Report

Identification of an oncogenic RAB protein

See allHide authors and affiliations

Science  09 Oct 2015:
Vol. 350, Issue 6257, pp. 211-217
DOI: 10.1126/science.aaa4903

Cancer as a case of uncontrolled traffic

Healthy cells are like skilled air traffic controllers. They continually move proteins to and from the cellular destinations where they are needed, usually without mishap, through an elaborate system of endomembranes. Wheeler et al. show that a glitch in the traffic control system can help propel a cell toward malignancy (see the Perspective by Ferguson). RAB35, a protein previously implicated in endomembrane trafficking, is a key regulator of a well-known oncogenic signaling pathway. Mutations in RAB35 found in certain human tumors aberrantly activate this pathway and cause mislocalization of a factor that promotes cell growth.

Science, this issue p. 211, see also p. 162

Abstract

In a short hairpin RNA screen for genes that affect AKT phosphorylation, we identified the RAB35 small guanosine triphosphatase (GTPase)—a protein previously implicated in endomembrane trafficking—as a regulator of the phosphatidylinositol 3′-OH kinase (PI3K) pathway. Depletion of RAB35 suppresses AKT phosphorylation in response to growth factors, whereas expression of a dominant active GTPase-deficient mutant of RAB35 constitutively activates the PI3K/AKT pathway. RAB35 functions downstream of growth factor receptors and upstream of PDK1 and mTORC2 and copurifies with PI3K in immunoprecipitation assays. Two somatic RAB35 mutations found in human tumors generate alleles that constitutively activate PI3K/AKT signaling, suppress apoptosis, and transform cells in a PI3K-dependent manner. Furthermore, oncogenic RAB35 is sufficient to drive platelet-derived growth factor receptor α to LAMP2-positive endomembranes in the absence of ligand, suggesting that there may be latent oncogenic potential in dysregulated endomembrane trafficking.

The phosphatidylinositol 3′-OH kinase (PI3K)/AKT pathway is a key regulator of cell survival, proliferation, and growth and is commonly activated in human cancers by receptor tyrosine kinase (RTK) signaling (1, 2). To identify previously unknown regulators of PI3K/AKT signaling, we performed an RNA interference (RNAi) screen using lentiviruses that express short hairpin RNAs (shRNAs) (3) targeting genes coding for all known G proteins and lipid or protein kinases. Because AKT phosphorylation is a faithful indicator of PI3K activity, we used an immunofluorescent approach to quantitatively measure phosphorylation of AKT at serine 473 (S473) (4) in HeLa cells, which display robust growth factor–induced AKT phosphorylation and lack mutations in core PI3K signaling pathway genes (Fig. 1A). We screened a collection of 7450 shRNAs in an arrayed format in triplicate to identify kinases or guanosine triphosphatases (GTPases) whose depletion could alter PI3K-dependent AKT phosphorylation.

Fig. 1 An arrayed lentiviral loss-of-function RNA interference screen identifies known and previously unrecognized regulators of the PI3K/AKT axis.

(A) An immunofluorescent assay for phospho-S473 AKT quantitatively reflects PI3K activity. HeLa cells grown in 384-well plates were serum-starved overnight, treated with PIK-90 and insulin as indicated, and then fixed and processed for immunofluorescence with a whole-cell stain (red) and an anti–phospho-S473 AKT monoclonal antibody (green) (top). Signals were imaged and quantified with a near-infrared scanner, and the phospho-AKT signal intensity was divided by the whole-cell stain signal intensity to calculate a normalized phospho-AKT value for each well (bottom). (B) Averaged (n = 3 replicates) phospho-AKT z scores of screened shRNAs. HeLa cells were plated in 384-well plates, transduced with lentiviral reagents expressing control shRNAs or shRNAs targeting human kinases and GTPases, and then processed as in (A). Phospho-AKT z scores for each shRNA were calculated, averaged, and arranged as indicated. (C) Functional grouping of the 26 genes whose knockdown lowered AKT phosphorylation, that are not known to regulate PI3K/AKT signaling, and that are mutated in human cancers. Numbers indicate the number of genes in each functional group. MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; cAMP, adenosine 3′,5′-monophosphate. (D) Representation of somatic mutations in RAB35 in cancer found in oncogenomic databases. Mutations predicted by the cBioPortal for Cancer Genomics to have a medium effect on protein function are noted in black, whereas mutations predicted to have a high effect are noted in red. Table S7 contains a full list of somatic mutations of RAB35 found in cancers. aa, amino acids. (E) shRNAs targeting RAB35 deplete RAB35 protein levels and inhibit AKT phosphorylation. HeLa cells were transduced with lentiviruses expressing the indicated shRNAs, starved overnight and treated with serum as indicated, and lysed; cell lysates were analyzed by immunoblotting. Red asterisks indicate shRNA “hits” from our screen. FBS, fetal bovine serum.

