Melanoma has an unusual capacity to spread in early-stage disease, prompting aggressive clinical intervention in very thin primary tumors. Despite these proactive efforts, patients with low-risk, low-stage disease can still develop metastasis, indicating the presence of permissive cues for distant spread. Here, we show that constitutive activation of the small GTPase ARF6 (ARF6Q67L) is sufficient to accelerate metastasis in mice with BRAFV600E/Cdkn2aNULL melanoma at a similar incidence and severity to Pten loss, a major driver of PI3K activation and melanoma metastasis. ARF6Q67L promoted spontaneous metastasis from significantly smaller primary tumors than PTENNULL, implying an enhanced ability of ARF6-GTP to drive distant spread. ARF6 activation increased lung colonization from circulating melanoma cells, suggesting that the prometastatic function of ARF6 extends to late steps in metastasis. Unexpectedly, ARF6Q67L tumors showed upregulation of Pik3r1 expression, which encodes the p85 regulatory subunit of PI3K. Tumor cells expressing ARF6Q67L displayed increased PI3K protein levels and activity, enhanced PI3K distribution to cellular protrusions, and increased AKT activation in invadopodia. ARF6 is necessary and sufficient for activation of both PI3K and AKT, and PI3K and AKT are necessary for ARF6-mediated invasion. We provide evidence for aberrant ARF6 activation in human melanoma samples, which is associated with reduced survival. Our work reveals a previously unknown ARF6-PI3K-AKT proinvasive pathway, it demonstrates a critical role for ARF6 in multiple steps of the metastatic cascade, and it illuminates how melanoma cells can acquire an early metastatic phenotype in patients.

Significance:

These findings reveal a prometastatic role for ARF6 independent of tumor growth, which may help explain how melanoma spreads distantly from thin, early-stage primary tumors.

A hallmark of melanoma is early, aggressive spread of disease when primary tumors are as thin as 1 mm (1). The small GTPase adenosine diphosphate (ADP)-ribosylation factor 6 (ARF6) controls invasion of cutaneous melanoma (2–5) and other cancers (6). In its active, GTP-bound state, ARF6 promotes invasion whereas inactive, GDP-bound ARF6 reduces invasion (3–5). The critical role for ARF6 in cutaneous melanoma appears to be common, as knockdown of ARF6 uniformly inhibits invasion in a broad panel of human melanoma cell lines (2). Consistent with the in vitro phenotypes, our previous work suggest that ARF6 is necessary for metastasis. Specifically, pharmacologic inhibition of ARF6 activation reduced spontaneous metastasis in an orthotopic xenograft model of BRAF-mutant melanoma (2). In cutaneous melanoma, ARF6 is activated by WNT5A (2) and HGF (3), both of which result in increased invasion. In uveal melanoma, ARF6 is activated by mutant GNAQ to control tumor growth (7). Beyond melanoma, ARF6 is activated by and coordinates signaling from a variety of signals including EGFR (6), HER2/ERBB2 (8), c-MET (9), VEGFR2 (6), Frizzled (2, 10), inflammatory receptors (6), and G-Protein–coupled receptors (6). In addition, ARF6 has a critical role in the functional output of mutant p53 (11) and RAS (12). Despite compelling evidence that ARF6 is necessary for invasion and metastasis, where ARF6 functions in the metastatic cascade and how it shapes the course of disease are unknown.

Aberrant PI3K pathway activation is a known driver of melanoma disease progression, with up to 70% of melanomas showing reduced PTEN expression or activation of the kinase AKT (13–16). Phosphorylated AKT (pAKT) levels increase significantly from benign nevi to primary melanoma, and from primary melanoma to metastasis. Furthermore, primary melanomas with increased pAKT levels have a worse prognosis (14). PTEN deletion or activation of PI3K or AKT1 accelerates metastasis in genetically engineered mouse models of BRAF-mutant melanoma (17–19). Approximately 20% of BRAF-mutant melanomas show genetic loss of PTEN or rare gain-of-function mutations in PIK3CA or AKT (20). Overall, genomic alterations predicted to activate the PI3K pathway have been detected in approximately 50% of all molecular subtypes of cutaneous melanoma (20). Importantly, activation of the PI3K pathway can also occur in the absence of mutation. The tumor microenvironment can be a rich source of stimulatory signals that promote disease progression and therapy resistance (21). Tumor stroma can generate growth factor ligand(s) that act in a paracrine fashion to stimulate MAPK/ERK and PI3K-AKT, causing resistance to BRAF inhibition in melanoma and other cancers (22). Thus, understanding mechanisms of PI3K activation in cancer may help improve treatment strategies.

In this study, we investigated the role of activated ARF6 in melanoma progression using clinically relevant, immunocompetent genetic models. Our work uncovered novel mechanisms of ARF6-mediated invasion via increased levels and activity of PI3K, and support a role for activated ARF6 in multiple steps of metastasis. Furthermore, we provide evidence for aberrant ARF6 activation in patient samples.

RCAS virus propagation and delivery in vivo

The RCAS-Cre ± ARF6Q67L plasmids were transfected into DF-1 cells and these cells were prepared for subcutaneous flank injection as described previously (17). HA-tagged ARF6Q67L expression was confirmed with anti-HA Western blot.

Mouse husbandry, genotyping, and phenotyping

Animal studies were performed in accordance with a protocol approved by the University of Utah Institutional Animal Care and Use Committee. The DCT:TVA; BrafCA; Cdkn2af/f and DCT:TVA; BrafCA; Cdkn2af/f; Ptenf/f mice were described previously (17). Tumor growth was measured by caliper three times weekly. Mice were euthanized once the primary tumor reached a size of 2 cm in any direction, when skin ulceration occurred at the primary tumor site, or when the primary tumor caused symptoms due to deep local invasion. No mice necessitated sacrifice based on overall health. Metastases were evaluated by routine histology, performed by a board-certified pathologist (A.H. Grossmann) who was blinded to genotypes/experimental group assignments. Metastatic volume in lungs was measured as cross-sectional tumor area (ImageJ pixels) from hematoxylin and eosin (H&E)-stained slides. Metastatic tumors were imaged at ×20 magnification for cross-sectional area. The cross-sectional area of spherical tumors larger than the 20× image capture field (6.5 mm tumor diameter threshold), which occurred in Ptenf/f+Arf6Q67L mice, was derived from a diameter ratio, generating a radius (r) equivalent, where r = diameter of the large Ptenf/f+Arf6Q67L tumor/6.5 mm. The calculated cross-sectional surface area = r2 × cross-sectional area (in pixels of a 6.5-mm tumor). Mice that failed to grow primary tumors were included in the tumor incidence calculations but were excluded from tumor growth, onset/latency, survival, and the metastatic incidence analyses. pAKT and Ki67 IHC stains from primary tumors were evaluated in a blinded fashion.

Mouse tumor cell lines and experimental metastasis model

Early passage tumor cell lines from mice 6431, 6455, 7657, 5588, and 5523 were derived from primary melanomas removed from BrafCA;Cdkn2af/f mice that had been injected with DF-1 cells producing either Cre or Cre-ARF6Q67L virus. 6431, 6455, 7657, and 5523 melanoma cell lines were tested for Mycoplasma by IDEXX, pretreated with plasmocin (InvivoGen) as needed prior to injection into tail veins of NOD SCID mice at 5.0 × 105 cells per mouse. The mice were monitored for up to 4 weeks and sacrificed when the health of the animal necessitated euthanasia according to the guidelines of the University of Utah Institutional Animal Care and Use Committee. The lungs were harvested and fixed in 10% neutral buffered formalin overnight, transferred to 70% ethyl alcohol, and paraffin-embedded. Five-micron–thick sections were mounted to glass slides and stained with H&E by the BMP research histology laboratory located at the Huntsman Cancer Institute, University of Utah (Salt Lake City, UT).

Cell lines and compounds

LOX IMVI were acquired from the NCI-60 Human Cancer Cell Line Repository. SK-MEL-28, Yugen-8, and Yusac-2 cells were provided by D. Grossman (Huntsman Cancer Institute). DF-1 cells and A375-TVA (pDEST12.2-dsRED-LOXp eGFP) cells were provided by S.L. Holmen (University of Utah, Salt Lake City, UT). SK-MEL-147, SK-MEL-5, SK-MEL-103, CACL, A375, HEY-T30, UACC.257, and UACC.62 were provided by M. VanBrocklin (Huntsman Cancer Institute). A2058, MeWo, SK-MEL-2, and WM266-4 cells were purchased from the ATCC. LOX IMVI, Yugen-8, and Yusac-2 cells were maintained in DMEM-high glucose media supplemented with 5% FBS (Invitrogen), penicillin/streptomycin (Invitrogen), and maintained at 37°C, with 5% CO2. A375, A2058, MeWo, SK-MEL-2, and WM266-4 cells were maintained in DMEM-high glucose media supplemented with 10% FBS (Invitrogen) + penicillin/streptomycin (Invitrogen), and incubated at 37°C, with 5% CO2. All other human melanoma cell lines were maintained in RPMI1640-high glucose media supplemented with 10% FBS (ATCC) + penicillin/streptomycin (Invitrogen), and maintained at 37°C, with 5% CO2. NIH3T3 cells were maintained in DMEM + sodium pyruvate + penicillin/streptomycin (Invitrogen) and 10% calf serum (ATCC). DF-1 cells were grown in DMEM-high glucose media supplemented with 10% FBS (Invitrogen), gentamicin (Invitrogen), and maintained at 39°C, with 5% CO2. Mouse melanoma primary cells were cultured in DMEM with nutrient mixture F-12, supplemented with 10% FBS (ATCC), penicillin/streptomycin (Invitrogen), 0.25 μg/mL Amphotericin B (Invitrogen), and MEM Non-Essential Amino Acids Solution (Invitrogen). For Fig. 5B, human melanoma cell lines were grown in RPMI1640-high glucose media supplemented with 10% FBS (ATCC), penicillin/streptomycin (Invitrogen), and maintained at 37°C, with 5% CO2. A375-TVA (pDEST12.2-dsRED-LOXp eGFP) cells were grown in DMEM-high glucose media supplemented with 10% FBS (Invitrogen), gentamicin (Invitrogen), and maintained at 37°C, with 5% CO2 and used to confirm expression of RCAS constructs. Human cell lines were authenticated using ATCC's human short tandem repeat identity profiling cell authentication service and the BRAF, NRAS, and KRAS mutation status was sequenced to confirm the expected (published) profile. GDC-0941 and MK2206 were purchased from Selleck Chemicals.

