We have previously reported a model for melanoma progression in which oscillation between melanoma cell phenotypes characterized by invasion or proliferation is fundamental to tumor heterogeneity and disease progression. In this study we examine the possible role of hypoxia as one of the microenvironmental influences driving metastatic progression by promoting a switch from a proliferative to an invasive phenotype. Immunohistochemistry on primary human cutaneous melanoma biopsies showed intratumoral heterogeneity for cells expressing melanocytic markers, and a loss of these markers correlated with hypoxic regions. Furthermore, we show that the downregulation of melanocytic markers is dependent on hypoxia inducible factor 1α (HIF1α), a known regulator of the hypoxic response. In vitro invasion assays showed that a hypoxic environment increases the invasiveness of proliferative melanoma cell cultures in a HIF1α-dependent manner. In contrast, invasive phenotype melanoma cells showed no increase in invasive potential upon exposure to hypoxia. Thus, exposure of proliferative melanoma cells to hypoxic microenvironments is sufficient, in a HIF1α-dependent manner, to downregulate melanocytic marker expression and increase their invasive potential.

Many metastatic melanoma patients experience durable responses to anti-PD1 and/or anti-CTLA4; however, a significant proportion (over 50%) do not benefit from the therapies. In this study, we sought to assess pretreatment liquid biopsies for biomarkers that may correlate with response to checkpoint blockade. We measured the combinatorial diversity evenness of the T-cell receptor (TCR) repertoire (the DE 50 , with low values corresponding to more clonality and lack of TCR diversity) in pretreatment peripheral blood mononuclear cells from melanoma patients treated with anti-CTLA4 (n ¼ 42) or anti-PD1 (n ¼ 38) using a multi-N-plex PCR assay on genomic DNA (gDNA). A receiver operating characteristic curve determined the optimal threshold for a dichotomized analysis according to objective responses as defined by RECIST1.1. Correlations between treatment outcome, clinical variables, and DE 50 were assessed in multivariate regression models and confirmed with Fisher exact tests. In samples obtained prior to treatment initiation, we showed that low DE 50 values were predictive of a longer progression-free survival and good responses to PD-1 blockade, but, on the other hand, predicted a poor response to CTLA4 inhibition. Multivariate logistic regression models identified DE 50 as the only independent predictive factor for response to anti-CTLA4 therapy (P ¼ 0.03) and anti-PD1 therapy (P ¼ 0.001). Fisher exact tests confirmed the association of low DE 50 with response in the anti-CTLA4 (P ¼ 0.041) and the anti-PD1 cohort (P ¼ 0.0016). Thus, the evaluation of basal TCR repertoire diversity in peripheral blood, using a PCRbased method, could help predict responses to anti-PD1 and anti-CTLA4 therapies.

BackgroundMelanoma is the most fatal skin cancer displaying a high degree of molecular heterogeneity. Phenotype switching is a mechanism that contributes to melanoma heterogeneity by altering transcription profiles for the transition between states of proliferation/differentiation and invasion/stemness. As phenotype switching is reversible, epigenetic mechanisms, like DNA methylation, could contribute to the changes in gene expression.ResultsIntegrative analysis of methylation and gene expression datasets of five proliferative and five invasion melanoma cell cultures reveal two distinct clusters. SOX9 is methylated and lowly expressed in the highly proliferative group. SOX9 overexpression results in decreased proliferation but increased invasion in vitro. In a B16 mouse model, sox9 overexpression increases the number of lung metastases. Transcriptional analysis of SOX9-overexpressing melanoma cells reveals enrichment in epithelial to mesenchymal transition (EMT) pathways. Survival analysis of The Cancer Genome Atlas melanoma dataset shows that metastatic patients with high expression levels of SOX9 have significantly worse survival rates. Additional survival analysis on the targets of SOX9 reveals that most SOX9 downregulated genes have survival benefit for metastatic patients.ConclusionsOur genome-wide DNA methylation and gene expression study of 10 early passage melanoma cell cultures reveals two phenotypically distinct groups. One of the genes regulated by DNA methylation between the two groups is SOX9. SOX9 induces melanoma cell invasion and metastasis and decreases patient survival. A number of genes downregulated by SOX9 have a negative impact on patient survival. In conclusion, SOX9 is an important gene involved in melanoma invasion and negatively impacts melanoma patient survival.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0594-4) contains supplementary material, which is available to authorized users.