RAB7 Controls Melanoma Progression by Exploiting a Lineage-Specific Wiring of the Endolysosomal...
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Please cite this article in press as: Alonso-Curbelo et al., RAB7 Controls Melanoma Progression by Exploiting a Lineage-Specific Wiring of the Endo-lysosomal Pathway, Cancer Cell (2014), http://dx.doi.org/10.1016/j.ccr.2014.04.030
Cancer Cell
Article
RAB7 Controls Melanoma Progressionby Exploiting a Lineage-Specific Wiringof the Endolysosomal PathwayDirena Alonso-Curbelo,1 Erica Riveiro-Falkenbach,1 Eva Perez-Guijarro,1 Metehan Cifdaloz,1 Panagiotis Karras,1
Lisa Osterloh,1 Diego Megıas,2 Estela Canon,1 Tonantzin G. Calvo,1 David Olmeda,1 Gonzalo Gomez-Lopez,3
Osvaldo Grana,3 Vıctor Javier Sanchez-Arevalo Lobo,4 David G. Pisano,3 Hao-Wei Wang,5 Pablo Ortiz-Romero,6
Damia Tormo,1,8 Keith Hoek,7 Jose L. Rodrıguez-Peralto,6 Johanna A. Joyce,5 and Marıa S. Soengas1,*1Melanoma Laboratory, Molecular Oncology Programme2Confocal Microscopy Unit, Biotechnology Programme3Bioinformatics Unit, Structural Biology and Biocomputing Programme4Epithelial Carcinogenesis Laboratory, Molecular Pathology ProgrammeSpanish National Cancer Research Centre (CNIO), Madrid 28029, Spain5Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA6Instituto de Investigacion i+12, Hospital 12 de Octubre, Universidad Complutense, Madrid 28041, Spain7Department of Dermatology, University Hospital of Zurich, Zurich 8091, Switzerland8Present address: BiOncoTech Therapeutics, Valencia 46980, Spain
*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.ccr.2014.04.030
SUMMARY
Although common cancer hallmarks are well established, lineage-restricted oncogenes remain less under-stood. Here, we report an inherent dependency of melanoma cells on the small GTPase RAB7, identifiedwithin a lysosomal gene cluster that distinguishes this malignancy from over 35 tumor types. Analyses inhuman cells, clinical specimens, and mouse models demonstrated that RAB7 is an early-induced melanomadriver whose levels can be tuned to favor tumor invasion, ultimately defining metastatic risk. Importantly,RAB7 levels and function were independent of MITF, the best-characterized melanocyte lineage-specifictranscription factor. Instead, we describe the neuroectodermal master modulator SOX10 and the oncogeneMYC as RAB7 regulators. These results reveal a unique wiring of the lysosomal pathway that melanomasexploit to foster tumor progression.
INTRODUCTION
Most solid tumors that have been scrutinized in detail contain
a vast array of (epi)genetic alterations (Garraway and Lander,
2013). Therefore, the identification of tumor drivers represents
a main challenge in oncology. This is remarkably daunting
in cutaneous melanoma, where over 85,000 coding mutations
have been reported (Lawrence et al., 2013). Gene discovery
in melanoma is further hampered by the inherently dynamic
behavior of these tumor cells (Javelaud et al., 2011; Roesch
Significance
Mechanisms underlying lineage specificity can help explain wtypes. The discovery of oncogenic dependencies is particularnumber of mutations and epigenetic alterations. Here, we showa developmental memory that reflects a unique wiring of vesiclenriched endolysosomal gene cluster that revealed unanticipapotential of these aggressive tumors. Our data illustrate how lysal feature of eukaryotic cells, can be hijacked in a tumor-type
et al., 2010). In particular, gene expression changes linked to
epithelial-to-mesenchymal-like transition have been described
whereby melanomas deregulate proliferation, lineage specifica-
tion, and embryonic differentiation genes to acquire invasive
properties (Alonso et al., 2007; Caramel et al., 2013; Gupta
et al., 2005; Widmer et al., 2012). However, although expression
analyses, spheroid cultures, andmouse xenografts support mel-
anoma cell plasticity (Caramel et al., 2013; Pinner et al., 2009;
Widmer et al., 2012), the underlying mechanisms are still not fully
understood.
hy particular genes drive malignancy in only certain tumorly challenging in melanoma, a disease with a strikingly highthat despite their complex genotype, melanoma cells retain
e trafficking pathways. Specifically, we identify a melanoma-ted roles of RAB7 in controlling the proliferative and invasivesosomal-associated degradation, otherwise a rather univer-- and stage-dependent manner.
Cancer Cell 26, 1–16, July 14, 2014 ª2014 Elsevier Inc. 1
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Selective Dependence of Melanoma Cells on RAB7
2 Cancer Cell 26, 1–16, July 14, 2014 ª2014 Elsevier Inc.
Please cite this article in press as: Alonso-Curbelo et al., RAB7 Controls Melanoma Progression by Exploiting a Lineage-Specific Wiring of the Endo-lysosomal Pathway, Cancer Cell (2014), http://dx.doi.org/10.1016/j.ccr.2014.04.030
Cancer Cell
Selective Dependence of Melanoma Cells on RAB7
Please cite this article in press as: Alonso-Curbelo et al., RAB7 Controls Melanoma Progression by Exploiting a Lineage-Specific Wiring of the Endo-lysosomal Pathway, Cancer Cell (2014), http://dx.doi.org/10.1016/j.ccr.2014.04.030
A less explored strategy to identify drivers of tumor progres-
sion is to focus on lineage-specific traits, instead of on genes
or pathways that may be deregulated across multiple cancer
types (Garraway and Lander, 2013). The underlying hypothesis
is that malignant cells may potentiate or rewire signaling cas-
cades that are specifically required for the survival and/or phys-
iological functions of their normal cell precursors (Garraway and
Sellers, 2006). In melanoma, for example, studies of the micro-
phthalmia-associated transcription factor (MITF) have illustrated
how cancer cells can hijack lineage specification genes to pro-
mote tumor development (Tsao et al., 2012). Thus, protumori-
genic targets of MITF include a variety of factors involved in
the control of cell death, DNA replication, repair, and mitosis,
as well as microRNA production, membrane trafficking, and
mitochondrial metabolism, among others (Cheli et al., 2010;
Haq et al., 2013a; Vazquez et al., 2013). Nevertheless, MITF is
a dynamically regulated rheostat, favoring or dampening malig-
nant features of melanoma cells in a context-dependent manner
(Koludrovic and Davidson, 2013). Moreover, although MITF is
amplified in a subset of melanomas, it can also be inhibited post-
transcriptionally (Bell and Levy, 2011; Javelaud et al., 2011;
Thurber et al., 2011). Consequently, MITF and its targets, such
as the trafficking modulator RAB27A or the antiapoptotic
BCL2A1 protein, are overexpressed (and required) only by a
fraction of melanomas (Akavia et al., 2010; Haq et al., 2013b).
It is unclear whether there are alternative lineage-restricted
drivers common to a broader spectrum of melanomas.
Here, we have performed a comprehensive computational,
genetic, and functional characterization of melanoma-enriched
pathways to identify lineage-specific tumor drivers that define
the inherent plasticity of this aggressive malignancy.