Proliferation assay

Cell proliferation was measured by CyQUANT (Invitrogen) as described previously (7).

Apoptosis assay

Apoptosis assays were performed using RealTime-Glo Annexin V Apoptosis Assay kit (Promega). A total of 5,000 cells in 10% FBS or 0% FBS-containing medium were plated into each of four wells of duplicate 96-well plates. Annexin V luminescence and CyQUANT fluorescence (Invitrogen) were measured 24 hours later after cell attachment. Apoptosis index was calculated by Annexin V values normalized to cell numbers (CyQUANT values).

ARF6-GTP expression signature analysis

RNA was extracted from fresh-frozen mouse tumors using Qiagen RNAeasy Plus Universal Mini Kit after frozen section histologic confirmation of high tumor content. RNA-sequencing was performed using Illumina TruSeq Stranded mRNA Library Preparation Kit with polyA selection followed by lllumina HiSeq 2500 125-cycle paired-end sequencing, performed by the High Throughput Genomics laboratory at the Huntsman Cancer Institute. The mouse GRCm38 FASTA and GTF files were downloaded from Ensembl release 90 and the reference database was created using STAR version 2.5.2b with splice junctions optimized for 125 base pair reads. Reads were trimmed of adapters using cutadapt version 1.16 and then aligned to the reference database using STAR in two-pass mode to output a BAM file sorted by coordinates. Mapped reads were assigned to annotated genes in the GTF file using featureCounts version 1.5.1. The output files from cutadapt, FastQC, STAR, and featureCounts were summarized using MultiQC to check for any sample outliers. Differentially expressed genes were identified using a 10% false discovery rate with DESeq2 version 1.16.0. Two BRAFV600E/Cdkn2ANULL control tumor samples were outliers in principal component analysis and were excluded from further analysis. Pathway analysis was performed with Illumina Base Space Correlation Engine. The RNAseq data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE129392 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GExxx).

Proteomics

Protein extraction and reverse-phase protein array of frozen mouse tumors was performed by the MD Anderson Cancer Center RPPA Core Facility. Analysis of human melanoma A2058 cells was accomplished with the Proteome Profiler Human Phospho-Kinase Array Kit, (R&D Systems) according to the manufacturer's instructions, using 600 μg of total cell lysates incubated with the phosphokinase membrane array.

Western blotting and antibodies

Cell lysates were prepared, protein was quantified, and Western blots were performed as described previously (2). Antibodies dilutions for Western blots include ARF6 (Cell Signaling Technology-5740) 1:1,000, HA tag (Cell Signaling Technology-3724) 1:1,000, MYC tag (Cell Signaling Technology-2276) 1:5,000, AKT (Cell Signaling Technology-9272) 1:1,000, pAKT S473 (Cell Signaling Technology-4060) 1:2,000, p44/42 MAPK (Erk1/2;Cell Signaling Technology-9107) 1:1,000, phospho-p44/42 MAPK (Erk1/2 Thr202/Tyr204; Cell Signaling Technology-4377) 1:2,000. PI3K p85 (Cell Signaling Technology-4292) 1:1,000, PI3K p110α (Cell Signaling Technology-4249), and GAPDH (Cell Signaling Technology-5174) 1:5,000.

Viral vector constructs

The retroviral vector system used for in vivo delivery of Cre and ARF6Q67L has been described previously (17). ARF6Q67L cDNA was provided in pCDNA3.1 by Zongzong Tong (Navigen, Inc.). RCAS-ARF6Q67L-HA-IRES-Cre was cloned by PCR amplification of ARF6Q67L using primers with EcoRI and NotI sites and then inserting the PCR product into a pENTR3c (Invitrogen) vector containing HA-IRES-Cre. This was followed by recombination into RCASBP(A) DV using LR Clonase Enzyme Mix (Invitrogen), as per the manufacturer's specifications. pENTR3c, Gateway reagents, and RCAS-Cre were provided by S. Holmen's laboratory (Huntsman Cancer Institute). The adenoviral constructs ARFQ67L and ARFT27N were provided by Zongzhong Tong (Navigen, Inc.) and then amplified by Vector Biolabs. Ad-CMV-Null empty vector control was purchased directly from Vector Biolabs.

Matrigel invasion assay

Twenty-four hours after adenoviral transduction, invasion was performed as described previously (2) with minor modifications. A total of 5 × 104 infected cells were seeded into chambers. Either vehicle (0.1% DMSO), MK2206 (1 μmol/L), or GDC-0941 (1 μmol/L) was added to both chambers. After 48-hour incubation, media were replaced in the bottom chamber with Calcein AM (BD Biosciences) according to the manufacturer's instructions. Calcein-stained cells were visualized with a fluorescent microscope and quantified with plate reader at 485/530 nm.

Gelatin invasion assay

Invasion into gelatin labeled with Oregon Green 488 was performed as described previously (23, 24), with minor modifications. In 8-well chambered coverglass, 25 μL casts of gelatin were dried, fixed, and quenched in PBS containing 0.5 mol/L glycine and then overlayed with 10 μL Matrigel polymerized for 30–60 minutes at 37°C. The matrix was equilibrated in DMEM +10% FBS for 30 minutes at 37°C before adding cells. Prior to invasion, A2058 cells were transduced for 24 hours with 109 pfu/mL adenoviruses per 10-cm2 dish. Cells were then treated with mitomycin C in preparation for invasion as described previously (2).

Immunofluorescence and confocal microscopy of invading cells

Prior to invasion, A2058 cells were cultured to 80% confluence and transduced for 24 hours with 109 pfu/mL adenoviruses per 10-cm2 dish. Cells were then treated with mitomycin C in preparation for invasion as described previously (2). Twenty-four hours prior to Matrigel invasion, Matrigel GFR (B-D) was thawed on ice overnight at 4°C. Transduced cells were harvested, washed three times in high glucose DMEM + Pyruvate + 0.1% FBS and resuspended to 1.5 × 105/mL. During the cell preparation period, 40 μL ice-cold Matrigel was pipetted to the center wells of an 8-well chambered coverglass (Thermo Fisher Scientific) on ice. Four microliters ice-cold PBS was added to each 40 μL dome of Matrigel. The domes were polymerized at 37°C/5% CO2 for 30 minutes. A total of 350 μL of A2058 cell suspension were added to each well and cells were incubated at 37°C/5% CO2 for 48 hours. Media were gently aspirated and wells were rinsed once in 10% neutral buffered formalin, then fixed for 30 minutes at room temperature in same, followed by three rinses in “PBSA” (1× PBS +0.1% sodium azide.) Matrigels were permeablized for 40 minutes in PBSA + 0.02% Triton X-100 at room temperature, followed by a blocking in PBSA + 3% v/v BSA for 60 minutes. Primary antibodies to pAKT S473 (Cell Signaling Technology-4060) and cortactin (Abcam 33333) were diluted 1:100 and 1:300, respectively, in PBSA + 3% BSA, and incubated on Matrigels overnight at 4°C. Antibodies were then aspirated and the wells were washed 4 × 20 minutes in PBSA. Signals were detected by fluorescently conjugated secondary antibodies; Donkey anti-rabbit IgG Alexa 488, (pAKT) and donkey anti-mouse IgG, Alexa 594 (cortactin), diluted to 10 μg/mL in PBSA + 0.1% BSA and incubated for 50 minutes at room temperature in a darkened box. Wells were aspirated by gentle vacuum and washed 4 × 20 minutes at room temperature in PBSA. A total of 5 μg/mL DAPI in PBSA for added for 30 minutes, followed by 5-minute washing in PBSA, and mounting in 40% w/v polyvinyl pyrrolidone + 4% v/v glycerol+0.1% sodium azide dissolved in 1 mol/L Tris, pH 8.0. Images were collected on an Olympus Fluoview1000 scanning laser confocal microscope at ×600 magnification and ×1,200 magnification. Invasion into gelatin labeled with Oregon Green 488 was performed as described previously (23, 24), with minor modifications. In 8-well chambered coverglass, 25 μL casts of gelatin were dried, fixed, quenched in PBS containing 0.5 mol/L glycine and then overlayed with 10 μL Matrigel polymerized for 30–60 minutes at 37°C. The layered matrix was equilibrated in DMEM + 10% FBS for 30 minutes at 37°C before adding cells. Staining of A2058 cells in gelatin followed 20 hours of invasion, fixation for 30 minutes at room temperature in freshly prepared 10% neutral buffered formalin, four washes in PBSA, and storage, tightly wrapped, at 4°C. Wells were permeablized for 10 minutes in PBSA + 0.1% Triton X-100, followed by three washes in PBSA. Blocking, washing, and primary and secondary antibodies were applied as described above. A glycerol-based antifade mount was applied. Images were collected with oil immersion at ×1,200 and ×1,800 magnification on an Olympus FV1000 laser scanning confocal microscope. For orthogonal views of invasion, 85 × 0.2 μm, Z stacks were generated, (17-μm depth) and 3D animation images were produced in the FluoView software.

