Neoantigen Targetability in Progressive Advanced Melanoma.

PURPOSE: The availability of (neo)antigens and the infiltration of tumors by (neo)antigen-specific T cells are crucial factors in cancer immunotherapy. In this study, we aimed to investigate the targetability of (neo)antigens in advanced progessive melanoma and explore the potential for continued T-cell-based immunotherapy. EXPERIMENTAL DESIGN: We examined a cohort of eight patients with melanoma who had sequential metastases resected at early and later time points. Antigen-presenting capacity was assessed using IHC and flow cytometry. T-cell infiltration was quantified through multiplex immunofluorescence. Whole-exome and RNA sequencing were conducted to identify neoantigens and assess the expression of neoantigens and tumor-associated antigens. Mass spectrometry was used to evaluate antigen presentation. Tumor recognition by autologous T cells was assessed by coculture assays with cell lines derived from the metastatic lesions. RESULTS: We observed similar T-cell infiltration in paired early and later metastatic (LM) lesions. Although elements of the antigen-presenting machinery were affected in some LM lesions, both the early and later metastasis-derived cell lines were recognized by autologous T cells. At the genomic level, the (neo)antigen landscape was dynamic, but the (neo)antigen load was stable between paired lesions. CONCLUSIONS: Our findings indicate that subsequently isolated tumors from patients with late-stage melanoma retain sufficient antigen-presenting capacity, T-cell infiltration, and a stable (neo)antigen load, allowing recognition of tumor cells by T cells. This indicates a continuous availability of T-cell targets in metastases occurring at different time points and supports further exploration of (neo)antigen-specific T-cell-based therapeutic approaches for advanced melanoma.

Epithelial-Mesenchymal Plasticity Induced by Discontinuous Exposure to TGFß1 Promotes Tumour Growth.

Transitions between epithelial and mesenchymal cellular states (EMT/MET) contribute to cancer progression. We hypothesize that EMT followed by MET promotes cell population heterogeneity, favouring tumour growth. We developed an EMT model by on and off exposure of epithelial EpH4 cells (E-cells) to TGFß1 that mimics phenotypic EMT (M-cells) and MET. We aimed at understanding whether phenotypic MET is accompanied by molecular and functional reversion back to epithelia by using RNA sequencing, immunofluorescence (IF), proliferation, wound healing, focus formation and mamosphere formation assays as well as cell xenografts in nude mice. Phenotypic reverted epithelial cells (RE-cells) obtained after MET induction presented epithelial morphologies and proliferation rates resembling E cells. However, the RE transcriptomic profile and IF staining of epithelial and mesenchymal markers revealed a uniquely heterogeneous mixture of cell subpopulations with a high self-renewal ability. RE cell heterogeneity was stably maintained for long periods after TGFß1 removal both in vitro and in large tumours derived from the nude mice. Overall, we show that phenotypic reverted epithelial cells (RE cells) do not return to the molecular and functional epithelial state and present mesenchymal features related to aggressiveness and cellular heterogeneity that favour tumour growth in vivo. This work strengthens epithelial cell reprogramming and cellular heterogeneity fostered by inflammatory cues as a tumour growth-promoting factor in vivo.

Altered propionate metabolism contributes to tumour progression and aggressiveness.

The alteration of metabolic pathways is a critical strategy for cancer cells to attain the traits necessary for metastasis in disease progression. Here, we find that dysregulation of propionate metabolism produces a pro-aggressive signature in breast and lung cancer cells, increasing their metastatic potential. This occurs through the downregulation of methylmalonyl coenzyme A epimerase (MCEE), mediated by an extracellular signal-regulated kinase 2-driven transcription factor Sp1/early growth response protein 1 transcriptional switch driven by metastatic signalling at its promoter level. The loss of MCEE results in reduced propionate-driven anaplerotic flux and intracellular and intratumoral accumulation of methylmalonic acid, a by-product of propionate metabolism that promotes cancer cell invasiveness. Altogether, we present a previously uncharacterized dysregulation of propionate metabolism as an important contributor to cancer and a valuable potential target in the therapeutic treatment of metastatic carcinomas.