To identify shRNAs that altered AKT phosphorylation (i.e., “hits”), we translated the phospho-AKT signal in each screened well to a z score (5). shRNAs targeting genes that positively regulate AKT phosphorylation (pAKT)—RICTOR, PIK3CA, PIK3CB, AKT1, and AKT3—all diminished AKT phosphorylation and scored as “pAKT-low” hits (Fig. 1B and tables S1 to S3). Conversely, shRNAs targeting genes whose protein products inhibit AKT signaling—such as RAPTOR and RHEB—elevated phospho-AKT levels and scored as “pAKT-high” hits. Thus, our screen successfully identified known regulators of PI3K/AKT signaling, as well as a number of genes not previously implicated in regulation of the pathway (table S4). We prioritized hits for genes that had no reported link to PI3K/AKT signaling (table S5) and searched oncogenomic databases to identify those with somatic mutations reported in human cancers (6, 7). This left us with a list of 29 genes (table S6), 26 of which were pAKT-low hits. Next, we grouped the 26 pAKT-low hits by their annotated functions from the literature and found that the best-represented functional group was the RAB GTPases, which are well known to regulate intracellular protein trafficking (Fig. 1C). This finding suggests that dysregulated protein trafficking by mutant RAB proteins could play a role in oncogenesis.

Of the five RAB proteins that appeared in our list in table S6 (see also Fig. 1C), we pursued RAB35 because, in addition to meeting all of the criteria mentioned previously, it is a well-studied GTPase for which reagents and tools are readily available. RAB35 is a regulator of cytoskeletal organization and trafficking at the recycling endosome (811). Endomembrane trafficking by RAB35 is regulated by the DENND1 family of RAB35 guanine nucleotide exchange factors (9, 1214) and by the TBC1D10 family of RAB35 GTPase activating proteins (1517). Because RAB35 was a robust hit in our screen (fig. S1A), has not been implicated as a regulator of PI3K/AKT signaling, and has somatic mutations that have been reported in human cancers mapping to residues conserved in other small GTPases (Fig. 1D and table S7), we prioritized RAB35 for further study.

Next, we confirmed that depletion of RAB35 with the same shRNAs that scored in the original screen suppressed AKT phosphorylation to a similar extent as depletion of the mTORC2 component RICTOR (Fig. 1E). Furthermore, additional shRAB35 hairpins not present in the original screen also suppressed AKT phosphorylation, as well as the AKT substrate FOXO1/3A (fig. S1B). Thus, multiple independent shRNAs targeting RAB35 consistently impair AKT phosphorylation in HeLa cells.

To ensure that the effect of RAB35 knockdown on AKT signaling was not cell-type specific, we depleted RAB35 protein in human embryonic kidney (HEK) 293 E cells and in murine NIH-3T3 cells (Fig. 2, A and B). Knockdowns of RAB35 with two different shRNAs diminished AKT phosphorylation at S473 in response to serum stimulation in both human and mouse cells and did so more potently than RICTOR. Furthermore, the PI3K-dependent phosphorylation of FOXO1/3A and the SGK1 substrate NDRG-1 was also decreased in cells depleted of RAB35. We expanded this analysis to seven additional cancer cell lines with different mutational backgrounds (oncogenic PI3KCA or KRAS, deleted PTEN, mutated NF2 or TP53) and generally found reduced AKT phosphorylation after RAB35 depletion, with some modest differences that are likely attributable to distinct hairpins in distinct cellular contexts (fig. S2, A to C). Furthermore, AKT phosphorylation at the PDK1 phosphorylation site T308 was consistently reduced by RAB35 knockdown, suggesting that RAB35 may function upstream of PDK1. Collectively, these data indicate that RAB35 is broadly necessary for the efficient activation of PI3K/AKT signaling in response to serum stimulation.