RESULTS
Selective Enrichment of Lysosomal-Associated Traits inMelanomaTo identify signaling cascades unique to melanoma, gene set
enrichment analysis (GSEA) was performed on transcriptional
data sets (Barretina et al., 2012; Scherf et al., 2000; Shanka-
varam et al., 2007) covering over 35 different tumor types. As
expected, pigmentation-related Gene Ontology (GO) gene sets
were enriched in melanoma cells (false discovery rate [FDR] <
3.04 3 10�5; Table S1 available online). However, the highest
enrichment in melanoma was found for the GO lysosome, vacu-
Figure 1. Melanoma-Selective Overexpression of a Lysosomal Gene C(A) GSEA heatmap showing a selective enrichment of the Lysosome Gene Ontolo
tumor cell lines in the CCLE.
(B) Enrichment plot for the lysosome GO gene set described in (A) in melanoma
(C) Effect of chloroquine (20 mM, 48 hr) on the viability of the indicated tumor c
triplicate. Data are represented as mean ± SEM. ***p < 0.001.
(D) Box plots showing the relative RAB7A mRNA levels across the different tumo
centric robust multiarray analysis-normalized mRNA expression data. The numb
(E) Western blot analysis showing the relative levels of RAB7, MITF, and RAB2
cell lines.
(F) Quantification of RAB7 protein expression by immunohistochemical staining o
(n = 121).
(G) Representative immunohistochemical staining of RAB7 protein (pink) in human
magnification images and 20 mm in the insets.
See also Figure S1 and Tables S1 and S2.
ole, and lytic vacuole gene sets (FDR < 1.0 3 10�8; Table S1). In
particular, a cluster of lysosome-associated factors was coex-
pressed within all 55 melanoma cell lines of the Cancer Cell
Line Encyclopedia (CCLE) in a distinct manner from >750
examples of other tumor types (see heatmaps in Figure 1A, the
corresponding enrichment plots in Figure 1B, and a ranked list
of melanoma-enriched genes in Figure S1A). Lysosomes share
common precursors with melanosomes (Raposo and Marks,
2007). However, additional GSEA also confirmed a specific
enrichment of ‘‘lysosome-only’’ genes in melanoma (including
proteases, lipases, acid ceramidases, acid phosphatases, and
other lytic enzymes), with no link to melanosome biogenesis or
function (Figure S1B). Consistent with these results, representa-
tive melanoma cell lines (n = 8) were found to have an overall
higher lysosomal-associated proteolytic activity compared to
cells of other tumor types (n = 7), as reflected by the cleavage
of the DQ-Green BSA probe (Figure S1C). Moreover, melanoma
cell lines were found to be highly sensitive to the lysosomotropic
agent chloroquine under conditions in which neither the viability
nor proliferation rate of other nonmelanoma cell lines was signif-
icantly affected (Figure 1C; Figures S1D–S1F). Importantly, chlo-
roquine effectively inhibited global lysosomal function in all cell
lines tested, as defined by the accumulation of the autophagic
flux markers LC3-II, p62, and NBR1 (Figures S1G and S1H).
Together, these data support a specific wiring of lysosomal-
associated degradative pathways in melanoma cells, which is
required for their maintenance.
Preferential Accumulation of RAB7 in Melanoma Cellsand Tumor SpecimensHistological and functional analyses were then performed across
different tumor types to identify putative lineage-specific cancer
drivers within the melanoma-enriched lysosomal signature.
We selected the RAB7A GTPase for validation based on the
following criteria. (1) RAB7A (hereafter referred to as RAB7 for
simplicity) showed the highest enrichment in melanoma (Fig-
ure 1D), exceeding other RABs such as RAB27A (Figure S1I),
known tomodulate theproliferation of a subset ofmelanomacells
(Akavia et al., 2010). (2) RAB7 has key roles in lysosome biogen-
esis and lysosomal-associated degradation of cytoplasmic ves-
icles (Bucci et al., 2000; Zerial and McBride, 2001; Zhang et al.,
2009), processes also found by GSEA to be upregulated in
melanoma (Table S1). (3) RAB7 maps within a genomic region
frequently amplified in melanoma (see representative array
luster Containing the Small GTPase RAB7gy gene set (GO:0005764) in melanoma cell lines compared to nonmelanoma
versus nonmelanoma tumor cell lines (GSEA FDR = 0.018).
ell lines. Shown is an example of two independent experiments performed in
r types, extracted from CCLE_Expression_Entrez_2012-10-18.res, with gene-
er of cell lines of each tumor type analyzed is indicated in parentheses.
7A protein in the indicated melanoma (blue) and nonmelanoma (black) tumor
n multitumor tissue microarrays containing the indicated human cancer types
biopsies from the indicated cancer types. Scale bars represent 200 mm in low-
Cancer Cell 26, 1–16, July 14, 2014 ª2014 Elsevier Inc. 3
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Cancer Cell
Selective Dependence of Melanoma Cells on RAB7
4 Cancer Cell 26, 1–16, July 14, 2014 ª2014 Elsevier Inc.
Please cite this article in press as: Alonso-Curbelo et al., RAB7 Controls Melanoma Progression by Exploiting a Lineage-Specific Wiring of the Endo-lysosomal Pathway, Cancer Cell (2014), http://dx.doi.org/10.1016/j.ccr.2014.04.030
Cancer Cell
Selective Dependence of Melanoma Cells on RAB7
Please cite this article in press as: Alonso-Curbelo et al., RAB7 Controls Melanoma Progression by Exploiting a Lineage-Specific Wiring of the Endo-lysosomal Pathway, Cancer Cell (2014), http://dx.doi.org/10.1016/j.ccr.2014.04.030
comparative genomic hybridization [CGH] data in Figure S1J and
additional information in Table S2), consistent with computa-
tional algorithms that suggested a putative driver role in this dis-
ease (Akavia et al., 2010). (4) There was no previous indication
that RAB7 could be regulated or acting in a tumor-type-specific
manner (Wang et al., 2011; Zhang et al., 2009). Moreover, the
specific rolesofRAB7 in cancer areunclear, as it can support pro-
tumorigenic (Wang et al., 2012; Williams and Coppolino, 2011) or
suppressive effects (Edinger et al., 2003; Steffan and Cardelli,
2010; Steffan et al., 2014) depending on the cellular context.
Western blot and tissue microarray (TMA) protein expression
analyses across 17 different cancer types confirmed a selective
enrichment of RAB7 in melanoma cell lines (Figure 1E) and tumor
specimens (Figures 1F and 1G; p < 0.001, n = 121), even in cases
with negligible levels of MITF (Figure 1E). RAB7 levels were
significantly elevated in melanoma cells as compared to normal
skinmelanocytes (see Figure S1K for representative examples of
costaining of RAB7 and the S100 melanocytic marker in normal
skin and primary melanoma human specimens). Together, these
results suggest that RAB7 could represent anMITF-independent
lineage-specific melanoma driver.
