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Cancer metastasis requires the transient activation of cellular programs enabling dissemination and seeding in distant organs. Genetic, transcriptional and translational intra-tumor heterogeneity contributes to this dynamic process. Beyond this, metabolic intra-tumor heterogeneity has also been observed, yet its role for cancer progression remains largely elusive. Here, we discovered that intra-tumor heterogeneity in phosphoglycerate dehydrogenase (PHGDH) protein expression drives breast cancer cell dissemination and metastasis formation. Specifically, we observed intra-tumor heterogeneous PHGDH expression in primary breast tumors, with low PHGDH expression being indicative of metastasis in patients. In mice, Phgdh protein, but not mRNA, expression is low in circulating tumor cells and early metastatic lesions, leading to increased dissemination and metastasis formation. Mechanistically, low PHGDH protein expression induces an imbalance in glycolysis that can activate sialic acid synthesis. Consequently, cancer cells undergo a partial EMT and show increased p38 as well as SRC phosphorylation, which activate cellular programs of dissemination. In turn, inhibition of sialic acid synthesis through knock-out of cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS) counteracts the increased cancer cell dissemination and metastasis induced by low PHGDH expression. In conclusion, we find that heterogeneity in PHGDH protein expression promotes cancer cell dissemination and metastasis formation.

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In cancer cells, microRNAs (miRNAs) are often aberrantly expressed resulting in impaired mRNA translation. In this study we show that miR-193b and miR-30c-1* inhibit, whereas miR-576-5p accelerates invasion of various human melanoma cell lines. Using Boyden chamber invasion assays the effect of selected miRNAs on the invasive capacity of various human melanoma cell lines was analyzed. Upon gene expression profiling performed on transfected A375 cells, CTGF, THBS1, STMN1, BCL9, RAC1 and MCL1 were identified as potential targets. For target validation, qPCR, Western blot analyses or luciferase reporter assays were applied. This study reveals opposed effects of miR-193b / miR-30c-1* and miR-576-5p, respectively, on melanoma cell invasion and on expression of BCL9 and MCL1, possibly accounting for the contrasting invasive phenotypes observed in A375 cells transfected with these miRNAs. The miRNAs studied and their targets identified fit well into a model proposed by us explaining the regulation of invasion associated genes and the observed opposed phenotypes as a result of networked direct and indirect miRNA / target interactions. The results of this study suggest miR-193b and miR-30c-1* as tumor-suppressive miRNAs, whereas miR-576-5p appears as potential tumor-promoting oncomiR. Thus, miR-193b and miR-30c-1* mimics as well as antagomiRs directed against miR-576-5p might become useful tools in future therapy approaches against advanced melanoma.

Background The regulatory functions of microRNAs (miRNAs) in anti‐tumour immunity have been mainly described in immune effector cells. Since little is known about miRNA effects on the susceptibility of target cells during T cell—target cell interaction, this study focused on the identification of miRNAs expressed in tumour cells controlling their susceptibility to CD8 + T cell‐mediated cytotoxicity. Methods Luciferase expressing B16F10 melanoma (B16F10 Luci + ) cells transfected with individual miRNAs covering a comprehensive murine miRNA library were screened for their susceptibility to lysis by an established cytotoxic T lymphocyte (CTL) line (5a, clone Nβ) specific for the melanoma‐associated antigen tyrosinase‐related protein 2. miRNAs with the most pronounced effects on T cell‐mediated lysis were validated and stably expressed in B16F10 cells. In silico analyses identified common targets of miRNA sets determined by the screen, which were further confirmed by small interfering RNA (siRNA)‐mediated silencing experiments modulating immune surveillance. The Ingenuity Pathway Analysis (IPA) software and RNA sequencing (RNA‐seq) data from miRNA‐overexpressing cell lines were applied to investigate the underlying mechanisms. The Cancer Genome Atlas (TCGA)‐derived miRNA sequencing data were used to assess the correlation of miRNA expression with melanoma patients’ survival. Results The miRNA screen resulted in the selection of seven miRNAs enhancing CTL‐mediated melanoma cell killing in vitro. Upon stable overexpression of selected miRNAs, hsa‐miR‐320a‐3p, mmu‐miR‐7037‐5p and mmu‐miR‐666‐3p were determined as most effective in enhancing susceptibility to CTL lysis. In silico analyses and subsequent siRNA‐mediated silencing experiments identified Psmc3 and Ndufa1 as common miRNA targets possibly involved in the functional effects observed. The analyses of RNA‐seq data with IPA showed pathways, networks, biological functions and key molecules potentially involved in the miRNA‐mediated functional effects. Finally, based on TCGA data analysis, a positive correlation of the conserved miRNAs among the panel of the seven identified miRNAs with overall survival of melanoma patients was determined. Conclusions For the first time, this study uncovered miRNA species that affect the susceptibility of melanoma cells to T cell‐mediated killing. These miRNAs might represent attractive candidates for novel therapy approaches against melanoma and other tumour entities.

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Cytotoxic T cells dynamically rewire their metabolism during the course of an immune response. While T cell metabolism has been extensively studied at phenotypic endpoints of activation and differentiation, the underlying dynamics remain largely elusive. Here, we leverage on single-cell RNA-sequencing (scRNA-seq) measurements of in vitro activated and differentiated CD8+ T cells cultured in physiological media to resolve these metabolic dynamics. We find that our scRNA-seq analysis identifies most metabolic changes previously defined in in vivo experiments, such as a rewiring from an oxidative to an anabolism-promoting metabolic program during activation to an effector state, which is later reverted upon memory polarization. Importantly, our scRNA-seq data further provide a dynamic description of these changes. In this sense, our data predict a differential time-dependent reliance of CD8+ T cells on the synthesis versus uptake of various non-essential amino acids during T cell activation, which we corroborate with additional functional in vitro experiments. We further exploit our scRNA-seq data to identify metabolic genes that could potentially dictate the outcome of T cell differentiation, by ranking them based on their expression dynamics. Among the highest-ranked hits, we find asparagine synthetase (Asns), whose expression sharply peaks for effector CD8+ T cells and further decays towards memory polarization. We then confirm that these in vitro Asns expression dynamics are representative of an in vivo situation in a mouse model of viral infection. Moreover, we find that disrupting these expression dynamics in vitro, by depleting asparagine from the culture media, delays central-memory polarization. Accordingly, we find that preventing the decay of ASNS by stable overexpression at the protein level in vivo leads to a significant increase in effector CD8+ T cell expansion, and a concomitant decrease in central-memory formation, in a mouse model of viral infection. This shows that ASNS expression dynamics dictate the fate of CD8+ T cell differentiation. In conclusion, we provide a resource of dynamic expression changes during CD8+ T cell activation and differentiation that is expected to increase our understanding of the dynamic metabolic requirements of T cells progressing along the immune response cascade.