Fig. 2 RAB35 is necessary and sufficient for serum-induced PI3K/AKT signaling and associates with PI3Kα.

(A and B) Depletion of RAB35 inhibits serum-induced activation of AKT. (A) HeLa and HEK-293E cells were transduced with lentiviral reagents expressing the indicated shRNAs. Cells were serum-starved overnight, treated as indicated with FBS, and lysed; cell lysates were then analyzed by immunoblotting. (B) Murine NIH-3T3 cells were serum-starved then treated as indicated with bovine calf serum (BCS) and analyzed as in (A). (C) GTPase-deficient RAB35Q67L activates PI3K/AKT signaling. HeLa and HEK-293E cells were stably transduced with lentiviruses expressing FLAG-RHEBwt, -RAB35wt, or -RAB35Q67L. Cells were serum-starved overnight, treated as indicated, and analyzed as in (A). (D) RAB35 associates with PI3K in a nucleotide-dependent manner. Cells stably expressing FLAG-RHEBwt, -RAB35wt, -RAB35Q67L, or -RAB35S22N were lysed, and immunoprecipitations (IPs) were performed using the indicated antibodies. Immunoprecipitates and total cell lysates were then analyzed by immunoblotting with the indicated antibodies. n.s., nonspecific bond.

To explore whether RAB35 is sufficient to activate the PI3K/AKT pathway, we generated cell lines that stably expressed either wild-type RAB35 (RAB35wt) or the dominant active GTPase-deficient, guanosine triphosphate (GTP)–bound RAB35 Q67→L67 (Q67L) mutant (RAB35Q67L). Cells expressing the wild-type RHEB GTPase (RHEBwt), which regulates mTORC1, served as a control. Stable expression of either RHEBwt or RAB35wt did not alter phosphorylation of AKT or extracellular signal–regulated kinase (ERK) in response to growth factor signaling (Fig. 2C and fig. S3). However, expression of the active RAB35Q67L mutant rendered AKT phosphorylation (at both T308 and S473) constitutively elevated and refractory to growth factor deprivation but did not affect ERK phosphorylation. FOXO1/3A phosphorylation was also consistently elevated in serum-deprived cells expressing RAB35Q67L. Therefore, the expression of GTP-bound RAB35 is sufficient to activate PI3K/AKT signaling in cells, even in the absence of growth factors.

The fact that RAB35Q67L results in constitutive phosphorylation of T308 as well as S473 suggests that RAB35 may function upstream of the T308 kinase PDK1 (18, 19). However, attribution of RAB35 function to PDK1 versus mTORC2 activation is challenging because the phosphorylation states of T308 and S473 are interdependent (2022). To address this issue, we took advantage of cells that stably express alleles of murine AKT1 (mAkt1) in which either T308 or S473 were mutated to phosphomimetic aspartate residues (23) (fig. S4, A and B). As expected, mTOR blockade with the TORC1/2 inhibitor torin1 or blockade by RICTOR depletion (resulting in loss of mTORC2 function) suppressed phosphorylation at both sites in mAkt1wt-expressing cells (fig. S4C). In mAkt1S473D-expressing cells, which lack a regulated mTORC2 site, these same interventions either had no effect on T308 phosphorylation (RICTOR depletion) or enhanced T308 phosphorylation (torin 1).

Having confirmed the ability of this system to distinguish direct effects on T308 versus S473, we examined the impact of RAB35 knockdown. Unlike depletion of RICTOR or treatment with torin 1, depletion of RAB35 decreased T308 phosphorylation in mAkt1wt‑ and mAkt1S473D-expressing cells. RAB35 depletion also decreased S473 phosphorylation in mAkt1wt- and mAKTT308D-expressing cells (fig. S4C). These data suggest that RAB35 signals to AKT by functioning upstream of both PDK1 and mTORC2.

