Figure S1. Detection of HA-tagged ARF6Q67L in BrafCA;Cdkn2af/f melanoma. a) Anti-HA immunostain of FFPE sections of primary tumors with variable levels of detection of ARF6Q67L -HA. The strongest staining was observed in tumors with the highest level of mRNA detected by qRT-PCR (b). Absent staining of FFPE tumors was observed in tumors with low (6456) and undetectable (6457) levels of mRNA by qRT-PCR of frozen tumors. Scale bars = 20mm, 400x magnification. Control = BrafCA;Cdkn2af/f (Cre-only injection). b) qRT-PCR of Arf6 Q67L-HA from fresh, frozen primary tumor fragments. Bars represent the absolute difference in crossing threshold (Ct) between Arf6Q67L-HA and Gapdh. The Ct threshold that defines detection was established above the background signal for controls in both the BrafCA;Cdkn2af/f and the BrafCA;Cdkn2af/f ;Ptenf/f cohorts (see Figures 1g and S3). With rare exception, positive tumors demonstrated Arf6 Q67L-HA expression levels lower than Gapdh.
ARTICLE ABSTRACT
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.
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.