Lineage-Specific Dependency of Melanoma Cells onRAB7Melanoma-restricted roles of RAB7 were investigated by stable
transduction of three independent short hairpin interfering RNAs
(shRNAs) in a panel of cell lines frommelanoma (n = 8) and from a
selection of other tumor types (n = 8). Melanoma cells responded
with a significant inhibition of cell proliferation (Figures 2A–2D;
Figures S2A and S2B) and colony-forming ability (Figure 2E). In
contrast, RAB7 downregulation hadminimal or moderate effects
on the proliferative capacities of the other tumor cell lines (Fig-
ures 2A–2E; Figures S2A and S2B). Controls for RAB7 shRNA
specificity included the overexpression of the well-characterized
dominant-negative RAB7 T22N mutant (Figure 2E; Figures S2C–
S2E) and of wild-type RAB7, the latter increasing melanoma
cell proliferation (Figures S2C and S2D). Importantly, the inhibi-
tory effects of RAB7 shRNA and the dominant-negative RAB7
mutant translated into a significantly reduced tumorigenic
potential in vivo (i.e., xenograft models) of both BRAFV600E-
and NRASQ61R-mutated melanoma cell lines (see examples in
Figures 2F and 2G). Of note, the selective dependency of mela-
noma cells on RAB7 was independent of their pigmentation sta-
tus and basal MITF levels (Figures 1E and 2C–2G and results not
shown), and was not recapitulated by other RAB proteins with
shared functions in melanosome transport (Figure S2E).
Figure 2. Lineage-Specific Dependency of Melanoma Cells on RAB7
(A) Western blot analysis showing RAB7 downregulation with three different RAB7
lines. Noninfected cells (-) or cells expressing scramble shRNA control (Ctrl) are
(B) Bright-field images of cell populations generated as in (A) and imaged at day
(C) RAB7 and b-actin immunoblots of total cell extracts isolated from the indica
shRNA3 (RAB7)-expressing lentiviruses.
(D) Impact of RAB7 downregulation on cell proliferation of the indicated cell popu
number was quantified during consecutive days. Data correspond to the mean ±
(E) Colony-formation assays with the indicated tumor cell lines expressing RAB7
corresponding controls. Scale bars represent 5 mm.
(F and G) Growth after subcutaneous implantation in nude mice of the indicat
dominant-negative mutant (RAB7 T22N) (G), or their corresponding controls. Re
***p < 0.001, **p < 0.01, *p < 0.05. See also Figure S2.
To define whether the high expression of RAB7 in melanoma
cells could be traced to normal melanocytes, human skin was
processed for the isolation ofmelanocytes, keratinocytes, and fi-
broblasts. Primary cultured melanocytes had intrinsically higher
levels of RAB7 (Figure S2F), and were more dependent on this
protein than their genetically matched nonmelanocytic counter-
parts (Figures S2G–S2I). Still, melanoma cells were more sensi-
tive than melanocytes to RAB7 depletion, responding with a
more marked inhibition of proliferation and acquisition of senes-
cence-like features (Figures S2G–S2K). These results suggest
that melanoma cells may exploit (and depend on) proliferative
roles of RAB7 present in normal precursors but acquire addi-
tional signals that impose a strict dependency on this GTPase.
Invasive and Metastatic Potential of Melanoma CellsControlled by RAB7Aberrant vesicle trafficking canmodulate cell shape, cell motility,
and metastasis (Goldenring, 2013). Therefore, we determined
whether the inhibition of melanoma cell proliferation was associ-
ated with cytoskeletal changes and alterations in cell motility. As
shown in Figure 3A, RAB7 depletion induced marked morpho-
logical changes in all melanoma cell lines tested. In particular,
prominent cellular vacuolization and increased dendricity were
characteristic of NRAS- and BRAF-mutated cases, respectively
(Figure 3A; see also Movie S1). RAB7 downregulation reduced
cell-cell contacts (Figure 3A), increased cell scattering (Fig-
ure S3A), and induced the disassembly of stress fibers and
cortical F-actin (see phalloidin staining of cells grown in mono-
layers in Figure S3B). Cytoskeletal alterations of shRAB7
melanoma cells were also evident in single-cell analysis of
cell polarization (Figure S3C), which also identified decreased
focal adhesions (paxillin staining; Figure S3C). These marked
phenotypic changes driven by RAB7 downregulation were not
observed in normal melanocytes and fibroblasts or in tumor cells
from seven other cancer types (Figure 3A).
Counterintuitively, although inhibition of RAB7 expression
reduced the proliferation and colony formation of melanoma
cells, the phenotypic changes described above suggested
enhanced rather than reduced cell motility upon RAB7 downre-
gulation. Indeed, RAB7 shRNA increased the migration capacity
of melanoma cells in scratch assays (Figure S3D), increased
the invasive potential of moderately metastatic melanoma cells
in Matrigel invasion assays (Figure 3B), and favored their lung
extravasation ability after tail vein injection in immunosup-
pressed mice (Figure 3C). Furthermore, melanoma cell lines
with the highest invasive and metastatic potential in vitro and
shRNAs (1–3) in the indicated melanoma (blue) and nonmelanoma (black) cell
also shown as reference controls.
6 posttransduction. Scale bars represent 50 mm.
ted tumor cell lines, noninfected (-) or infected shRNA control (Ctrl), or RAB7
lations. Cells were plated at equal cell numbers at day 6 postinfection and cell
SEM of three experiments performed in triplicate.
shRNA3 (shRAB7), RAB7 T22N dominant-negative mutant (RAB7DN), or their
ed melanoma cell lines expressing RAB7 shRNA3 (shRAB7) (F), RAB7 T22N
sults represent the mean ± SEM of ten mice per condition.
Cancer Cell 26, 1–16, July 14, 2014 ª2014 Elsevier Inc. 5
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Cancer Cell
Selective Dependence of Melanoma Cells on RAB7
6 Cancer Cell 26, 1–16, July 14, 2014 ª2014 Elsevier Inc.
Please cite this article in press as: Alonso-Curbelo et al., RAB7 Controls Melanoma Progression by Exploiting a Lineage-Specific Wiring of the Endo-lysosomal Pathway, Cancer Cell (2014), http://dx.doi.org/10.1016/j.ccr.2014.04.030
Cancer Cell
Selective Dependence of Melanoma Cells on RAB7
Please cite this article in press as: Alonso-Curbelo et al., RAB7 Controls Melanoma Progression by Exploiting a Lineage-Specific Wiring of the Endo-lysosomal Pathway, Cancer Cell (2014), http://dx.doi.org/10.1016/j.ccr.2014.04.030
in vivo were found to have lower basal levels of RAB7 (Figure 3D).
In a meta-analysis performed on six independently generated
melanoma transcriptomic data sets (Widmer et al., 2012),
RAB7 mRNA levels correlated negatively with a defined proinva-
sive melanoma gene signature (Hoek et al., 2008) but positively
with proproliferative gene sets (Figure S3E). Therefore, RAB7
has different dose- and tumor-type-dependent roles in cancer
cell proliferation and invasion.