We reasoned that if RAB35 acts upstream of both PDK1 and mTORC2, it may be controlling their common regulator, PI3K. Although we could not detect an interaction between endogenous RAB35 and PI3K, we found that stably expressed RAB35, but not RHEB, copurified with PI3K when the kinase was immunoprecipitated by its p85 subunit (Fig. 2D). Interestingly, more FLAG-tagged RAB35 (FLAG-RAB35) copurified with PI3K in immunoprecipitates from cells expressing constitutively GTP-bound RAB35Q67L than from cells expressing RAB35wt. Conversely, we found that the same experiment performed using lysates from cells stably expressing a constitutively guanosine diphosphate–bound allele of RAB35 (RAB35S22N) did not recover any FLAG-RAB35. Thus, the physical association of RAB35 and PI3K is nucleotide-dependent. We also asked whether RAB35 is necessary for PI3K kinase activity in vitro. As expected, PI3K immunopurified from cells depleted of PI3Kα had significantly reduced in vitro kinase activity toward phosphatidylinositol 4,5-bisphosphate (fig. S5A). PI3K purified from cells depleted of RAB35 had a ~50% reduction in PI3K kinase activity in vitro. Further, we found that depletion of RAB35 did not reduce the amount of p110α recovered in p85 immunoprecipitates, which suggests that RAB35 depletion does not disrupt the p85-p110α interaction (fig. S5B). Thus, RAB35 copurifies with PI3K in a nucleotide-dependent manner and may be required for optimal PI3K enzymatic function.

Given that RAB35 functions upstream of PI3K, we investigated whether RAB35 acts downstream of any particular growth factor receptor. Depletion of RICTOR, PI3Kα, or RAB35 in HeLa cells reduced S473 phosphorylation of AKT in response to treatment with either insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF), platelet-derived growth factor AA (PDGF-AA), or vascular endothelial growth factor (VEGF) (fig. S6A). RAB35 depletion did not dramatically alter phosphorylation of ERK or tyrosine phosphorylation of receptors for IGF, EGF, PDGF, or VEGF (IGFR, EGFR, PDGFR, or VEGFR, respectively) (fig. S6, B and C). Collectively, these data suggest a model in which RAB35 functions upstream of PI3K and downstream of growth factor RTKs to activate AKT.

One criterion for prioritization of genes on our hit list (table S6) was evidence for mutation in human cancer databases (table S7). For RAB35, missense mutations have been reported at two residues that are conserved in RAS-like GTPases, A151T and F161L. We noticed that the A151T and F161L mutations in RAB35 were markedly similar to two well-documented KRAS mutations (A146T and F156L) that have been previously identified in human tumor samples (Fig. 3A) (24, 25). Although they are not canonical activating mutations, stable expression of either of these two KRAS mutants was sufficient to activate ERK signaling and transform NIH-3T3 cells in vitro. We therefore asked whether the two similar mutations in RAB35 might also be gain-of-function mutations that activate RAB35 signaling.

Fig. 3 RAB35 mutants identified in human tumors activate PI3K signaling, suppress apoptosis, and are transforming in vitro.

(A) Two mutations in RAB35 that code for amino acid changes A151T and F161L were identified in the MSKCC cBio and COSMIC data sets. Alignment of RAB35 with KRAS was performed using ClustalW2. (B) Mutant RAB35 alleles from human tumors can activate PI3K/AKT signaling. NIH-3T3 mouse fibroblasts stably expressing the indicated RAB35 alleles were serum-starved for 4 hours, left unstimulated or treated with BCS, and lysed; cell lysates were analyzed by immunoblotting for the indicated proteins. (C and D) Expression of RAB35 mutants suppresses apoptosis. (C) NIH-3T3 cells stably expressing the indicated proteins were plated and treated as indicated for 4 hours with or without serum-containing medium. Cells were then lysed, and the cell lysates were immunoblotted for the indicated proteins. (D) NIH-3T3 cells were treated as in (C) and trypsinized, and the number of viable cells was counted. Cell counts were normalized to the number of viable cells for each cell line from non–serum-starved conditions. Graph bars represent mean normalized cell count; error bars indicate SD (n = 3 replicates); and asterisks indicate statistical significance (P < 0.05), as determined by a Student’s t test. (E) RAB35 mutants can transform cells in vitro in a PI3K-dependent manner. NIH-3T3 cells stably expressing the indicated RAB35 alleles, HA-p110αH1047R, or myristoylated FLAG-AKT1myr were cultured for 4 weeks in medium containing 2% BCS with either dimethyl sulfoxide (DMSO) or 250 nM GDC-0941 (a pan-PI3K inhibitor), then fixed, stained with crystal violet, and imaged.