Tumor cells derived from BrafCA;Cdkn2af/f ± Arf6Q67L mice were plated to fibronectin-coated, chambered coverglass slides at a density of 6 × 104/mL or 5 × 103/well, and allowed to attach for 20 hours. Monolayers were fixed in 10% neutral buffered formalin for 20 minutes at room temperature, followed by washing × 3 in PBS + 0.1% NaZ. Wells were blocked and permeabilized × 45 minutes in PBS + 0.1% NaZ + 0.01% MgCl2 +0.01% CaCl2 (PNMC solution) + 0.1% Saponin + 1% BSA, followed by incubation in primary antibodies. Rabbit anti-HA (Cell Signaling Technology-3724) 1:400. Mouse anti-PI3K: anti-p85 (Abcam ab86714) 1:100 or anti-p110 (BD Biosciences 611398) 1:100, was diluted in PNMC solution and incubated overnight at 4°C. Slides were rinsed × 3 in PNMC solution. Secondary antibodies (donkey-anti-Rabbit IgG Alexa 594 conjugate, donkey anti-mouse IgG Alexa 488 conjugate, Thermo Fisher Scientific) were applied at 10 μg/mL each in blocking buffer for 2 hours in darkened chamber on ice. Slides were rinsed three times in PNMC solution and wells were mounted in a solution of 90% glycerol, 5 μg/mL DAPI, PBS + 0.1% NaZ + 0.05% DABCO + 0.5% n-Propyl Gallate + 0.01% p-Phenylenediamine, with storage at 4°C in darkened chamber. Z-stacked images in 5 × 0.5 μm slices were taken on an Olympus FV1000 scanning laser confocal microscope at ×1,200 magnification. Images were assessed by three independent, blinded investigators who evaluated a composite of random images from each condition. Quantification of p110α PI3K fluorescent signal on bulbo-spinous processes of mouse melanoma cells was performed according to standard protocols described in ImageJ tutorials (https://imagej.net/Image_Intensity_Processing#Getting_intensity_values_from_multiple_ROIs). Briefly, all regions of interest (ROI) were extracted from the RGB confocal images using the freehand selection tool. ROIs were uniformly thresholded and signal intensity was counted in ImageJ 64. Signal intensity was normalized to total pixels (surface area) in each ROI. All bulbo-spinous processes were quantified for all cells in eight distinct images per cell line.

RNA interference and viral transduction of cell lines

A total of 20 nmol/L siRNA duplexes were transfected into cell lines using RNAiMax (Invitrogen) following the manufacturer's recommendations. For A2058 cells, 70 μL of RNAiMax was used with 40 nmol/L siRNA. Forty-eight hours after transfection, the cell lines were split 1:2, allowed to adhere for 6 hours, retransfected, and allowed to grow for an additional 24 hours before harvesting. AllStars Negative Control siRNA (Qiagen) and ARF6-SMART pool (Dharmacon) siRNAs were used in these experiments. NIH3T3 cells were serum starved (0% FBS, DMEM) for 6 hours prior to adenoviral transduction and then incubated in virus-containing media for 18 hours prior to harvest of lysates. Transduction of A2058 cells was performed in whole media (10% FBS, DMEM) for 24 hours prior to harvest for invasion. Adenovirus was applied to A2058 and NIH3T3 at 1.0 × 107 pfu/10 mL media.

Quantitative RT-PCR

RNA was extracted from fresh frozen mouse tumor using RNeasy Mini Kit with RNase Free DNase set (Qiagen). cDNA was synthesized from 5 ng of total RNA using either qScript cDNA Super mix (Quanta) or RETROscript Reverse Transcription Kit (Invitrogen). Quantitative RT-PCR was performed with the Life Technologies QuantStudio 12K Flex instrument and QuantiTect SYBR Green PCR kit (Qiagen) with the following primers to ARF6-Q67L-HA: forward-GACAGGAACTGGTATGTGCA and reverse-TCGTATGGGTAAGATTTGTAG. Primer amplification efficiency was validated and relative expression of Arf6Q67L-HA was determined by the ΔΔCt method. Samples were run in triplicate and normalized to Gapdh using Mm_Gapdh_3_SG Primer (Qiagen).

Histology

Mouse tissues were flash frozen or fixed in 10% neutral buffered formalin overnight and stored in 70% ethyl alcohol. Paraffin-embedding and histologic sections were performed by the Research Histology Section of the Biorepository Molecular Pathology Laboratory in the Huntsman Cancer Institute.

IHC

Tissue sections were deparaffinized and rehydrated. Antigen retrieval was performed in citrate buffer (pH 6.0) at 120°C for 10 minutes. Sections were treated with 3% H2O2 and blocked with normal goat serum in 0.05% TBS-T for 60 minutes and washed three times with 0.05% TBS-T. Primary antibodies were diluted in SignalStain Ab Diluent (Cell Signaling Technology). Sections were incubated overnight at 4°C with primary antibody, washed for 5 minutes in 0.05% TBS-T, and then probed with SignalStain Boost Detection Reagent-rabbit or mouse (Cell Signaling Technology) for 30 minutes at room temperature in a humidified chamber. Visualization was carried out with SignalStain DAB (Cell Signaling Technology). Sections were counterstained with hematoxylin. Antibodies against the following antigens were used: HA-tag (Cell Signaling Technology-3724) 1:500; phospho-AKT (Ser 473;Cell Signaling Technology-4060) 1:100; Ki67 (MKI67; UMAB107)1:300.

PI3K catalytic assays

PI3K activity was measured from endogenous PI3K complex, as described previously (25). Cell lysates were prepared with IP lysis buffer (Thermo Fisher Scientific). PI3K complex from 1 mg of cell lysates was immunoprecipitated using protein A/G (Thermo Fisher Scientific) conjugated anti-p85 antibody (Cell Signaling Technology-4292) for 18 hours at 4°C, and then washed three times in IP lysis buffer and one time in KBZ buffer (Echelon). After washing, PI3K activity was measured with PI3-Kinase Activity ELISA: Pico kit (Echelon).

Procurement of patient samples

Excess tumor and paired nontumor tissues from routine surgical resections at the Huntsman Cancer Institute were collected and flash frozen in liquid nitrogen, under University of Utah Institutional Review Board–approved protocol (#10924), by the Biorepository Molecular Pathology shared resource at the Huntsman Cancer Institute. Quality control (viability, tumor content) was performed by routine histology by a board-certified pathologist (A.H. Grossmann).

ARF6-GTP pull-downs from patient samples

Human samples were prepared and ARF6-GTP pull-downs were performed and quantified as previously described (7).

The Cancer Genome Atlas melanoma survival analysis

ARF6 pathway genes (Supplementary Table S1) for differential expression query were selected on the basis of published literature. The Cancer Genome Atlas (TCGA) melanoma RNA-Seq data were extracted from all melanoma TCGA RNA-Seq data. Samples without survival data were excluded. Each gene was evaluated individually and those with statistical significance were then analyzed in the stage III subcohort. Specifically, stage III lymph node and skin metastases, as designated by TCGA, were selected for analysis. TCGA metadata were acquired from the GDC release 10.4, in which survival times were generated using days_to_death and days_to_last_follow_up data. Samples were stratified into ARF6-GAP-high (indicating higher overall ARF6-GAP gene expression) and ARF6-GAP low groups by median centering of expression levels for each gene. Survival P value were calculated by log-rank (Mantel–Cox) test.

Statistical analysis

For most experiments, statistical analyses were performed using GraphPad Prism 6.0f.

For statistical analyses of immunoblots from cell lines, the density of each band was normalized to an internal control protein and then the ratio of the normalized density of the band from the experimental treatment to the normalized density of the paired control treatment band was obtained. For patient samples, signals were normalized to total protein input as described previously (7). The 2 × 4 Fisher exact test and the Linear by Linear Association test was performed with IBM SPSS Statistics software. For animal studies, sample size estimates were based on results from pilot studies and a power analysis was performed with G*Power software.

ARF6-GTP accelerates melanoma metastasis and potentiates lung colonization in BrafCA;Cdkn2af/f mice

The impact of ARF6 on cancer progression in immunocompetent hosts is unknown, therefore we asked whether expression of activated ARF6 (ARF6-GTP) alters tumor incidence, growth, or metastasis in a clinically relevant genetically engineered mouse model of cutaneous melanoma (17). Invasion plays a role in multiple steps of the metastatic cascade, including early steps of primary tumor escape and intravasation into the lymphovascular system, and later extravasation. ARF6 is necessary for invasion in vitro, so we hypothesized that ARF6 activation would be sufficient to induce metastasis. To test our hypothesis, we utilized RCAS-mediated delivery of Cre recombinase specifically in the melanocytic lineage (17, 26) to induce constitutive activation of BRAFV600E (BrafCA) and deletion of Cdkn2a (Cdkn2af/f). BrafCA;Cdkn2af/f mice display a high incidence of primary tumors. Although Cdkn2a loss is expected to cause invasion and metastases (27), metastasis has not been observed in BrafCA;Cdkn2af/f mice without additional alterations such as Pten loss or AKT activation (17).