Global Impact of RAB7-Dependent Vesicular Traffickingon the Melanoma ProteomeNext, we sought to dissect the effector mechanisms by which
RAB7 levels could modulate the proliferative and invasive ca-
pacities of melanoma cells. First, we assessed autophagosome
maturation, as this is a prototypic function of RAB7 (Wang et al.,
2011; Zhang et al., 2009) and defective autophagy can compro-
mise melanoma cell proliferation per se (Checinska and Soen-
gas, 2011). Focal accumulation of the autophagosome marker
LC3 confirmed impaired autophagic degradation in RAB7
shRNA-treated melanoma cells (Figure S3F, left panels). None-
theless, a series of differences with canonical degradative pro-
grams was identified. (1) LC3-positive vesicles accumulating
upon RAB7 downregulation were unusually large and clearly
distinct from the ‘‘classical’’ compact LC3 foci induced by
treatment with rapamycin, a standard autophagy inducer (Fig-
ure S3F). (2) The formation of these large ring-shaped LC3
vesicles was independent of ATG7, a key autophagy regulator
(Figure S3G). (3) Instead, LC3 was loaded into pre-existing late
endosomes (matured from RAB5-decorated vesicles) that accu-
mulated when RAB7 was depleted (Figures S3H and S3I; Movie
S2). (4) The LC3-recruiting endosomes were identified asmacro-
pinosomes, because they originated frommembrane ruffles (not
shown) and were filled with fluid-phase endocytic tracers (Fig-
ure S3J). Together, these observations point to the existence
of an ATG7-independent route for lysosomal degradation of
autophagosome-endosome hybrids in melanoma cells, which
depends on RAB7 for its proper execution. Of note, normal
Figure 3. RAB7 Levels Inversely Modulate Cell Proliferation and Invasi
(A) Micrographs showing the selective impact of RAB7 downregulation by RAB7
nonmelanoma cell types. NRAS- and BRAF-mutated melanoma cells are labeled
(B) Invasiveness of SK-Mel-28 expressing scramble shRNA control (shControl) or
assay (48 hr). Data are presented as the average ± SEM of three independent ex
(C) Representative histological analysis of lung sections of nonobese diabetic/se
mCherry-labeled SK-Mel-28 expressing shControl or RAB7 shRNA3 (shRAB7). Ar
Cherry antibody. An average of 12 ± 2 cells were found extravasated per 10 mm
(D) Inverse correlation between RAB7 protein levels and the invasive capacity of
invasion assays, respectively. The percentage of invading cells is presented as th
RAB7 protein levels are presented as the average ± SD of two independent west
loading control.
(E) Pie chart representing the distribution of significantly down- and upregula
melanoma cells.
(F) GSEA plots showing representative examples of gene modules significantl
membrane trafficking; FDR = 0.016) upon RAB7 depletion (RAB7 KD) in UACC-6
HCT116 nonmelanoma cell lines are shown in Table S3.
(G) Opposing (and melanoma cell-specific) effect of RAB7 downregulation on th
invasion (i.e., CEACAM1), shown by immunoblotting in SK-Mel-28 and UACC-6
nonmelanoma reference.
(H) Immunoblot analyses of the indicated proteins in the conditioned media (CM)
cell lines, either noninfected (-) or infected to express scrambled shRNA (Ctrl) or
Additional information is included in Figure S3, Movies S1 and S2, and Table S3
melanocytes, which exhibited milder phenotypic changes upon
RAB7 downregulation, displayed an intrinsically silent macropi-
nocytic activity (Figure S3K).
RNA sequencing (RNA-seq) was performed at early time
points after transduction of shRNA-expressing vectors to
broadly dissect the molecular changes induced by deregulating
RAB7-mediated trafficking in representative melanoma cells.
HCT116 colon cancer cells were also analyzed as a nonmela-
noma reference. Notably, RAB7 depletion resulted in alterations
in the transcriptome of HCT116, but these changes were either
less significant or even opposite the effects in melanoma cells
(see GSEA results in Table S3). By contrast, depletion of RAB7
in melanoma cells led to a significant downregulation of multiple
gene sets involved in cell-cycle progression (Figure 3E;
Figure S3L; Table S3). The E2F1 signaling cascade (Figure 3F)
was particularly relevant, considering its requirement for mela-
noma cell proliferation (Halaban et al., 2000). Protein validation
analyses demonstrated that RAB7 shRNA induced the downre-
gulation of the E2F1 cofactor TFDP1, the E2F1/TFDP1 targets
CDC2 and CDC6, and various MCM proteins (Figure 3G and re-
sults not shown). Other key mitosis and cell-division-related
pathways were also identified (e.g., involving aurora kinases or
PLK1-associated programs; see Figure S3L and Table S3),
which interestingly have not previously been linked to RAB7.
Of note, the downregulation of cell-cycle factors (see TFDP1 in
Figure S3M, top panel) and the phenotypic changes induced
by RAB7 shRNA (Figure S3M, bottom panel and Figure S3N)
were rescued by transducing cells with a nontargetable
RAB7 construct, confirming the specificity of the depletion sys-
tem used.
RNA-seq also provided mechanistic insight into the proinva-
sive phenotype induced by RAB7 shRNA in melanoma cells
(see examples of pathways involved in cell motility, tumor inva-
sion, and extracellular matrix remodeling in Figures 3E and 3F,
Figure S3L, and Table S3). Western blot analyses validated
CEACAM1, a clinically relevant prometastatic melanoma gene
(Thies et al., 2002), as a RAB7-controlled factor (Figure 3G).
on-Related Pathways of Melanoma Cells
shRNA3 (shRAB7) on the morphology of melanoma cells versus the indicated
with an asterisk or a number sign (#), respectively. Scale bars represent 20 mm.
two different RAB7 shRNA (shRAB7) constructs, evaluated byMatrigel invasion
periments performed in duplicate. **p < 0.01.
vere combined immunodeficiency mice 48 hr after intravenous implantation of
rows point to tumor cells in the lung parenchyma, stained in brown with an anti-2 lung area in shControl versus 28 ± 3 in shRAB7 (p = 0.04, n = 5 mice/group).
the indicated melanoma cell lines, evaluated by western blotting and Matrigel
e average ± SEM of three independent Matrigel invasion experiments. Relative
ern blots quantified with ImageJ software (NIH) and using a b-actin blot as the
ted genes (FDR < 0.05) upon transduction of RAB7 shRNA3 in UACC-62
y downregulated (left: E2F pathway; FDR = 5.48E-04) or upregulated (right:
2 melanoma cells. The complete GSEA data for both UACC-62 melanoma and
e levels of factors involved in cell proliferation (i.e., TFDP1, CDC2, CDC6) and
2 melanoma cell lines. HCT116 colon cancer cells (black) are included as a
or in total cell lysates isolated from nonmelanoma (black) and melanoma (blue)
RAB7 shRNA3 (RAB7).
.