To test this possibility, we generated NIH-3T3 cells stably expressing either RAB35wt, GTPase-deficient RAB35Q67L, or the RAB35A151T and RAB35F161L alleles reported in human tumors. As expected from our earlier experiments in human cells, expression of RAB35wt did not activate AKT phosphorylation upon serum deprivation, whereas expression of the GTPase-deficient RAB35Q67L did (Fig. 3B). Notably, expression of the two naturally occurring RAB35A151T and RAB35F161L mutants also elevated AKT phosphorylation levels during serum deprivation, indicating that both are gain-of-function alleles sufficient to activate PI3K/AKT signaling.

Because PI3K/AKT signaling inhibits apoptosis, we next examined whether cells expressing mutant RAB35 alleles are resistant to apoptosis triggered by growth factor withdrawal. Four hours of serum deprivation of NIH-3T3 cells stably expressing RAB35wt were sufficient to elevate cleaved levels of the apoptotic markers PARP and caspase 3 (Fig. 3C). In comparison, cells stably expressing one of the three RAB35 mutants or oncogenic p110αH1047R had decreased levels of cleaved PARP and cleaved caspase 3 after serum withdrawal. Further, although cells expressing RAB35wt died in response to serum deprivation, cells expressing mutant alleles of RAB35 or p110αH1047R had significantly improved cell viability when deprived of growth factors (Fig. 3D). Taken together, these data suggest that mutant RAB35 proteins can mitigate cell death in response to growth factor deprivation.

To determine whether the mutant alleles of RAB35 are oncogenic, we examined their activity in a standard NIH-3T3 cell focus-formation assay—an in vitro model of density-independent growth that often correlates with tumorigenicity (26). NIH-3T3 cells expressing oncogenic p110αH1047R, myristoylated AKT1 (AKT1myr), or any of the three mutant alleles of RAB35, but not RAB35wt, formed foci (Fig. 3E and fig. S7). Moreover, we found that when cultured in medium containing 250 nM of the pan-class I PI3K inhibitor GDC-0941 (27), cells expressing RAB35 mutants and p110αH1047R were unable to form foci. Not surprisingly, PI3K blockade did not inhibit the growth of cells transduced with AKT1myr. Thus, the expression of the RAB35 mutants identified in human cancers can transform NIH-3T3 cells in vitro in a PI3K-dependent manner.

Because RAB proteins regulate endomembrane trafficking, we considered whether our evidence implicating RAB35 in AKT activation, downstream of RTKs and upstream of PI3K, is linked to this more established function of RAB proteins. The fact that many RTKs undergo ligand-stimulated internalization and recycling through endosomes, while remaining competent to signal (2832), suggests that RAB35 may function in this process. In considering this possibility, we noted highly elevated levels of tyrosine phosphorylation of the PDGFRα/β—but not of IGF1R, fibroblast growth factor receptor, VEGFR, or MET—in cells expressing the constitutively active RAB35Q67L allele (Fig. 4A and fig. S8). This increase in PDGFRα/β phosphorylation was markedly reduced by treatment of cells with crenolanib (CP-868596), a PDGFR inhibitor that affects both PDGFRα and PDGFRβ with similar potency (3335) (Fig. 4B and fig. S9). Notably, crenolanib treatment also diminished phosphorylation of AKT and the downstream substrate FOXO1/3A, suggesting that the RAB35Q67L allele activates AKT through PDGFR. Treatment with a more β-selective PDGFR inhibitor (CP-673451) only moderately affected AKT phosphorylation, implicating PDGFRα as the most likely RAB35Q67L target. The pan-PI3K inhibitor (GDC-0941) and the PI3Kα-specific inhibitor (BYL719) both suppressed AKT phosphorylation but not PDGFRα/β phosphorylation, providing further evidence that PDGFR/RAB35Q67L functions upstream of PI3K.