Metabolic Stress Triggers Immune Escape by Tumors.

There is major interest in understanding the role of the tumor microenvironment in immune escape. In a recent report, Marijt et al. shed light on the signaling cascade that leads to decreased surface expression of MHC class I by tumor cells triggered by low oxygen and glucose availability, revealing new opportunities for therapeutic intervention.

OXPHOS dysfunction regulates integrin-ß1 modifications and enhances cell motility and migration.

Mitochondria are central organelles for cellular metabolism. In cancer cells, mitochondrial oxidative phosphorylation (OXPHOS) dysfunction has been shown to promote migration, invasion, metastization and apoptosis resistance. With the purpose of analysing the effects of OXPHOS dysfunction in cancer cells and the molecular players involved, we generated cybrid cell lines harbouring either wild-type (WT) or mutant mitochondrial DNA (mtDNA) [tRNAmut cybrids, which harbour the pathogenic A3243T mutation in the leucine transfer RNA gene (tRNAleu)]. tRNAmut cybrids exhibited lower oxygen consumption and higher glucose consumption and lactate production than WT cybrids. tRNAmut cybrids displayed increased motility and migration capacities, which were associated with altered integrin-ß1 N-glycosylation, in particular with higher levels of ß-1,6-N-acetylglucosamine (GlcNAc) branched N-glycans. This integrin-ß1 N-glycosylation pattern was correlated with higher levels of membrane-bound integrin-ß1 and also with increased binding to fibronectin. When cultured in vitro, tRNAmut cybrids presented lower growth rate than WT cybrids, however, when injected in nude mice, tRNAmut cybrids produced larger tumours and showed higher metastatic potential than WT cybrids. We conclude that mtDNA-driven OXPHOS dysfunction correlates with increased motility and migration capacities, through a mechanism that may involve the cross talk between cancer cell mitochondria and the extracellular matrix.

GNAQ and BRAF mutations show differential activation of the mTOR pathway in human transformed cells.

Somatic mutations in GNAQ gene were described as being the main oncogenic activation in uveal melanomas, whereas mutations in BRAF gene have been described as a key genetic alteration that contributes to skin melanoma development. We have previously reported differential activation of the MAPK and AKT/mTOR signalling pathways in uveal and skin melanomas harbouring, respectively, GNAQ and BRAF mutations. The aim of this work was to compare the functional effect of GNAQ and BRAF mutations in mTOR and MAPK pathway activation, cell proliferation and apoptosis. In this work, we performed transient transfection of HEK293 cells with BRAF(WT), BRAF(V 600E), GNAQ(WT), GNAQ(Q209P) and GNAQ(Q209L) vectors. We treated melanoma cell lines displaying different BRAF and GNAQ mutational status with the mTOR inhibitor RAD001 and with the MEK1/2 inhibitor U0126 and evaluated the effects in the growth of the cell lines and in mTOR and MAPK pathway effectors expression. At variance with the significant increase in the level of pmTOR Ser2448 and pS6 Ser235/236 proteins observed in cells transfected with BRAF vectors, no significant alteration in mTOR pathway effectors was observed in cells transfected with the three GNAQ expressing vectors. Also, GNAQ overexpression enhances Stat3 activation, which might mediate GNAQ oncogenic effects. None of the vectors led to significant differences in proliferation or apoptosis in the transfected cell lines. Cell lines harbouring a BRAF mutation were more sensitive to RAD001 treatment. U0126 leads to the reduction of MAPK and mTOR pathways activation in all cell lines tested. Our results indicate that GNAQ and BRAF activation drive distinct intracellular signalling pathways that may be useful for therapeutic decisions in human melanomas.