Cancer Cell 26, 1–16, July 14, 2014 ª2014 Elsevier Inc. 7
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5 YEAR FOLLOW-UP
10 YEAR FOLLOW-UP
(legend on next page)
Cancer Cell
Selective Dependence of Melanoma Cells on RAB7
8 Cancer Cell 26, 1–16, July 14, 2014 ª2014 Elsevier Inc.
Please cite this article in press as: Alonso-Curbelo et al., RAB7 Controls Melanoma Progression by Exploiting a Lineage-Specific Wiring of the Endo-lysosomal Pathway, Cancer Cell (2014), http://dx.doi.org/10.1016/j.ccr.2014.04.030
Cancer Cell
Selective Dependence of Melanoma Cells on RAB7
Please cite this article in press as: Alonso-Curbelo et al., RAB7 Controls Melanoma Progression by Exploiting a Lineage-Specific Wiring of the Endo-lysosomal Pathway, Cancer Cell (2014), http://dx.doi.org/10.1016/j.ccr.2014.04.030
Furthermore, proteomic analyses of conditioned media, guided
by the deregulation of membrane trafficking and protein secre-
tion pathways revealed by RNA-seq (Figure 3F; Table S3),
demonstrated that RAB7 downregulation ultimately promotes
the secretion of fibronectin and multiple lysosomal cathepsins
(CTS) (Figure 3H and data not shown), all with well-known
roles in tumor metastasis (Mason and Joyce, 2011). Although
cathepsin secretion has been associated with RAB7 downregu-
lation (Steffan et al., 2014), these effects are likely cell-type-
dependent, as they were not found in the nonmelanoma
HCT116 cell line (even in the case of the secretion of CTS-X,
which is equally expressed in all cell types analyzed; Figures
3G and 3H). Immunofluorescence analyses demonstrated that
the increased protein secretion of RAB7-depleted melanoma
cells was a direct consequence of changes in the fate and distri-
bution of endocytic vesicles. CTS-B accumulated in RAB5-
decorated early endosomes that, instead of being transferred
to perinuclear lysosomes for degradation, relocalized toward
the plasma membrane when RAB7 was reduced (Figure S3O).
Together, these data provide mechanistic insight into the broad
effector mechanisms whereby RAB7 influences melanoma cell
proliferation and invasion, and further underscore a cancer-
type-dependent impact of deregulated vesicle trafficking.
RAB7 Is an Early-InducedMelanomaDriver Tuned Downat Invasive Stages of Tumor Progression In VivoThe dual roles of RAB7 found in cultured melanoma cells (i.e.,
a requirement for proliferation but an enhancement of invasive
phenotypes when tuned down) predicted that this protein would
be regulated in a dynamic manner in vivo, with a selective down-
modulation favoring the switch to metastatic stages. To validate
our findings, RAB7 expression was analyzed in a comprehensive
series of TMAs containing human benign intradermal nevi and bi-
opsies collected from radial growth phase (RGP), vertical growth
phase (VGP), and metastatic melanomas (n = 152 cases). Mela-
noma specimens showed significantly higher levels of RAB7
compared to surrounding unaffected normal skin and benign
nevi (Figures 4A and 4B), with the highest enrichment in the
early-stage RGP specimens (p < 0.001). High expression of
RAB7 positively correlated with high Cyclin D1 levels (Figures
S4A and S4B), further supporting a role for this GTPase in mela-
noma cell proliferation. However, RAB7 levels did not remain
constant, as a significant reduction of RAB7 levels was observed
at the RGP-to-VGP transition (Figure 4B), the stage at whichmel-
Figure 4. Stage-Dependent Expression and Prognostic Value of RAB7(A) Representative IHC of RAB7 (pink) in the indicated human benign (a) and ma
micrographs of the corresponding upper panels (a–e). Asterisks mark stromal ce
(B) Quantification of RAB7 IHC staining on TMAs containing the indicated humanm
in parentheses at the bottom of the bar graph (total n = 152). ***p < 0.001.
(C) Confocal immunofluorescence (IF)-based quantification of the mean RAB7
melanoma specimen. Blue color represents stromal cells.
(D) Staining of RAB7 and Cyclin D1 by IHC in consecutive slides from the same
(E) Inverse correlation between RAB7 protein levels and Breslow index or dep
each group.
(F) Kaplan-Meier curves showing 10-year metastasis-free survival upon resectio
RAB7 protein levels in primary tumor specimens (n = 112, p = 0.001).
(G) Cox regression models indicating the predictive value of RAB7 expression on
depth. HR, hazard ratios; 95% CI, 95% confidence intervals.
See Figure S4 for additional information.
anoma cells are known to acquire invasive properties. Single-cell
confocal analyses of RAB7 levels in whole-tissue sections of pri-
mary melanomas further revealed decreasing RAB7 expression
toward the dermal-invading front of primary tumors (see a repre-
sentative example in Figures 4C and 4D). Similarly, human mel-
anoma cells grown in mice also showed a decreasing gradient
of RAB7 expression toward the deeper areas of the xenograft tu-
mors (see arrows in Figure S4C). The inverse correlation between
RAB7 expression and depth of melanoma invasion (Breslow
scoring) was confirmed in an independent cohort of primary
human melanomas (Figure 4E; p < 1.0 3 10�3, n = 116). Impor-
tantly, no RAB7-negative tumor was identified, and even the
‘‘low-expressing’’ melanomas retained RAB7 at levels invariably
higher than the surrounding stromal counterparts (marked with
asterisks in Figure 4A), in agreement with a lineage-specific
dependence of melanomas on this factor.
To assess the physiological relevance of the downmodulation
of RAB7 in melanoma metastasis, retrospective 10-year follow-
up analyses were performed using clinically annotated TMAs
containing a total of 112 primary melanoma specimens. Patients
with reduced expression of RAB7 in primary tumors showed
an increased risk for the development of metastasis (p = 0.001,
Figure 4F; Cox hazard ratio of 2.52 at 10 years after diagnosis,
Figure 4G) and a poor overall survival (Figures S4D and S4E).
Low RAB7 levels were predictive of metastatic risk even when
adjusted for Breslow depth (Figure 4G). Together, these data
identify RAB7 as a dynamically regulated and physiologically
relevant risk factor that might underlie the phenotypic plasticity
of aggressive melanoma cells.
Oncogene-Dependent Regulation of RAB7 by MYCComputational analyses of the RAB7 promoter revealed the
presence of eight E boxes (Figure 5A, top panel) characteristi-
cally recognized by the oncogenic transcription factor MYC,
well known for its involvement in melanoma progression (Polsky
and Cordon-Cardo, 2003). This oncogene was also attractive to
investigate further, because its depletion leads to melanoma cell
vacuolization and dendricity in amanner similar to RAB7 (Zhuang
et al., 2008). Histological analyses revealed a marked hetero-
geneity of MYC expression in melanocytic lesions. Nonetheless,
MYC was enriched in melanocytic nests close to the epidermal/
dermal interface, where RAB7 also accumulated (Figure 5B).