Fig. 4 Expression of dominant active RAB35Q67L activates PI3K signaling via PDGFRα.

(A) Stable expression of dominant active RAB35Q67L activates the PDGFR. HEK-293E cells stably expressing the indicated FLAG-tagged GTPases were serum-starved, treated as indicated, lysed, and analyzed by immunoblotting. (B) Pharmacologic inhibition of PDGFRα inhibits PI3K/AKT activity in cells expressing dominant active RAB35Q67L. Cells stably expressing RAB35Q67L were treated with DMSO, a pan-PI3K inhibitor (PI3Ki) (GDC-0941, 0.5 μM), a PI3Kα-specific inhibitor (PI3Kαi) (BYL719, 1 μM), a PDGFRβ inhibitor (CP-673451, 1 μM), or a PDGFRα/β inhibitor (crenolanib, CP-868596, 1 μM) in serum-free media overnight then lysed and analyzed as in (A). (C) PDGFRα is trafficked to LAMP2-positive endomembranes after stimulation with PDGF. HEK-293E cells stably expressing FLAG-RAB35wt were transfected with cDNA expressing PDGFRα-myc, serum-starved and then treated with PDGF-AA (100 ng/ml) for 15 min, and processed for immunofluorescence with anti-myc and anti-LAMP2 antibodies. (D) Expression of RAB35Q67L traffics PDGFRα to LAMP2-positive endomembranes in the absence of PDGF. HEK-293E cells stably expressing FLAG-RAB35Q67L were transfected with cDNA coding for PDGFRα-myc and then treated and processed for immunofluorescence as in (C). Scale bars in (C) and (D) represent 10 μm. (E) Cartoon depicting the trafficking of PDGFRα to LAMP2-positive endomembranes in cells expressing RAB35Q67L. In cells expressing RAB35wt, PDGFRα is activated at the cell membrane by PDGF-AA ligand and then internalized to EEA1-positive and LAMP2-positive endomembranes, where liganded PDGFRα drives PI3K/AKT signaling. Dashed lines reflect the fact that endomembrane-based signaling may not be as prominent as plasma membrane signaling in normal states (left). In cells expressing GTPase-deficient, GTP-bound RAB35Q67L, unliganded PDGFRα is internalized to EEA1- and LAMP2-positive compartments, where it drives constitutive activation of PI3K/AKT signaling (right).

We postulated that RAB35Q67L might initiate PDGFR-mediated AKT activation by altering PDGFRα localization. To explore this possibility, we transfected cells that stably expressed either RAB35wt or RAB35Q67L with cDNA encoding for myc-tagged PDGFRα and studied the localization of tagged PDGFRα in the presence or absence of the PDGFRα ligand PDGF-AA (fig. S10). As expected, in cells that expressed RAB35wt, stimulation with PDGF-AA relocalized PDGFRα into punctate intracellular structures. Notably, PDGFRα was constitutively localized to punctate structures in RAB35Q67L-expressing cells, even in the absence of PDGF-AA stimulation. This effect was specific to PDGFRα because internalization of EGFR was not altered by RAB35Q67L expression (fig. S11). By simultaneously visualizing PDGFRα-myc with the lysosomal protein LAMP2 or the early endosome marker EEA1, we next explored whether the punctate localization of PDGFRα in RAB35Q67L-expressing cells was the same as that seen in the normal physiologic context of PDGFRα activation by PDGF-AA. In both PDGF-AA-stimulated RAB35wt cells and unstimulated RAB35Q67L-expressing cells, transfected PDGFRα-myc colocalized with endogenous LAMP2 (Fig. 4, C and D, and fig. S12) and EEA1 (fig. S13). We also found that FLAG-RAB35 is present in endomembranes that contain LAMP2 (fig. S14A) but not in those that contain EEA1 (fig. S14B). This suggests that PI3K activity downstream of RAB35Q67L may originate on LAMP2-positive membranes. Finally, because it has been established that PI3K activity is required for the internalization and trafficking of PDGFRs (36, 37), we asked whether the localization of PDGFRα-myc in cells expressing RAB35Q67L was a result of the increased PI3K signaling in these cells. Although PI3K was required for ligand-induced PDGFRα internalization in cells that express RAB35wt (fig. S15A), treatment of cells expressing RAB35Q67L with a pan-PI3K inhibitor did not diminish the localization of PDGFRα-myc to LAMP2-positive compartments (fig. S15B). PI3K inhibitor treatment resulted in changes in LAMP2 vesicle morphology that may merit further investigation (fig. S15, A and B). Together, these data establish that activated PDGFRα is normally trafficked through LAMP2-positive endomembranes and that constitutively active RAB35 likely drives unliganded PDGFRα to this location, where it is active and can signal to PI3K/AKT (Fig. 4E). Further study is required to determine whether mutant RAB35 diverts newly synthesized PDGFRα from the cell membrane to LAMP2-positive endomembranes or prevents the exit of existing PDGFRα from LAMP2 compartments.