To model activated ARF6 (ARF6-GTP), we delivered a constitutively active mutant form, Arf6Q67L, along with Cre in RCAS, injected subcutaneously into the flank of Dct::TVA;BrafCA;Cdkn2af/f mice. We confirmed expression of HA-tagged ARF6Q67L by IHC (Supplementary Fig. S1A) and/or by qRT-PCR (Supplementary Fig. S1B). Tumor incidence, growth, and survival were similar between BrafCA;Cdkn2af/f (control) and BrafCA;Cdkn2af/f + Arf6Q67L mice (Supplementary Fig. S2A–S2C), indicating that ectopic ARF6-GTP does not alter primary tumorigenesis in this model. There was, however, an increase in spontaneous metastatic disease. Overall, the metastatic incidence increased from 4.3% to 11.1% in the ARF6Q67L cohort (Supplementary Fig. S2D), consistent with ARF6 driving metastasis. In mice with metastases, there was a dramatic increase in metastatic volume in the ARF6Q67L cohort (Fig. 1A–C). Control mice showed negligible metastatic disease with infrequent and very early microscopic foci of tumor in the lungs (Fig. 1A and C), whereas mice with ARF6Q67L demonstrated bulky metastatic disease (Fig. 1B and C). Thus, ARF6-GTP was sufficient to expedite the metastatic process, leading to accelerated disease progression.

Figure 1.

ARF6-GTP is sufficient to potentiate metastasis of BRAFV600E/Cdkn2aNULL tumors, and is equipotent as PTENNULL. A–C, Spontaneous pulmonary metastatic burden. A, Micrometastases (inset, outlined) from BrafCA;Cdkn2af/f (control) mouse. B, Multiple, large, spontaneous metastases in BrafCA;Cdkn2af/f + Arf6Q67L mouse. C, Metastatic volume in mice with metastases; Mann–Whitney test, two-tailed. D–F, Lung colonization of tumor cells derived from BrafCA;Cdkn2af/f ± Arf6Q67L mice, injected into tail veins of NOD SCID mice. D, Benign lung, control mouse. E, Lung with tumor colonies. F, Number of mice with pulmonary tumor colonies by 4 weeks postinjection, Fisher exact test, two-tailed. H&E images, ×20 magnification (larger images, scale bar, 500 μm), with ×400 magnification of metastatic focus (A, inset, scale bar, 20 μm). G, Metastatic incidence and volume from BRAFV600E/CDKN2ANull tumors ± ARF6-GTP or PTEN or both. Histogram (right), overall significance was evaluated by 2 × 4 Fisher exact test, P = 0.042. 2 × 2 Fisher exact test, two-tailed, P = 0.0498 for PTENNull versus PTENNull+ ARF6-GTP. Arrow, Linear by Linear Association test, based on the order shown. Diagram illustrates the overall phenotypic patterns for each cohort. The left column reflects the overall incidence of metastatic mice (red). The right column reflects the volume of disease burden (red) in metastatic mice. See also Supplementary Fig. S4E.

Figure 1.

ARF6-GTP is sufficient to potentiate metastasis of BRAFV600E/Cdkn2aNULL tumors, and is equipotent as PTENNULL. A–C, Spontaneous pulmonary metastatic burden. A, Micrometastases (inset, outlined) from BrafCA;Cdkn2af/f (control) mouse. B, Multiple, large, spontaneous metastases in BrafCA;Cdkn2af/f + Arf6Q67L mouse. C, Metastatic volume in mice with metastases; Mann–Whitney test, two-tailed. D–F, Lung colonization of tumor cells derived from BrafCA;Cdkn2af/f ± Arf6Q67L mice, injected into tail veins of NOD SCID mice. D, Benign lung, control mouse. E, Lung with tumor colonies. F, Number of mice with pulmonary tumor colonies by 4 weeks postinjection, Fisher exact test, two-tailed. H&E images, ×20 magnification (larger images, scale bar, 500 μm), with ×400 magnification of metastatic focus (A, inset, scale bar, 20 μm). G, Metastatic incidence and volume from BRAFV600E/CDKN2ANull tumors ± ARF6-GTP or PTEN or both. Histogram (right), overall significance was evaluated by 2 × 4 Fisher exact test, P = 0.042. 2 × 2 Fisher exact test, two-tailed, P = 0.0498 for PTENNull versus PTENNull+ ARF6-GTP. Arrow, Linear by Linear Association test, based on the order shown. Diagram illustrates the overall phenotypic patterns for each cohort. The left column reflects the overall incidence of metastatic mice (red). The right column reflects the volume of disease burden (red) in metastatic mice. See also Supplementary Fig. S4E.

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The metastatic cascade is a multistep process involving invasion and escape from the primary tumor, intravasation (invasion into lymphovascular system), survival in the circulation, extravasation (invasion into a distant organ), and survival and growth in distant organs (28). The proinvasive role of ARF6 in cancer cells in vitro is well-established and consistent with our observation that ARF6 activation increases metastatic disease burden. A role for ARF6 in proliferation or survival is also possible. Although ARF6Q67L primary tumors did not show a growth advantage over controls, ARF6Q67L primary tumor cells showed increased proliferation and decreased apoptosis in vitro (Supplementary Fig. S2E and S2F), demonstrating context-dependent functions of activated ARF6. In contrast, ARF6-GTP had no impact on proliferative activity in primary tumors (Supplementary Fig. S2G). Together these data suggest that ARF6-GTP might convey a proliferative or survival advantage specifically to tumor cells en route to or colonizing distant organs. In addition to facilitating primary tumor escape of a metastatic cell, ARF6 may function later in the metastatic cascade. The increase in metastatic volume suggests a role for ARF6 after initial primary tumor escape.

To test the hypothesis that activated ARF6 promotes a late step in metastasis, we employed a tail-vein injection model of experimental metastasis. This approach monitors survival in the circulation, extravasation, invasion into distant organs, and/or end-organ colonization. We derived four mouse melanoma cell lines from BrafCA;Cdkn2af/f ± Arf6Q67L mouse tumors, delivered them separately to the circulation by tail-vein injection into NOD SCID mice, and evaluated lung histology for metastases within 4 weeks postinjection. We found that ARF6-GTP dramatically enhanced metastatic lung colony formation in this model (Fig. 1D–F). Specifically, circulating tumor cells expressing ARF6Q67L colonized the lungs in 100% of mice, whereas controls colonized the lungs in 33% of mice (Fig. 1F). These data are consistent with previous work showing that expression of an inactive, GDP-bound form of ARF6 (T27N) in human LOX melanoma cells, injected into tail veins of Nude mice, reduces lung colonization (5). Thus, ARF6 activation provides a selective advantage for survival of circulating tumor cells, extravasation, end-organ invasion, and/or establishment of metastatic colonies while inactivation of ARF6 curbs this prometastatic phenotype.

ARF6-GTP is sufficient to potentiate metastasis and exacerbates melanoma metastasis upon PTEN loss

To determine how ARF6 affects other known drivers of metastatic melanoma, we directly compared the BrafCA;Cdkn2af/f + Arf6Q67L mice to BrafCA;Cdkn2af/f;Ptenf/f mice (17). All mice engineered to express HA-tagged ARF6Q67L in tumors were confirmed by anti-HA immunostaining (see Supplementary Fig. S1A for examples) and/or by qRT-PCR (Supplementary Fig. S3). The overall incidence of primary tumor development was significantly higher and tumor onset (disease latency) was dramatically shorter in Ptenf/f mice (Supplementary Fig. S4A and S4B). Individual tumor growth rates were highly heterogeneous in Ptenf/f mice, causing erratic growth curves in the cohort, but trended higher than Arf6Q67L mice (Supplementary Fig. S4C). The difference in tumor growth was best represented in the survival data, where larger primary tumors in Ptenf/f mice translated into reduced survival after tumor onset (Supplementary Fig. S4D). No mice were sacrificed due to symptoms from metastasis. Rather, our experimental endpoint was limited by primary tumor growth and related symptoms that mandated animal sacrifice. These data suggest that PTEN loss accelerates tumorigenesis in BRAFV600E/Cdkn2aNull melanoma, in contrast to ectopic expression of ARF6-GTP.

Despite lagging behind in tumor incidence, disease latency, and tumor growth, the Arf6Q67L mice demonstrated a metastatic incidence that is equivalent to the Ptenf/f mice (Fig. 1G). Both cohorts showed an increase in overall metastatic incidence to 10%–11% from about 4%, and both showed equivalent bulky metastatic disease (Supplementary Fig. S4E). No brain metastases were detected in the Ptenf/f mice, as reported previously (17), nor the Arf6Q67L mice. These phenotypes indicate that while PTEN loss incites aggressive primary tumor growth as well as progression to metastasis, ARF6-GTP function in this model specifically promotes metastasis. This is an important distinction, because it demonstrates that activation of ARF6 can promote a prometastatic state that is independent of tumor growth, possibly increasing the risk of distant spread in smaller tumors, a notorious clinical feature of melanoma.