Chromatin immunoprecipitation demonstrated that the three
most proximal E boxes upstream of the RAB7 transcriptional
in Human Melanoma Biopsieslignant melanocytic lesions (b–e). Lower panels (f–h) show high-magnification
lls.
elanocytic lesions. The number of samples per category analyzed is indicated
protein expression per melanoma cell in a representative whole-tissue VGP
VGP melanoma specimen shown in (C).
th of invasion (p < 0.001). n indicates the total number of cases analyzed in
n of primary melanomas. Patients were stratified as a function of high and low
5- and 10-year metastasis-free survival, with or without adjusting for Breslow
Cancer Cell 26, 1–16, July 14, 2014 ª2014 Elsevier Inc. 9
A B
C D E
F G
(AFU)
(AF
U)
(legend on next page)
Cancer Cell
Selective Dependence of Melanoma Cells on RAB7
10 Cancer Cell 26, 1–16, July 14, 2014 ª2014 Elsevier Inc.
Please cite this article in press as: Alonso-Curbelo et al., RAB7 Controls Melanoma Progression by Exploiting a Lineage-Specific Wiring of the Endo-lysosomal Pathway, Cancer Cell (2014), http://dx.doi.org/10.1016/j.ccr.2014.04.030
Cancer Cell
Selective Dependence of Melanoma Cells on RAB7
Please cite this article in press as: Alonso-Curbelo et al., RAB7 Controls Melanoma Progression by Exploiting a Lineage-Specific Wiring of the Endo-lysosomal Pathway, Cancer Cell (2014), http://dx.doi.org/10.1016/j.ccr.2014.04.030
start site were bound by MYC (Figure 5A, bottom panel). When
MYC was depleted by validated small interfering RNA (siRNA)
(Doe et al., 2012), RAB7 expression was inhibited by >60%
(Figure 5C).
Lineage-Specific but MITF-Independent Regulation ofRAB7 by SOX10We next interrogated lineage-restricted transcription factors that
may confer specificity to the melanoma-enriched expression
of RAB7 found by GSEA. Although the best-characterized mela-
nocyte developmental pathways involve MITF, melanoma cells
with low or undetectable MITF retained high levels of RAB7 in
culture (Figure 1E; Figure S5A) and in tumor biopsies (Figure 5D;
see quantification in Figure 5E and additional information in Fig-
ure S5B). Indeed, downregulation of MITF by validated siRNAs
had nomeasurable impact on RAB7 levels in different melanoma
cell lines (Figure 5F) or at different time points (Figure S5C) in
which the expression of other known MITF targets, such as
RAB27A, was effectively blocked. Of note, depletion of TFEB,
a transcription factor of the MITF/TFE family that controls the
expression of various autophagy-associated factors (Settembre
et al., 2011), had no significant effect on RAB7 (although it
did exert a modest reduction [30%] of RAB27A expression;
Figure 5G).
Having excluded a role for MITF in the control of RAB7 mRNA
or protein levels, we next interrogated the contribution of PAX3
and SOX10, transcription factors acting early in the specification
of the melanocytic lineage. No significant changes in RAB7
levels were found upon depletion of PAX3 (Figure 6A). However,
siRNA against SOX10 effectively reduced RAB7 mRNA and pro-
tein (Figures 6A–6D). Of note, SOX10 expression mimicked that
of RAB7, being retained in MITF-negative melanoma cells and
expressed at lower levels in highly invasive cell lines (Figure 6E).
A significant positive correlation was found between RAB7 and
SOX10 mRNAs in frozen human melanoma specimens (Fig-
ure 6F; r = 0.6, n = 15), as further supported by analysis of protein
expression in consecutive sections of benign andmalignant mel-
anocytic biopsies (Figure 6G). Thus, gradients of SOX10 were
identified within human melanocytic lesions, although less
marked than for RAB7 (likely reflecting additional mechanisms
of regulation of both factors). Nonetheless, intraepidermal nests
Figure 5. Control of RAB7 Transactivation by MYC, but Not by MITF o
(A) The top panel shows eight putative Myc-binding site E boxes (E1–E8) flanking
panels summarize quantitative (q)ChIP data for theMYC antibody (blue bars), pres
IgG control (gray bars). Acetylcholine receptor and nucleolin (ACHR and NCL, res
MYC binding, respectively.
(B) IHC for RAB7 and MYC in intradermal nevi (a and b) and VGP melanoma (c an
stromal compartments (the latter are marked with asterisks). T marks the tum
magnification on the right.
(C) Downregulation of RAB7 protein expression shown by immunoblotting 72 hr
levels in control (Ctrl) siRNA-transduced cells are included as a reference.
(D) Confocal microphotographs of melanoma biopsies stained for RAB7 (red)
40,6-diamidino-2-phenylindole. Shown are lymph node (LN) metastasis and prim
example of a metastasis positive for RAB7 only (right).
(E) Confocal-based quantification of the mean immunofluorescence signal per ce
fluorescence units.
(F) Immunoblots showing relative levels of MITF, RAB27A, RAB7, and b-actin (l
control or MITF siRNA. Here and in (G), data are shown at 72 hr posttransfection
(G) RT-PCR analyses of RAB7, RAB27A, TFEB, and 18S (loading control) mRNA
See Figure S5 for additional information.
in nevi or primary melanomas with strong RAB7 expression were
highly positive for SOX10 (Figure 6G, b and c). Parallel expres-
sion of SOX10 and RAB7 was also found in metastatic mela-
nomas (see Figure 6G, d and e for examples of specimens in
which these proteins are coordinately expressed at low or high
levels, respectively).
Together, our results identify two levels of regulation of RAB7,
by the oncogenic transcription factor MYC and by the melano-
cytic lineage specifier SOX10. These data illustrate how endoly-
sosomal trafficking can be controlled in a lineage-specific
manner in cancer, distinguishing melanoma from other tumor
types.
DISCUSSION
Here, we have demonstrated that melanoma cells exploit a
lineage-specific wiring of the endolysosomal pathway to drive
tumor progression. Specifically, we have shown that the small
GTPase RAB7 is selectively upregulated in melanoma as part
of a lysosomal-associated signature that distinguishes this
malignancy from over 35 cancer types. This induction occurs
at early stages of melanoma development and sustains mela-
noma cell proliferation. However, expression studies in clinical
biopsies combined with functional studies in cultured cells re-
vealed that this GTPase can be partially downregulated in highly
invasive melanoma cells (see model in Figure 7). This pattern of
expression reflects MITF-independent roles of RAB7 regulating
the fate of endolysosomal traffic in melanoma cells, modulating
their transcriptome, proteome, and secretome and ultimately
influencing the proliferation and invasion of these aggressive
cells. The physiological relevance of these data was demon-
strated in 10-year follow-up studies of patient prognosis, which
support RAB7 as a risk factor for melanoma metastasis and
overall poor survival.
Lysosomes are essential organelles in normal and tumor cells
(de Duve, 2005). Similarly, RAB7 has been described to modu-
late endosomal maturation and autophagosome resolution in
various cell types (Wang et al., 2011; Zhang et al., 2009). Why
then are endo/lysosomal pathways (and, more specifically,
RAB7) wired (and required) in such a specific manner in mela-
noma? An attractive scenario to account for the coregulation
r TEFB
the transcriptional start site (TSS) of the RAB7 gene as indicated. The bottom
ented as themean ± SEMof three independent experiments with respect to the
pectively) promoter amplicons were used as negative and positive controls for
d d). The staining was optimized for optimal visualization of melanocytic versus
oral compartment. Areas contained in the dashed boxes are shown at high
after transfection of the indicated concentrations of MYC siRNA. Basal RAB7
and MITF (green). In the merged figure, nuclei appear counterstained with
ary melanoma specimens coexpressing both factors (left and middle) and an
ll for RAB7 and MITF in the same patient biopsies shown in (D). AFU, arbitrary
oading control) protein levels in the indicated melanoma cell lines expressing
.
levels in the indicated melanoma cell lines expressing control or TFEB siRNAs.