Here we demonstrate a previously unappreciated role for the small GTPase RAB35 in regulating growth factor signaling to PI3K/AKT. We also characterize two RAB35 missense mutations found in human tumors that confer gain of function in standard transformation assays, displaying phenotypes similar to well-characterized oncogenic KRAS alleles. Searches of existing human tumor sequencing databases reveal that RAB35 mutations are rare (table S7) and are therefore unlikely to appear on current lists of human cancer genes that rely solely on statistical methods to distinguish between driver and passenger mutations. Nonetheless, the conserved location of the RAB35 mutations to analogous residues in KRAS and their oncogenic phenotype in functional assays provide strong evidence that these are true driver mutations and are perhaps clinically actionable on the basis of their sensitivity to PI3K inhibition. Considering the broad role of RAB family proteins in endomembrane trafficking, it is intriguing to speculate that the oncogenic RAB35 phenotype reported here may be a consequence of dysregulated membrane trafficking. Our studies showing that the constitutively active RAB35Q67L allele is sufficient to drive PDGFRα to LAMP2-positive endomembranes, in a pattern that mirrors that seen with native PDGF-AA ligand, are consistent with this model. Alternatively, these mutations may be neomorphic, conferring a novel molecular function to RAB35 that results in signaling in a RAS-like manner. The fact that other RAB proteins (RAB1B, RAB39, RABL3, RASEF) emerged as hits in our screen and have rare missense mutations reported in human tumors (table S6) begs a broader examination of this question.

Supplementary Materials

www.sciencemag.org/content/350/6257/211/suppl/DC1

Materials and Methods

Figs. S1 to S15

Tables S1 to S7

References (3855)

References and Notes

  1. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
  2. See supplementary materials and methods for a detailed explanation of z score calculation and hit criteria.
  3. Acknowledgments: We thank all of the members of the Sabatini and Sawyers laboratories for critical readings of this manuscript and helpful discussions. We also thank N. D. Novikov, M. A. Domser, K. C. Woods D. J. Spencer, T. R. Peterson, C. Thoreen, S. Kinkel, M. J. Evans, R. Bose, P. Watson, O. S. Andersen, S. C. Blanchard, T. E. McGraw, N. Rosen, and A. Smogorzewska for helpful discussions; S. Silver for assistance with the RNAi screens; V. Boyko and Y. Romin of the MSKCC Molecular Cytology Core Facility for assistance with confocal microscopy; A. Kazlauskas for helpful discussions about PDGFR biology; and the late A. Hall for helpful input about GTPase biology. Funds for the RNAi screen were provided by the Starr Cancer Consortium Award I2-A117. D.B.W. was supported by the Department of Defense Prostate Cancer Research Program via the Office of the Congressionally Directed Medical Research Programs Predoctoral Prostate Cancer Training Award PC094483, as well as by NIH Medical Scientist Training Program grant GM07739. R.Z. was supported by the Glenn Foundation for Medical Research, the NIH Director’s New Innovator Award 1DP2CA195761-01, and the Pew-Stewart Scholarship for Cancer Research. This work was partially supported by NIH grants CA103866 and AI47389 (to D.M.S.) and CA155169 and CA092629 (to C.L.S.). Data from the RNAi screen described in this manuscript can be found in the supplementary materials.
View Abstract

Navigate This Article