We previously reported that PTEN loss cooperates with activated AKT1 in the BrafCA;Cdkn2af/f model to induce a more aggressive metastatic phenotype than each allele individually (17). Similarly, we found that combining PTENNull with ARF6-GTP dramatically and significantly increased the metastatic incidence and volume compared with each allele alone (Fig. 1G; Supplementary Fig. S4E). A linear-by-linear association test revealed a statistically significant increase in disease severity along an ordered spectrum. Thus, similar to AKT (17), activated ARF6 cooperates with PTEN loss to promote metastatic melanoma.

ARF6 activation leads to increased PI3K expression and AKT activation in BRAFV600E/CDKN2ANull tumors

ARF6 facilitates signaling of multiple proteins with direct and indirect transcriptional output, including ERK (3, 7, 10), PLC (7), Hedgehog (29), β-catenin (2, 10), and YAP (7). These data suggest that ARF6 may impact global expression changes. To identify novel ARF6-regulated target genes and pathways, we analyzed transcriptomes from our BRAFV600E/CDKN2ANull ± ARF6Q67L mouse melanoma tumors. We detected a unique gene expression signature in the ARF6Q67L tumors (Fig. 2A). This signature included upregulation of gene biogroups related to known ARF6-dependent cell functions in trafficking, cell adhesion, motility, and providing an internal positive control that helps to validate the utility of RNA sequencing as a discovery tool for ARF6.

Figure 2.

ARF6-GTP upregulates PI3K expression and AKT activation. A, RNA sequencing of mouse tumors reveals differential expression between melanomas from control (n = 4) and Arf6Q67L mice (n = 6). Left, heatmap of relative expression is shown, range of 2 to −2 (log2). Right, upregulated gene biosets in ARF6Q67L tumors. * denotes gene sets that include Pik3r1. B, ARF6Q67L induces 1.4503-fold increase in Pik3r1 expression in tumors. Unpaired t test, two-tailed. C, In mouse tumor cell lines derived from a distinct group of mice from those sequenced, ARF6Q67L increases both p85 and p110 PI3K protein levels. Ratio paired t test, two-tailed. HA immunoblot detects ectopic HA-tagged ARF6Q67L. D, Heatmap representing differences in signaling protein levels detected with RPPA. Columns represent individual mouse tumors. Unpaired t test, two-tailed.

Figure 2.

ARF6-GTP upregulates PI3K expression and AKT activation. A, RNA sequencing of mouse tumors reveals differential expression between melanomas from control (n = 4) and Arf6Q67L mice (n = 6). Left, heatmap of relative expression is shown, range of 2 to −2 (log2). Right, upregulated gene biosets in ARF6Q67L tumors. * denotes gene sets that include Pik3r1. B, ARF6Q67L induces 1.4503-fold increase in Pik3r1 expression in tumors. Unpaired t test, two-tailed. C, In mouse tumor cell lines derived from a distinct group of mice from those sequenced, ARF6Q67L increases both p85 and p110 PI3K protein levels. Ratio paired t test, two-tailed. HA immunoblot detects ectopic HA-tagged ARF6Q67L. D, Heatmap representing differences in signaling protein levels detected with RPPA. Columns represent individual mouse tumors. Unpaired t test, two-tailed.

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Interestingly, mTOR signaling components were upregulated in ARF6Q67L tumors (Fig. 2A), including Pik3r1, which encodes the p85 regulatory subunit of PI3K. This finding is provocative because the PI3K pathway is strongly linked to melanoma disease progression and because our ARF6Q67L mice showed a comparable metastatic phenotype to mice with PTEN loss. Transcript levels of Pik3r1 were increased in ARF6Q67L tumors compared with BrafCA;Cdkn2af/f controls (Fig. 2B). Likewise, p85 protein levels were significantly increased in primary tumor cell lines derived from ARF6Q67L mouse melanomas (Fig. 2C). The p85 regulatory subunit facilitates PI3K signaling by stabilizing the p110 catalytic subunit of PI3K (30), and through recruitment of p110 to the plasma membrane (reviewed in ref. 31). Consistent with this function, we detected increased p110α protein levels in BrafCA;Cdkn2af/f + ARF6Q67L melanoma tumor cell lines (Fig. 2C). Pik3ca mRNA levels were not altered in the ARF6Q67L tumors, suggesting that the increased p110 protein is secondary to ARF6-GTP–mediated increases in p85.

To broadly query for signaling proteins altered by ARF6 activation, we tested our mouse tumors with reverse-phase protein array (RPPA). Compared with BRAFV600E/Cdkn2aNULL controls, ARF6Q67L tumors showed the most dramatic increase in phosphorylated AKT (pAKT) levels (Fig. 2D). Other pathways upregulated include components of receptor tyrosine kinase signaling (HER3 and GAB2), calcium signaling (PKCa), autophagy (LC3A-B), dynamic chromatin structure (histone H3 and di-methyl histone H3), and cell motility (myosin IIa phosphorylation and α-tubulin; refs. 32–35). Interestingly, myosin IIa upregulation distinguishes ARF6Q67L tumors from PTENNULL tumors (Fig. 2D). ARF6Q67L tumors also show a stronger signal for tuberin T1462 phosphorylation than PTENNULL tumors. As Tuberin T1462 is a PI3K-regulated AKT substrate and constitutively phosphorylated in PTENNULL cells (36), these data suggest that ARF6-GTP is a potent activator of PI3K and/or AKT. Compared with ARF6Q67L, PTENNULL tumors show upregulation of cyclin-D1 and phosphorylation of RB, which could explain how the PTENNULL primary tumor growth rate exceeds ARF6Q67L tumors. Together, these findings help explain the overlapping but distinct phenotypes between PTENNULL and ARF6Q67L. ARF6-GTP may compensate for the lag in tumor growth, compared with PTEN loss, by activating cell motility pathways, ultimately reaching the same metastatic endpoint.

ARF6-GTP enhances PI3K distribution to cellular protrusions

Because ARF6 is critical for intracellular trafficking, we next evaluated the localization of p85 and p110 in these tumor cells. BrafCA;Cdkn2af/f control mouse melanoma cells grown in vitro were polygonal shaped, whereas BrafCA;Cdkn2af/f+Arf6Q67L tumor cells were strikingly elongated/spindle shaped (Fig. 3; Supplementary Fig. S5). Both groups demonstrated prominent spinous/dendritic processes. Immunofluorescent staining confirmed that p85 and p110α levels increased in the presence of ARF6Q67L (Fig. 3), consistent with transcriptome and Western blot analysis (Fig. 2C and D). The localization pattern of endogenous p85 did not appear to change with ARF6 activation (Fig. 3A), remaining mostly in the cell body. In contrast, endogenous p110α was more detectable in peripheral, protrusive structures of ARF6Q67L cells: in both bulbous and spinous processes (Fig. 3B and C). These data suggest that ARF6-GTP is sufficient to control, either directly or indirectly, the subcellular localization of the catalytic subunit of PI3K to specific peripheral compartments within melanoma cells.

Figure 3.

ARF6-GTP increases the distribution of p110α PI3K in cellular protrusions in melanoma. Primary melanoma cells derived from mouse tumors. Magnification, ×1,200. Scale bars, 30 μm. Left, BrafCA;Cdkn2af/f control (5588). Right, BrafCA;Cdkn2af/f + Arf6Q67L (6431), where arrows highlight bulbous and spinous cellular projections. A, Green, anti-p85 PI3K. B, Green, anti-p110a PI3K. A and B, Red, anti-HA (HA-tagged ARF6Q67L). Blue, DAPI. C, Quantification of p110α PI3K (green) in bulbs and spines (outlined regions; right). ROI, regions of interest selected for quantification of p110α signal intensity. All bulbo-spinous processes were quantified for all cells in eight distinct images per cell line. Representative images are shown. Magnification, ×1,800. Scale bars, 20 μm. Mann–Whitney U test.

Figure 3.

ARF6-GTP increases the distribution of p110α PI3K in cellular protrusions in melanoma. Primary melanoma cells derived from mouse tumors. Magnification, ×1,200. Scale bars, 30 μm. Left, BrafCA;Cdkn2af/f control (5588). Right, BrafCA;Cdkn2af/f + Arf6Q67L (6431), where arrows highlight bulbous and spinous cellular projections. A, Green, anti-p85 PI3K. B, Green, anti-p110a PI3K. A and B, Red, anti-HA (HA-tagged ARF6Q67L). Blue, DAPI. C, Quantification of p110α PI3K (green) in bulbs and spines (outlined regions; right). ROI, regions of interest selected for quantification of p110α signal intensity. All bulbo-spinous processes were quantified for all cells in eight distinct images per cell line. Representative images are shown. Magnification, ×1,800. Scale bars, 20 μm. Mann–Whitney U test.

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ARF6-GTP is sufficient for PI3K and AKT activation

Thus far, our data suggest that ARF6 activation upregulates expression and distribution of PI3K. Whether ARF6 also controls PI3K activation in melanoma, or any other cancer, is unknown. We asked whether ARF6-GTP is sufficient for activation of PI3K, or of its effector AKT, in our melanoma mouse model. We measured endogenous p110 PI3K catalytic activity in anti-p85 PI3K immunoprecipitates, as described previously (25). We found that the catalytic activity of endogenous PI3K increased in the presence of ARF6Q67L (Fig. 4A). Furthermore, we observed higher levels of activated AKT (pAKT) in ARF6Q67L mouse melanoma cells relative to controls (Fig. 4B). Ectopic expression of ARF6Q67L was also sufficient to activate AKT in NIH3T3 cells (Fig. 4C), suggesting a broader phenomenon.