Cancer Cell 26, 1–16, July 14, 2014 ª2014 Elsevier Inc. 11
A B C
FED
G
(legend on next page)
Cancer Cell
Selective Dependence of Melanoma Cells on RAB7
12 Cancer Cell 26, 1–16, July 14, 2014 ª2014 Elsevier Inc.
Please cite this article in press as: Alonso-Curbelo et al., RAB7 Controls Melanoma Progression by Exploiting a Lineage-Specific Wiring of the Endo-lysosomal Pathway, Cancer Cell (2014), http://dx.doi.org/10.1016/j.ccr.2014.04.030
Figure 7. Model Summarizing Key Aspects of the Regulation and
Function of RAB7 Described in This Study
RAB7 was found to be selectively enriched in melanoma, expressed in an
MITF-independentmanner and controlled by the lineage-specific transcription
factor SOX10 (1). A second level of regulation was found linked toMYC, a well-
known oncogenic transcription factor activated at early stages of melanoma
development (2). Importantly, RAB7 levels were not constant duringmelanoma
progression, being selectively downmodulated (but never abrogated) to favor
invasive phenotypes (3), and ultimately increasing the risk for metastatic
development (4). RAB7-dependent vesicles are depicted as red dots. See the
text for additional details.
Cancer Cell
Selective Dependence of Melanoma Cells on RAB7
Please cite this article in press as: Alonso-Curbelo et al., RAB7 Controls Melanoma Progression by Exploiting a Lineage-Specific Wiring of the Endo-lysosomal Pathway, Cancer Cell (2014), http://dx.doi.org/10.1016/j.ccr.2014.04.030
of the lysosomal-related genes found here enriched in mela-
noma is that they are coordinately involved in functions that
are unique to this tumor type. For example, various lysosomal
factors including RAB7 are well known for participating in mela-
nosome maturation (Gomez et al., 2001). Nevertheless, our data
revealed an enrichment of ‘‘lysosome-only’’ genes in melanoma
cells that transcends cellular pigmentation. Moreover, these
cells showed a particular dependency on lytic activities of the
lysosome that rely on RAB7 function. In particular, the inherently
active influx of ATG7-independent autophagosome-macropino-
some hybrids found here in melanoma cells may impose a
requirement for RAB7 that cannot be compensated by other
RAB members with more restricted roles in membrane traf-
Figure 6. Lineage-Specific Modulation of RAB7 by SOX10
(A) Quantitative RT-PCR analyses of RAB7, PAX3, and SOX10 expression (relativ
Here and in (B)–(D), data are shown at 72 hr posttransfection. Error bars in (A) a
triplicate.
(B) RAB7, SOX10, and MITF mRNA levels in total cell extracts isolated from the i
levels are included as a loading reference.
(C) RAB7 and MITF mRNA levels (relative to 18S mRNA levels) determined by qR
(D) Impact of siSOX10 on RAB7 and MITF protein levels monitored by immunob
(E) Immunoblots of total cell extracts isolated from the indicated melanoma cell lin
invasive melanoma cell lines (as shown in Figure 3D) are marked in green.
(F) Positive correlation between relative mRNA levels of RAB7 versus SOX10 (es
correlation coefficient, r = 0.6).
(G) Representative IHC for RAB7 (upper panels, pink) and SOX10 (lower pane
melanocytic lesions. Stromal cells can be distinguished by negative SOX10 stain
ficking. By modulating the fate of these vesicles (i.e., from lyso-
somal targeting to recycling or secretion), RAB7 may control the
levels and/or localization of other key protumorigenic proteins,
exemplified here by lysosomal cathepsins and other prometa-
static factors.
Finding that RAB7 is controlled by SOX10 and MYC in an
MITF-independent manner has important basic and translational
implications. Thus, these results separate RAB7 from key mod-
ulators of proliferation and survival such as RAB27A, BCL2A1,
or PGC1a, which are strictly controlled by MITF (Cheli et al.,
2010; Haq et al., 2013a, 2013b). Moreover, SOX10 is not in-
hibited by mechanisms that downregulate MITF, some of which,
including BRAF mutations, are relatively frequent in malignant
melanomas (Haq et al., 2013a; Vazquez et al., 2013). This may
ensure a ‘‘developmental memory’’ in the expression of RAB7.
Nevertheless, RAB7 does not lead to the apoptotic collapse
recently described for SOX10 depletion (Shakhova et al.,
2012), suggesting that there are nonoverlapping functions for
both factors. The direct regulation of RAB7 by MYC is also rele-
vant, as this oncogene can bemodulated by a variety of signaling
cascades (Dang, 2012), opening the possibility of a broader
oncogenic regulation of RAB7 in melanoma. In this context,
it is tempting to speculate that the downregulation of RAB7
(and/or its upstream drivers) in the invasive front of aggressive
melanomas is modulated by epithelial-to-mesenchymal-like
mechanisms, such as those recently described to underlie the
transcriptional switch associated with prometastatic pheno-
types (Caramel et al., 2013).
The spatiotemporal regulation of RAB7 is central to under-
standing its role in melanomagenesis and metastasis. Studies
limited to either primary lesions or to late stages of the disease
would have missed the downregulation of this protein occurring
at the RGP-to-VGP transition, predictingmetastatic risk. It would
therefore be interesting to determine whether the levels of RAB7
fluctuate during the development of other tumor types. Further
lineage-specific analyses of RAB7 may also be relevant in he-
matopoietic and lymphoid tumors, which intriguingly showed a
systematic downregulation of the endolysosomal gene cluster
found here upregulated in melanoma (Figure 1A). Whether line-
age specificity may account for modest effects of RAB7 deple-
tion on the viability of normal murine T cells (Roy et al., 2013),
or even for suppressive roles in murine hematopoietic lines
(Edinger et al., 2003) or prostate cancer (Steffan et al., 2014),
deserves further investigation. Although this study focused on
sporadic cancers, the protumorigenic roles herein described
e to 18S mRNA) in UACC-62 melanoma cells expressing the indicated siRNAs.
nd (C) correspond to the SEM of two independent experiments performed in
ndicated melanoma cell lines expressing control or SOX10 siRNA. 18S mRNA
T-PCR in the indicated melanoma cell lines transduced with siC or siSOX10.
lotting in the indicated cell lines. b-actin is shown as the loading control.
es and probed for RAB7, SOX10, MITF, and a-tubulin (loading control). Highly
timated with respect to HPRT mRNA) in human melanoma biopsies (Pearson
ls, brown) in normal skin (a) and the indicated benign (b) or malignant (c–e)
ing. Scale bars represent 200 mm.
Cancer Cell 26, 1–16, July 14, 2014 ª2014 Elsevier Inc. 13
Cancer Cell
Selective Dependence of Melanoma Cells on RAB7
Please cite this article in press as: Alonso-Curbelo et al., RAB7 Controls Melanoma Progression by Exploiting a Lineage-Specific Wiring of the Endo-lysosomal Pathway, Cancer Cell (2014), http://dx.doi.org/10.1016/j.ccr.2014.04.030
for RAB7 might offer a mechanistic framework to explain the
development of melanomas in a subset of patients with Char-
cot-Marie-Tooth 2B, a hereditary disease associated with acti-
vating mutations in the RAB7 gene (Greene et al., 1980; McCray
et al., 2010).
