB7A/ lysosomal function might be working in melanoma. Like other Rabs, RAB7A functions by regulating intracellular vesicular transport, specifically by controlling the fusion of endosomes and autophagosomes with lysosomes (Hyttinen et al., 2013). Previous studies have suggested a link between autophagy and the survival of melanoma and other tumours; therefore, they investigated whether RAB7A links these processes (Mathew et al., 2007). Interestingly, RAB7A knockdown caused the accumulation of a novel class of autophagosomes of macropinocytic origin, suggesting that RAB7A-dependent trafficking normally consumes these organelles. Transcriptomic and proteomic analyses of knockdown cells and conditioned medium revealed reduced expression of cell cycle progression genes and

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

News and Views increased release of lysosome stored pro-metastatic factors, respectively. This is consistent with the increased motility of knockdown cells. These observations suggested that at high expression, RAB7A might support proliferation by enhancing the rate of cellular nutrition while at lower expression, it might favour metastasis by enhancing release of pro-metastatic factors. Given the remarkable differences in the behaviour of different cell lines, how might the differences in RAB7A expression contribute to melanoma pathogenesis? To address this issue, AlonsoCurbelo et al. used histological methods to investigate the spatial distribution of differences in RAB7A expression in biopsies of benign nevi and a range of malignant melanomas. Interestingly, they found that RAB7A expression was most prominent in early-stage radial growth phase melanoma and nevi (i.e. premetastatic) compared with vertical growth phase and metastatic tumours. Accordingly, confocal imaging of sections of primary melanoma revealed a gradient of RAB7A expression decreasing from the central tumour mass towards the invading edge of the tumour. These observations support the idea that RAB7A expression might be ‘tuned’ to the local needs of populations of cells within the tumour, with downregulation in the

periphery of larger late-stage primary tumours to facilitate metastasis to new niches. In striking agreement with this idea, analysis of the 10-year survival of a large cohort of ”unlike other melanoma patients corregrowth lated inversely promoting with Rab7 melanoma expression in the genes, for primary tumour. example Finally, as a RAB27A, RAB7A first step to was not understand how regulated by this switch in MITF” expression might be regulated during tumour development, Alonso-Curbelo et al. investigated how RAB7A expression in melanoma could be regulated. Their studies focused on knockdown of established pigment cell transcription factors and revealed that, Instead, they found evidence that SOX10 and MYC regulate RAB7A expression independently of MITF. Overall, this study reveals a novel role for RAB7A in regulating the distribution of cargo, for example lysosomal hydrolases, through the post-Golgi endocytic and secretory pathways that are particularly important in the pathogenesis of melanoma. Furthermore, the finding that RAB7A is regulated by a mechanism dis-

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

creet from other melanoma oncogenes might provide the basis for future treatments of melanoma directed at modulation the activities of lysosomes and RAB7A. It should be of some interest for future studies to better understand the mechanism by which the position of cells within a melanoma may regulate RAB7A during progression of the disease.

References Goldenring, J.R. (2013). A central role for vesicle trafficking in epithelial neoplasia: intracellular highways to carcinogenesis. Nat. Rev. Cancer 13, 813–820. Hyttinen, J.M., Niittykoski, M., Salminen, A., and Kaarniranta, K. (2013). Maturation of autophagosomes and endosomes: a key role for Rab7. Biochim. Biophys. Acta 1833, 503–510. Mathew, R., Karantza-Wadsworth, V., and White, E. (2007). Role of autophagy in cancer. Nat. Rev. Cancer 7, 961–967. Peinado, H., Aleckovic, M., Lavotshkin, S. et al. (2012). Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat. Med. 18, 883–891. Recchi, C., and Seabra, M.C. (2012). Novel functions for Rab GTPases in multiple aspects of tumour progression. Biochem. Soc. Trans. 40, 1398–1403.


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Melanoma goes on a diet to get moving: toning down phagocytic Rab7 expression helps melanoma to metastasise.

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