Figure 4.

ARF6-GTP is sufficient for PI3K and AKT activation. A, Catalytic activity of endogenous PI3K in mouse tumor cell lines; one-way ANOVA with Tukey multiple comparisons test. B, Mouse melanoma cells BrafCA;Cdkn2af/f control (5588), BrafCA;Cdkn2af/f + Arf6Q67L (6431 and 6455). C, Adenoviral delivery of MYC-tagged ARF6Q67L to serum-starved NIH3T3 cells. B and C, Graphs show individual data points normalized to control along with geometric means, 95% CI, ratio paired t test, two-tailed. D, Phosphorylated AKTS473 detected by IHC in primary tumors from Arf6Q67L mice but not BrafCA;Cdkn2af/f controls. Scale bars, 20 μm; ×400 magnification. E, pAKT stains tended to highlight tumor cells at the invasive front (arrows). Mouse tumors 8445, 6807, and 6808: scale bars, 20 μm, Magnification, ×400. In 6807, pAKT highlights tumor cells invading edematous subcutaneous tissue (top right of image). In 6808, pAKT highlights tumor cells invading adipose (left portion of image). Mouse 7760 shows pAKT highlighting tumor invading skeletal muscle. Scale bar, 50 μm. Magnification, ×100. F, Heterogeneous pAKT staining of metastatic tumors. Pulmonary metastasis (met), scale bars, 20 μm. Magnification, ×400. Lymph node met, scale bars, 50 μm. Magnification, ×200. BrafCA;Cdkn2af/f mice are known to develop benign pulmonary adenomas (unrelated to melanoma) and these fail to stain with pAKT, providing a negative internal control (right; scale bar, 100 μm; magnification, ×100).

Figure 4.

ARF6-GTP is sufficient for PI3K and AKT activation. A, Catalytic activity of endogenous PI3K in mouse tumor cell lines; one-way ANOVA with Tukey multiple comparisons test. B, Mouse melanoma cells BrafCA;Cdkn2af/f control (5588), BrafCA;Cdkn2af/f + Arf6Q67L (6431 and 6455). C, Adenoviral delivery of MYC-tagged ARF6Q67L to serum-starved NIH3T3 cells. B and C, Graphs show individual data points normalized to control along with geometric means, 95% CI, ratio paired t test, two-tailed. D, Phosphorylated AKTS473 detected by IHC in primary tumors from Arf6Q67L mice but not BrafCA;Cdkn2af/f controls. Scale bars, 20 μm; ×400 magnification. E, pAKT stains tended to highlight tumor cells at the invasive front (arrows). Mouse tumors 8445, 6807, and 6808: scale bars, 20 μm, Magnification, ×400. In 6807, pAKT highlights tumor cells invading edematous subcutaneous tissue (top right of image). In 6808, pAKT highlights tumor cells invading adipose (left portion of image). Mouse 7760 shows pAKT highlighting tumor invading skeletal muscle. Scale bar, 50 μm. Magnification, ×100. F, Heterogeneous pAKT staining of metastatic tumors. Pulmonary metastasis (met), scale bars, 20 μm. Magnification, ×400. Lymph node met, scale bars, 50 μm. Magnification, ×200. BrafCA;Cdkn2af/f mice are known to develop benign pulmonary adenomas (unrelated to melanoma) and these fail to stain with pAKT, providing a negative internal control (right; scale bar, 100 μm; magnification, ×100).

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To understand the role of ARF6-GTP in PI3K-AKT activation in vivo, we evaluated pAKT in histologic sections of our primary mouse tumors. IHC staining revealed that pAKT is detectable in more than half of primary tumors from ARF6Q67L mice, whereas pAKT was not detected in primary tumors from control mice (Fig. 4D). Overall, pAKT staining was highly heterogeneous, detectable in only a fraction of the tumor cells (Fig. 4D and E). Activated AKT was often limited to cells at the invasive front, at the interface with subcutaneous fibro-edematous connective tissue, skeletal muscle, and/or adipose (Fig. 4E). Activated AKT was also detected in metastatic tumors from BrafCA;Cdkn2af/ + Arf6Q67L mice (Fig. 4F). Together these results demonstrate that ARF6-GTP is sufficient for activation of the PI3K pathway and suggest that ARF6-GTP may engage PI3K signaling in specific cell populations within a tumor.

ARF6 is necessary for PI3K and AKT activation in human cancer cells

To test whether ARF6 is required for PI3K/AKT signaling and to extend our observations to human melanoma, we expressed a dominant negative ARF6 (ARF6T27N) in human melanoma A2058 cells. Ectopic expression of ARF6T27N significantly reduced PI3K catalytic activity in A2058 cells (Fig. 5A). Furthermore, ectopic expression of ARF6T27N reduced pAKT levels in 10 of 15 melanoma cell lines, independent of the primary driver gene mutation status (Fig. 5B and C; Supplementary Fig. S6A). ARF6T27N also reduced pAKT levels in a KRAS-mutant carcinoma cell line (HEY-T30), suggesting that ARF6 also regulates PI3K signaling in nonmelanoma cancer cells (Fig. 5B; Supplementary Fig. S6A). Knockdown of ARF6 also reduced pAKT levels (Fig. 5D; Supplementary Fig. S6B). In a proteomic phosphokinase array, ARF6T27N incited the greatest impact on AKT and its substrate PRAS40, reducing phosphorylation of both (Supplementary Fig. S6C). Importantly, this proteomic survey corroborates our findings in mouse melanoma cells whereby AKT phosphorylation is one of the strongest signals in ARF6Q67L tumors (Fig. 2E). Together, our in vitro and in vivo findings demonstrate that ARF6 is necessary and sufficient for PI3K activation and AKT signaling.

Figure 5.

ARF6 is necessary for PI3K and AKT activation. A–C, Adenoviral delivery of MYC-tagged ARF6T27N, a GDP-bound, inactive form of ARF6. A, Catalytic activity of endogenous PI3K in A2058 human melanoma cells. Unpaired t test, two-tailed. B, Fifteen melanoma and one carcinoma (HEY-T30) cell lines were screened for AKT activation. Eleven of 16 (68%) cell lines showed reduced pAKTS473 with ARF6T27N expression. C, Reproducible reduction in pAKT by ARF6T27N expression in A375 cells (n = 4), A2058 cells (n = 4), and HEY-T30 cells (n = 5). D, Reduced pAKT with siRNA knockdown of ARF6 in SK-MEL-147 (n = 5), A2058 (n = 4), and CACL (n = 4) and HEY-T30 (n = 6). See Supplementary Fig. S6 for quantitative data.

Figure 5.

ARF6 is necessary for PI3K and AKT activation. A–C, Adenoviral delivery of MYC-tagged ARF6T27N, a GDP-bound, inactive form of ARF6. A, Catalytic activity of endogenous PI3K in A2058 human melanoma cells. Unpaired t test, two-tailed. B, Fifteen melanoma and one carcinoma (HEY-T30) cell lines were screened for AKT activation. Eleven of 16 (68%) cell lines showed reduced pAKTS473 with ARF6T27N expression. C, Reproducible reduction in pAKT by ARF6T27N expression in A375 cells (n = 4), A2058 cells (n = 4), and HEY-T30 cells (n = 5). D, Reduced pAKT with siRNA knockdown of ARF6 in SK-MEL-147 (n = 5), A2058 (n = 4), and CACL (n = 4) and HEY-T30 (n = 6). See Supplementary Fig. S6 for quantitative data.

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The PI3K/AKT pathway is necessary for ARF6-mediated invasion

In the presence of ARF6-GTP, p110 PI3K localized to peripheral cellular protrusions (Fig. 3B), As ARF6 has a prominent role in invadopodia function (3, 5), our data suggest that ARF6 engages the PI3K–AKT pathway to facilitate invasion. The PI3K–AKT pathway has been implicated in cell motility/invasion (37), including in melanoma (17). We previously reported that knockdown of ARF6 or pharmacologic inhibition of ARF6 universally reduces invasion of human cutaneous melanoma cell lines in vitro (2). Muralidharan-Chari and colleagues showed that ectopic ARF6T27N inhibits invasion, whereas ARF6Q67L increases invasion of LOX melanoma cells (4). Consistent with these findings, ectopic ARF6Q67L increased Matrigel invasion of A2058 melanoma cells in vitro (Fig. 6). With this system, we tested whether PI3K and AKT kinase activity are necessary for ARF6-mediated invasion. Indeed, the pan-AKT inhibitor MK2206 (Fig. 6A and B) and the pan-Class I PI3K inhibitor GSK-0941 (Fig. 6C and D) both abrogated ARF6Q67L-dependent Matrigel invasion, demonstrating that PI3K signaling is necessary for ARF6-mediated invasion.

Figure 6.

PI3K and AKT are necessary for ARF6-GTP–dependent melanoma invasion. A and C, Matrigel invasion of A2058 cells is increased by ectopic expression of Myc-tagged ARF6Q67L (A–D). The AKT inhibitor MK2206 (1 μmol/L; A) or the PI3K inhibitor GDC0941 (1 μmol/L; C) eliminates ARF6Q67L-induced invasion. Error bars, SD; ****, P < 0.0001, ANOVA with Tukey multiple comparisons test. B and D, MYC-tagged ARF6Q67L expression relative to endogenous ARF6. E, pAKT levels and localization in invading A2058 cells, plated on Matrigel dome, serum starved in PBS. Top row, 20 hours after plating. Magnification, ×600. Scale bars, 60 μm. Middle and bottom rows, 48 hours after plating. Magnification, ×1,200. Scale bars, 30 μm. F, ARF6Q67L–induced pAKT and cortactin colocalization in A2058 cells invading and degrading gelatin (labeled with Oregon Green 488).