In summary, despite the massive number of genetic and
epigenetic alterations thatmelanoma cells accumulate during tu-
mor progression, these cells wire endolysosomal programs (via
RAB7) in a rather uniform and unique manner. This lineage-spe-
cific wiring is exploited by melanoma cells to favor progression,
but it also serves as both a vulnerability and a potential marker
for patient prognosis.
EXPERIMENTAL PROCEDURES
Gene Expression Analyses in Cultured Cells and Tissue Specimens
Details of the human cancer cell lines used in this study, isolation of skin me-
lanocytes and fibroblasts, CGH analysis, RNA sequencing, protein immuno-
blotting and immunofluorescence in cultured cells, and histological analyses
in benign and malignant melanocytic biopsies (including single-cell confocal
analysis of gene expression) are described in Supplemental Experimental Pro-
cedures. Human tumor biopsies were obtained from the i+12 Biobank (RD09/
0076/00118) of the Hospital 12 de Octubre and the Spanish Hospital Biobank
Network, under appropriate ethical protocols by their Clinical Investigation
Ethical Committees.
Gene Set Enrichment Analysis in Multitumor Data Sets
GSEA was applied to previously reported data sets from the NCI-60 panel
(Scherf et al., 2000; Shankavaram et al., 2007) and the Cancer Cell Line
Encyclopedia (Barretina et al., 2012). GO terms (biological process, cellular
component, and molecular function) from levels 3–19 were also evaluated.
Genes were ranked using the t statistic. After Kolmogorov-Smirnoff testing,
those pathways showing false discovery rates <0.25 were considered en-
riched in the classes under comparison.
Estimation of the Prognostic Value of RAB7 Expression in Human
Melanomas
Complete 10-year follow-up survival data were available for 112 patients
(15 cases of RGP and 97 cases of VGP). Immunohistochemistry (IHC) scoring
was performed blind by two pathologists, and linkage to clinical data was per-
formed only after all analyses were completed. The specimens were classified
as low-intensity or high-intensity RAB7 staining. Metastasis-free survival and
overall survival were determined with a time-to-event analysis carried out by
computing time to these occurrences from the date of melanoma diagnosis.
For both metastasis and death, patients who did not experience the event at
the end of follow-up were considered to be right-censored observations.
Time-to-event distributions were estimated using the Kaplan-Meier method,
and log-rank homogeneity tests were carried out. To assess the possible prog-
nostic effect of Breslow index, American Joint Committee on Cancer stage,
and RAB7 labeling, Cox proportional hazards models were fitted for each
event to compute both crude (univariate) and adjusted (multivariate) hazard
ratios of each covariate with their corresponding 95% confidence limits. We
performed an automatic backward elimination procedure to keep in models
only variables with corresponding p values <0.2. For Breslow index, we sepa-
rated melanomas <1.5 mm from those R1.5 mm.
Protein Secretion Assays
For the analysis of changes in protein secretion by RAB7 depletion, cells
were transduced with lentiviruses coding for control or RAB7 shRNA. Seven
days after lentiviral infection, 2 3 106 cells were incubated in serum-free
DMEM for 18 hr. The corresponding conditioned medium was harvested,
clarified by centrifugation, filtered through a 0.45 mm filter, and then concen-
trated in Amicon Ultra-15 centrifugal filter devices with Ultracel-3 3 kDa
NMWL membrane (Millipore) by centrifugation at 4,000 3 g for 5 hr in a
swinging bucket rotor. Ten microliters of concentrated conditioned media
was subjected to protein immunoblotting.
14 Cancer Cell 26, 1–16, July 14, 2014 ª2014 Elsevier Inc.
Functional Assays of Tumorigenicity, Metastatic Potential,
Lysosome-Related Vesicle Trafficking, Expression Analyses,
Quantitative Chromatin Immunoprecipitation, and Microscopy
Functional inactivation of RAB7 (by shRNA, siRNA, and the dominant-negative
mutant RAB7 T22N), analyses of cell proliferation, colony formation,
wound-healing capacity, Matrigel invasion, lung colonization and cytoskeletal
alterations (CYTOOChips), and xenograft tumor models are described in
Supplemental Experimental Procedures. All experiments with mice were per-
formed in accordance with protocols approved by the Institutional Ethics
Committee of the Centro Nacional de Investigaciones Oncologicas (CNIO)
and the Instituto de Salud Carlos III. Fluid-phase endocytosis was visualized
by uptake of Lucifer yellow (Sigma) and 70 kDa rhodamine-dextran (Invitro-
gen), as summarized in Supplemental Experimental Procedures. Details of
lysosomal activity assays, drug treatments, microscopy, sh/siRNA-mediated
silencing, RNA sequencing, and autophagy are also provided in Supplemental
Experimental Procedures.
Statistical Analyses
Statistical tests performed to assess the significance of the computational,
histological, and functional data indicated above are described in Supple-
mental Experimental Procedures.
ACCESSION NUMBERS
The RNA-sequencing data discussed have been deposited in the National
Center for Biotechnology Information Gene Expression Omnibus under acces-
sion number GSE42735.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
five figures, three tables, and two movies and can be found with this article
online at http://dx.doi.org/10.1016/j.ccr.2014.04.030.
ACKNOWLEDGMENTS
The authors thank their colleagues in the CNIO Melanoma Group, particularly
Angel Colmenar for excellent technical assistance; Lionel Larribere, David
Saenz, and Francisco Real for their help and support; Jose A. Esteban,
F. Javier Carmona, Ashani Weeraratna, Xose Bustelo, and Charles Sherr for
critical reading of the manuscript; Beatriz Perez-Gomez for help with the sur-
vival analyses; the i+12 Biobank of the Hospital 12 de Octubre for providing
melanoma patient samples; Corine Bertolotto for providing MITF siRNA con-
structs; and Orlando Domınguez, Marta Canamero, Luis Lombardıa, and
Juan Cruz Cigudosa for their technical assistance. M.S.S. is funded by Pro-
jects SAF2011-28317 and Consolider RNAREG from the Spanish Ministry of
Economy and Innovation, R01CA125017 from the NIH, and a Team Science
Award by the Melanoma Research Foundation. J.L.R.-P. and P.O.-R. are
funded by grants FIS 11/025685 and FIS 11/1759, respectively, from the Span-
ishMinistry of Health. J.L.R.-P. was also supported by grant FMM-2008-106 of
Fundacion Mutua Madrilena, and P.O.-R. by Red Tematica de Investigacion
Cooperativa en Cancer. D.A.-C. and E.P.-G. are recipients of Scientists in
Training predoctoral fellowships from the Spanish Ministry of Science and
Innovation. M.C. and P.K. are funded by predoctoral fellowships of Fundacion
La Caixa. E.R.-F. is the recipient of a postdoctoral fellowship from Fundacion
Cientıfica de la Asociacion Espanola Contra el Cancer, and J.A.J. and H.-W.W.
are funded by the American Cancer Society (RSG-12-076-01-LIB).
Received: July 17, 2013
Revised: March 13, 2014
Accepted: April 28, 2014
Published: June 26, 2014
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