Tumour cells evade immune surveillance by upregulating the surface expression of programmed death-ligand 1 (PD-L1), which interacts with programmed death-1 (PD-1) receptor on T cells to elicit the immune checkpoint response. Anti-PD-1 antibodies have shown remarkable promise in treating tumours, including metastatic melanoma. However, the patient response rate is low. A better understanding of PD-L1-mediated immune evasion is needed to predict patient response and improve treatment efficacy. Here we report that metastatic melanomas release extracellular vesicles, mostly in the form of exosomes, that carry PD-L1 on their surface. Stimulation with interferon-γ (IFN-γ) increases the amount of PD-L1 on these vesicles, which suppresses the function of CD8 T cells and facilitates tumour growth. In patients with metastatic melanoma, the level of circulating exosomal PD-L1 positively correlates with that of IFN-γ, and varies during the course of anti-PD-1 therapy. The magnitudes of the increase in circulating exosomal PD-L1 during early stages of treatment, as an indicator of the adaptive response of the tumour cells to T cell reinvigoration, stratifies clinical responders from non-responders. Our study unveils a mechanism by which tumour cells systemically suppress the immune system, and provides a rationale for the application of exosomal PD-L1 as a predictor for anti-PD-1 therapy.

Targeted BRAF inhibition (BRAFi) and combined BRAF and MEK inhibition (BRAFi+MEKi) therapies have significantly improved clinical outcomes in patients with metastatic melanoma. Unfortunately, the efficacy is beset by the acquisition of drug resistance1–6. Here we investigated molecular mechanisms underlying acquired resistance to BRAFi (BRAFi resistance, “BR”) and BRAFi+MEKi (combination therapy resistance, “CR”). Consistent with previous studies, BR is mediated by ERK pathway re-activation. CR is, however, mediated by mechanisms independent of re-activation of ERK in many therapy-resistant cell lines and clinical samples. p21-activated kinases (PAKs) become activated in acquired drug resistant cells and play a pivotal role in mediating both BR and CR. Our screening using reverse phase protein array (RPPA) revealed distinct mechanisms by which PAKs mediate BR and CR. In BR, PAKs phosphorylate CRAF and MEK to reactivate ERK. In CR, PAKs regulate JNK and β-catenin phosphorylation, mTOR pathway activation, and inhibit apoptosis, thereby bypassing ERK. Together, our results provide new insights into molecular mechanisms underlying acquired drug resistance to current targeted therapies, and may help to direct novel drug development efforts to overcome acquired drug resistance.

The development of head and neck squamous cell carcinoma (HNSCC) is closely associated with inflammation. Tumor associated macrophages (TAMs), the largest population of inflammatory cells in the tumor stroma, serve an important role in accelerating cancer progression. The present study aimed to investigate the role of TAMs in the metastasis of HNSCC. TAM biomarkers and epithelial to mesenchymal transition (EMT)-associated proteins were detected using immunohistochemical and immunofluorescence staining in HNSCC. Then, direct and indirect co-culture systems of TAMs and HNSCC cells were established. The EMT-associated proteins and associated signaling pathways in HNSCC cells of the co-culture system were measured by reverse transcription-quantitative polymerase chain reaction and western blotting. Finally, hierarchical clustering was performed to analyze associations among TAM biomarkers, epidermal growth factor receptor (EGFR), activated extracellular signal-regulated protein kinase 1/2 (ERK1/2) and EMT-associated proteins in HNSCC tissues. The results indicated that the expression of EMT-associated proteins was positively associated with M2 macrophage biomarkers in HNSCC tissues. Cal27 cells were isolated from the co-culture system by fluorescence-activated cell sorting, and it was identified that E-cadherin was downregulated in Cal27 cells, while Vimentin and Slug were upregulated. Furthermore, the results indicated that EGF released by M2 macrophages in the co-culture served an important role by activating ERK1/2. The correlation and cluster analyses indicated that activated ERK1/2 was positively correlated with cluster of differentiation-163, EGFR, Vimentin and Slug. This suggested that TAMs may induce the EMT of cancer cells by activating the EGFR/ERK1/2 signaling pathway in HNSCC, which may be a promising approach to suppressing cancer metastasis.

SUMMARY Melanoma patients with oncogenic BRAFV600E mutation have poor prognoses. While the role of BRAFV600E in tumorigenesis is well established, its involvement in invasion that is clinically observed in melanoma patients, remains a topic of debate. Here we show that BRAFV600E melanoma cells have extensive invasion activity as assayed by degradation of extracellular matrix, and generation of F-actin and cortactin foci that mediate membrane protrusion. Inhibition of BRAFV600E blocks melanoma cell invasion. In a BRAFV600E-driven murine melanoma model or in patients’ tumor biopsies, cortactin foci decrease upon inhibitor treatment. In addition, genome-wide expression analysis shows that a number of invadopodia-related genes are down-regulated after BRAFV600E inhibition. Mechanistically, BRAFV600E induces phosphorylation of cortactin and the exocyst subunit Exo70 through ERK, which regulates actin dynamics and matrix metalloprotease secretion, respectively. Our results provide support for the role of BRAFV600E in metastasis, and suggest that inhibiting invasion is a potential therapeutic strategy against melanoma.