Figure 6.

PI3K and AKT are necessary for ARF6-GTP–dependent melanoma invasion. A and C, Matrigel invasion of A2058 cells is increased by ectopic expression of Myc-tagged ARF6Q67L (A–D). The AKT inhibitor MK2206 (1 μmol/L; A) or the PI3K inhibitor GDC0941 (1 μmol/L; C) eliminates ARF6Q67L-induced invasion. Error bars, SD; ****, P < 0.0001, ANOVA with Tukey multiple comparisons test. B and D, MYC-tagged ARF6Q67L expression relative to endogenous ARF6. E, pAKT levels and localization in invading A2058 cells, plated on Matrigel dome, serum starved in PBS. Top row, 20 hours after plating. Magnification, ×600. Scale bars, 60 μm. Middle and bottom rows, 48 hours after plating. Magnification, ×1,200. Scale bars, 30 μm. F, ARF6Q67L–induced pAKT and cortactin colocalization in A2058 cells invading and degrading gelatin (labeled with Oregon Green 488).

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To visualize the effect of ARF6-GTP on the PI3K–AKT pathway in invading melanoma cells, we plated A2058 cells on a dome of Matrigel and imaged cells penetrating and digesting the matrix. Cells were costained with anti-cortactin, a marker of invadopodia (38), and anti-pAKT (Fig. 6E). Ectopic expression of ARF6Q67L triggered an increase in peripheral and cytosolic pAKT levels above background. The untreated and vector control cells showed a rounded morphology on undigested Matrigel, whereas ARF6Q67L incited cell spreading into plump, polygonal shapes with extended cell processes (Fig. 6E, top row). Treatment with the pan-AKT inhibitor MK2206 reduced pAKT staining and restored the rounded cell shape. During active Matrigel digestion, cellular protrusions were more readily visualized and endogenous pAKT localized with cortactin in ring-shaped structures (Fig. 6E, middle and bottom rows, see arrows). This morphology is similar to the colocalization reported for cortactin and F-actin in invadopodia (38). ARF6Q67L expression led to an increase in these pAKT-rich invadopodia. ARF6-GTP induced condensation and colocalization of pAKT and cortactin in invadopodia and was also seen in melanoma cells invading and digesting gelatin matrix (Fig. 6F). Our data are consistent with findings in breast cancer cells in which PI3K and AKT mediate invadopodia formation and function (39).

Together these results demonstrate a previously unknown mechanism by which ARF6 facilitates invasion by engaging the PI3K–AKT pathway, which may help explain how ARF6 activation leads to metastasis. We propose a two-step mechanism of ARF6–PI3K–AKT signaling in invading melanoma cells. Activation of ARF6 induces (i) transcriptional upregulation of the p85 regulatory subunit of PI3K, causing stabilization of the p110 catalytic subunit, leading to (ii) p110 transport to invadopodia, where AKT is activated.

ARF6 is aberrantly activated in melanoma patient samples

We previously showed that pharmacologic inhibition of ARF6 limits metastasis, suggesting that ARF6-GTP is necessary for metastasis (17). Our current data reveal that ARF6-GTP is sufficient for metastasis and provide mechanistic insight. Yet, the clinical relevance of these findings remained uncertain because the activated, GTP-bound state of ARF6 in uncultured cancer specimens is unknown. Direct measurement of the activation state of small GTPases in patient samples is challenging due to the high instability of GTP binding and the significant input protein requirements for detection. Nevertheless, we asked whether ARF6-GTP levels are detectable and altered in tumors compared with nontumor tissue. We queried our frozen clinical biorepository for specimens of patient-matched melanoma and nontumor/normal tissue. Quality control analysis for tumor and tissue-type content was provided by H&E morphology evaluation. We limited our testing to samples with ample excess banked tissue and a high minimum viable tumor content of 70% for cancer samples. All nontumor, normal tissues were confirmed viable. With these inclusion criteria, we identified appropriate tissue pairs from 12 recent patients. Each pair was procured from a single surgical procedure (adjacent tissue in a wide resection) and processed simultaneously. GTP-bound ARF6 was detected in four of these pairs, including the normal tissue, reflecting the instability of the GTP-bound state and the variability in preanalytic processing of the tissues, such as time to freezing. Among these four pairs, ARF6-GTP levels were higher in the tumors compared with matched normal tissues (Fig. 7A). While these normal tissues do not contain a preponderance of the ideal control cell lineage, melanocytes, ARF6 activation has been reported in the predominant cell lineages of each of the matched normal tissues (uninvolved lymph node, fibroadipose, liver, and skin; refs. 40–44), as well as a variety of endothelial and epithelial cells (reviewed in ref. 6). These data support a biologic role for activated ARF6 specifically in tumor cell function.

Figure 7.

Evidence for aberrant ARF6 activation in melanoma. A, Western blot of ARF6-GTP pull downs from paired patient samples with quantified ARF6-GTP levels per 500 mg of total protein input. Paired ratio t test, two-tailed; bars, 95% CI. Each tumor sample is paired with its patient-matched, nontumor (normal) tissue type that was surgically resected and processed simultaneously. Normal tissues include lymph node, fibroadipose, liver, and skin (respectively, left to right). B and C, Reduced ARF6-GAP expression in skin (SK) and lymph node (LN) metastasis correlates with a reduced overall survival in stage III patients TCGA melanoma patients. The RNA expression ranks for ACAP1 and ARAP is shown. Higher rank indicates higher gene expression. Reduced ARAP2 and ACAP1 expression correlate with reduced overall survival from time of diagnosis. Log-rank (Mantel–Cox) test. See also Supplementary Fig. S7.

Figure 7.

Evidence for aberrant ARF6 activation in melanoma. A, Western blot of ARF6-GTP pull downs from paired patient samples with quantified ARF6-GTP levels per 500 mg of total protein input. Paired ratio t test, two-tailed; bars, 95% CI. Each tumor sample is paired with its patient-matched, nontumor (normal) tissue type that was surgically resected and processed simultaneously. Normal tissues include lymph node, fibroadipose, liver, and skin (respectively, left to right). B and C, Reduced ARF6-GAP expression in skin (SK) and lymph node (LN) metastasis correlates with a reduced overall survival in stage III patients TCGA melanoma patients. The RNA expression ranks for ACAP1 and ARAP is shown. Higher rank indicates higher gene expression. Reduced ARAP2 and ACAP1 expression correlate with reduced overall survival from time of diagnosis. Log-rank (Mantel–Cox) test. See also Supplementary Fig. S7.

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Biochemical measurement of small GTPase activation in patient samples is not scalable and, as demonstrated above, is limited by instability of the GTP-bound protein state and by significant tissue input requirements. These barriers to implementing biochemical interrogation of small GTPases in precious patient samples have limited our understanding of the contribution of many small GTPases to disease states. As an alternative to biochemical markers, we asked whether there is genomic evidence of ARF6 activation. ARF6 is expressed in a wide variety of cancer types, including melanoma (45), and high ARF6 expression correlates with reduced survival in pancreatic cancer (12). In prostate cancer, ARF6 expression is increased in tumor cells compared with normal prostate and ARF6 staining tends to increase with Gleason grade (46).

ARF6 is activated by a variety of extracellular cues and by oncogenic mutations in vitro (6, 7, 12, 29). Guanine nucleotide exchange factors (GEF) and GTPase-activating proteins (GAP) determine the active, GTP-bound state versus the inactive, GDP-bound state of small GTPases, respectively. Upregulated ARF6 GEF expression is linked to cancer progression. For example, in breast cancer, expression of GEP100 increases with pathologic grade and with the transition from in situ to invasive cancer (47). In lung adenocarcinoma, increased HER2 and GEP100 correlate with lymph node metastasis (8). In squamous cell carcinoma of the head and neck, high expression of AMAP1 correlates with reduced survival (48).

To investigate ARF6 pathway expression in a large melanoma cohort, we queried TCGA cutaneous melanoma RNAseq data (20) for alterations in the ARF6 pathway that correlate with overall survival from the time of diagnosis. We evaluated the expression levels of ARFs, ARF GEFs and GAPs, ARF6 effectors, and the ARF6-inhibiting ligand–receptor pair SLITs-ROBOs (Supplementary Table S1). We found that a distinct pattern emerged: in contrast to the patterns reported for carcinomas, neither ARF6 nor ARF6 GEF expression was associated with survival in melanoma. Rather, the expression of ARF GAPs, which inactivate ARF6, was prognostic in cutaneous melanoma. Specifically, low ARF GAP expression significantly correlated with reduced overall survival compared with high ARF GAP expression (Fig. 7B; Supplementary Fig. S7). The six ARF GAP genes that are prognostic include four ARF6-specific GAPs [ACAP1 (49), ADAP1 (50), ARAP2 (51), SMAP2 (52)], an ARF1/ARF5 GAP (AGAP2; ref. 53), and one predicted ARF GAP (AGFG2; refs. 54, 55). Low expression of each correlated with reduced overall survival in an individual gene analysis (Supplementary Fig. S7A). In lymph node and skin metastasis from stage III TCGA patients, a subset of patients who are at significant risk of metastatic progression, ARAP2 and ACAP1 expression were strongly prognostic for reduced overall survival (Fig. 7B and C). To a lesser extent, loss of ADAP1 and AGAP2 were associated with worse prognosis, although the association was not statistically significant (Supplementary Fig. S7B). Because the TCGA survival time begins at the date of diagnosis, this study is limited by a lack of primary tumors and may be biased by the lack of primary tumors that failed to metastasize. Nevertheless, downregulation of ARF6-specific GAPs (ARF6-suppressors) occurs during metastatic progression to stage III and correlates with reduced overall survival in patients with metastatic melanoma. These results suggest that ARF6 hyperactivation in melanoma is associated with metastatic progression and reveal the ARF6 pathway as a potential therapeutic target.

In this study, we shed new light on the potency, mechanism of action, and potential clinical impact of ARF6 activation in cutaneous melanoma. In the active, GTP-bound state, ARF6 is not only sufficient to accelerate metastases, it confers equal metastatic capacity, from smaller tumors, compared with deletion of the tumor suppressor Pten. In essence, ARF6 activation renders BRAFV600E/Cdkn2aNULL tumors more competent for metastasis, acting as a catalyst for primary tumor escape and/or end-organ colonization. Early metastasis from very thin primary tumors is a long-standing clinical conundrum in melanoma. Our data suggests that activation of ARF6 may explain how this occurs. Furthermore, our work suggests that ARF6 functions in multiple stages of metastasis, including later stages such as survival and end-organ colonization of circulating tumor cells. These data are significant because targeting ARF6 may have therapeutic utility in patients with a significant risk of metastatic progression. Pharmacologic inhibitors of ARF6 are in preclinical development and studies show efficacy in several in vivo disease models, including melanoma (2, 56–59). It will be important to test whether ARF6 pharmacologic inhibitors are effective in a variety of preclinical cancer model systems and to pair these studies with conditional genetic knockout models.

We have elucidated distinct roles for ARF6 in the metastatic cascade and uncovered a novel ARF6–PI3K–AKT pathway that is important for invasion. ARF6-GTP boosts expression of p85 PI3K, increasing p110 PI3K protein levels and enhancing localization of p110 PI3K to peripheral cellular compartments that are critical for melanoma invasion. This mechanism helps explain how ARF6 activation can synergize with Pten deletion in vivo to exacerbate metastasis. ARF6-induced PI3K expression and redistribution leads to increased pools of activated PI3K, while loss of PTEN eliminates the metabolism of its product PIP3, thus leading to increased PIP3 and activated AKT.

Our human data make a compelling case for the importance of ARF6 in cutaneous melanoma. ARF6-GTP is elevated in tumors compared with patient-matched, adjacent, nontumor tissue. Furthermore, in a large TCGA melanoma cohort, transcriptome data implicates ARF6 activation, via ARF6 GAP loss, in reduced overall survival. These human data are provocative because they suggest aberrant activation of ARF6 in melanoma patient samples and a pathologic role for ARF6 in this disease. Overall, our data strongly suggest that activation of ARF6, via tumor-intrinsic or tumor microenvironmental signals, incites disease progression in melanoma patients at least, in part, by engaging the PI3K pathway.

Our in vivo model provides a unique paradigm for understanding trafficking-specialized small GTPases in cancer. ARFs and RABs are distinct among the RAS superfamily in that they are critical for endocytic and exocytic membrane trafficking. These proteins often show overlapping functions, such as promoting receptor tyrosine kinase and MAPK signaling and cancer cell invasion (6, 60). Our work demonstrates that, similar to RAB35 (25); and RAB25 (61), ARF6 is also important for PI3K signaling. RAB35 appears to mediate late endosome-associated platelet-derived growth factor receptor activation of AKT (25). Ectopic RAB35Q67L associates with PI3K and increases pAKT levels in vitro. It is possible that ARF6 facilitates a similar RTK-PI3K endosomal signaling mechanism in melanoma. Using an in vivo approach, we discovered a distinct and unexpected mechanism of PI3K activation that begins with transcriptional upregulation of the p85 regulatory subunit of PI3K and ends with delivery of the p110 catalytic subunit of PI3K to invadopodia where PI3K and AKT are critical for ARF6-mediated invasion (graphical abstract).

While more work is needed to understand the role of ARF6 in fine-tuning the transcriptional output of signaling pathways, it is clear from our data and others that ARF6 can exert multiple levels of control over the PI3K pathway. A role for ARF6 in the PIP2 synthetic pathway was originally described by Honda and colleagues (62), who showed that synthesis of PIP2 by phosphatidylinositol 4-phosphate 5-kinaseα (PI4P5Kα) is stimulated by ARF proteins and phosphatidic acid. A more recent report shows that ARF6 controls PI4P5Kα-mediated PIP2 generation and AKT activation in benign hepatocytes (44). Unlike these previous studies, we show that ARF6 can control PI3K expression and, potentially, its catalytic activity. This is a unique role for ARF6 in PI3K–AKT signaling. In the future, it will be important to determine whether targeting ARF6 can inhibit the PI3K pathway in metastatic progression, particularly in the presence of PTEN loss.

Disruption of the metastatic cascade, as a therapeutic approach for patients with local, regional, or oligometastatic disease, remains an elusive but promising therapeutic goal in oncology (63). Reaching this goal begins with understanding the mechanistic underpinnings of metastasis and the development of preclinical metastatic models. ARF6 appears to be versatile in its capacity to facilitate distinct signaling pathways promoting cancer progression (6). In cutaneous melanoma, ARF6 has been linked to HGF-ERK–mediated invasion (3) and WNT5A-β-catenin–mediated invasion (2). Given that ARF6 is upstream of both MAPK/ERK (3, 7, 10, 59, 64) and now PI3K/AKT, it is possible that ARF6 regulates both of these pathways by exerting direct control over RAS. Regardless, the expanded reach of ARF6 in oncogenic signaling to both major arms of the RAS pathway has significant implications. RAS is an elusive pharmacologic target in cancer (65) while ARF6 inhibitors continue to show promise in preclinical models. In the future, it will be important to investigate a role for ARF6 in RAS-mediated oncogenesis and to test ARF6 pharmacologic inhibitors in cancers driven by unregulated RAS.

D.Y. Li is a senior vice president at Merck and has ownership interest (including stock, patents, etc.) in Merck, Recursion Pharmaceutical, and Navigen Pharmaceutical, and is a consultant/advisory board member for Recursion Pharmaceuticals. No potential conflicts of interest were disclosed by the other authors.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the NIH.

Conception and design: J.H. Yoo, D.Y. Li, A.H. Grossmann

Development of methodology: L. Acosta-Alvarez, A. Rogers, L.K. Sorensen, A.H. Grossmann

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.H. Yoo, L. Acosta-Alvarez, A. Rogers, J. Peng, L.K. Sorensen, D. Shin, S.J. Odelberg, A.H. Grossmann

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.H. Yoo, S.W. Brady, L. Acosta-Alvarez, A. Rogers, J. Peng, R.K. Wolff, D.A. Kircher, A. Bild, S.J. Odelberg, S.L. Holmen, A.H. Grossmann

Writing, review, and/or revision of the manuscript: J.H. Yoo, S.W. Brady, L. Acosta-Alvarez, A. Rogers, J. Peng, R.K. Wolff, S.J. Odelberg, D.Y. Li, S.L. Holmen, A.H. Grossmann

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J.H. Yoo, L. Acosta-Alvarez, A. Rogers, L.K. Sorensen, S.J. Odelberg, A.H. Grossmann

Study supervision: S.J. Odelberg, D.Y. Li, A.H. Grossmann

Other (illustration and graphic support): T. Mleynek

Other (performed in vitro assays): C.P. Rich

Other (provided critical reagents, technical support, scientific input and reading of the manuscript): S.L. Holmen

This study was supported by the National Cancer Institute K08 CA188563-01A1, R01 CA121118, K99 CA230312, the Melanoma Research Alliance MRA 347651, a Harold J. Lloyd Charitable Trust Career Development Award, NCI R01CA202778, NIAMS R01AR064788, NHLBI R01HL130541, T32HL007576-31, U54CA209978. We thank several shared resources at the Huntsman Cancer Institute: High-Throughput Genomics, Biorepository Molecular Pathology, and Bioinformatics are supported by award P30CA042014 from the National Cancer Institute and the Preclinical Research Resource, which provided experimental metastasis modeling services. We also thank Health Science Cores at the University of Utah: Genomics Core Facility; DNA Sequencing Core Facility; Fluorescence Microscopy Core Facility, supported by NCRR Shared Equipment Grant # 1S10RR024761-01. We thank the MDACC RPPA Core facility, funded by NCI CA16672. We thank Drs. R. Andtbacka, J. Hyngstrom, and R.D. Kim for patient specimen procurement. We thank C. Stubben for bioinformatics support and Dr. K. Boucher for biostatistics input in the TCGA survival correlates. We thank Drs. D. Grossman and M. VanBrocklin for human cell lines, Dr. M. McMahon for scientific and technical input, Drs. J. Kaplan and A. Anderson for manuscript editing, Dr. Z.Z. Tong and Navigen, Inc., for ARF6 plasmid constructs and D. Lim for graphics preparation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data