Metabolic reprogramming of stromal fibroblasts by melanoma exosome microRNA favours a pre-metastatic microenvironment.
ABSTRACT: Local acidification of stroma is proposed to favour pre-metastatic niche formation but the mechanism of initiation is unclear. We investigated whether Human Melanoma-derived exosomes (HMEX) could reprogram human adult dermal fibroblasts (HADF) and cause extracellular acidification. HMEX were isolated from supernatants of six melanoma cell lines (3 BRAF V600E mutant cell lines and 3 BRAF wild-type cell lines) using ultracentrifugation or Size Exclusion Chromatography (SEC). Rapid uptake of exosomes by HADF was demonstrated following 18 hours co-incubation. Exposure of HDAF to HMEX leads to an increase in aerobic glycolysis and decrease in oxidative phosphorylation (OXPHOS) in HADF, consequently increasing extracellular acidification. Using a novel immuno-biochip, exosomal miR-155 and miR-210 were detected in HMEX. These miRNAs were present in HMEX from all six melanoma cell lines and were instrumental in promoting glycolysis and inhibiting OXPHOS in tumour cells. Inhibition of miR-155 and miR-210 activity by transfection of miRNA inhibitors into HMEX reversed the exosome-induced metabolic reprogramming of HADF. The data indicate that melanoma-derived exosomes modulate stromal cell metabolism and may contribute to the creation of a pre-metastatic niche that promotes the development of metastasis.
Project description:BACKGROUND:Cancer-associated fibroblasts (CAFs) have been widely reported to promote tumor angiogenesis. However, the underlying mechanisms of the proangiogenic switch of CAFs remain poorly understood. This study aims to clarify the mechanisms underlying the proangiogenic switch of CAFs. METHODS:NIH/3T3 cells were treated with B16 and B16F10-derived exosomes. Then the CAFs markers and proangiogenic factors were detected by RT-PCR and Western blot. CCK-8 assay, transwell migration assay, tube formation assay, and in vivo Matrigel plug assay were conducted to determine the proangiogenic capability of CAFs. Western blot and AG490 were used to investigate the role of Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway in the proangiogenic switch of CAFs. Bioinformatics analysis, luciferase reporter assay, microRNA mimic and inhibitor, and xenograft models were used to investigate the role of mmu-miR-155-5p (miR-155) in the proangiogenic switch of CAFs. RESULTS:In this study, we show that melanoma cell-secreted exosomes can induce reprogramming of fibroblasts into CAFs and that exosomal miR-155 can trigger the proangiogenic switch of CAFs. Mechanistically exosomal miR-155 can be delivered into fibroblasts and promote the expression of proangiogenic factors, including vascular endothelial growth factor A (VEGFa), fibroblast growth factor 2 (FGF2), and matrix metalloproteinase 9 (MMP9), by directly targeting suppressor of cytokine signaling 1 (SOCS1). Downregulation of SOCS1 activates JAK2/STAT3 signaling pathway and elevates the expression levels of VEGFa, FGF2, and MMP9 in fibroblasts. Treatment with exosomes containing overexpressed miR-155 can promote angiogenesis, and the reduction of miR-155 in melanoma cell-secreted exosomes alleviates angiogenesis in vitro and in vivo. CONCLUSIONS:These results demonstrate that by promoting the expression of proangiogenic factors in recipient fibroblasts via SOCS1/JAK2/STAT3 signaling pathway, melanoma cell-secreted exosomal miR-155 can induce the proangiogenic switch of CAFs. Although tumor angiogenesis is modulated by various factors, exosomal miR-155 may be a potential target for controlling melanoma angiogenesis and used to set up novel strategies to treat melanoma.
Project description:Metabolic heterogeneity is a key factor in cancer pathogenesis. We found that a subset of BRAF- and NRAS-mutant human melanomas resistant to the MEK inhibitor selumetinib displayed increased oxidative phosphorylation (OxPhos) mediated by the transcriptional coactivator PGC1?. Notably, all selumetinib-resistant cells with elevated OxPhos could be resensitized by cotreatment with the mTORC1/2 inhibitor AZD8055, whereas this combination was ineffective in resistant cell lines with low OxPhos. In both BRAF- and NRAS-mutant melanoma cells, MEK inhibition increased MITF expression, which in turn elevated levels of PGC1?. In contrast, mTORC1/2 inhibition triggered cytoplasmic localization of MITF, decreasing PGC1? expression and inhibiting OxPhos. Analysis of tumor biopsies from patients with BRAF-mutant melanoma progressing on BRAF inhibitor ± MEK inhibitor revealed that PGC1? levels were elevated in approximately half of the resistant tumors. Overall, our findings highlight the significance of OxPhos in melanoma and suggest that combined targeting of the MAPK and mTORC pathways may offer an effective therapeutic strategy to treat melanomas with this metabolic phenotype.
Project description:DNA mutation-induced activation of RAS-BRAF-MEK-ERK signaling associated with intermittent or chronic ultraviolet (UV) irradiation cannot exclusively explain the excessive increase of malignant melanoma (MM) incidence since the 1950s. Malignant conversion of a melanocyte to an MM cell and metastatic MM is associated with a steady increase in microRNA-21 (miR-21). At the epigenetic level, miR-21 inhibits key tumor suppressors of the RAS-BRAF signaling pathway enhancing proliferation and MM progression. Increased MM cell levels of miR-21 either result from endogenous upregulation of melanocytic miR-21 expression or by uptake of miR-21-enriched exogenous exosomes. Based on epidemiological data and translational evidence, this review provides deeper insights into environmentally and metabolically induced exosomal miR-21 trafficking beyond UV-irradiation in melanomagenesis and MM progression. Sources of miR-21-enriched exosomes include UV-irradiated keratinocytes, adipocyte-derived exosomes in obesity, airway epithelium-derived exosomes generated by smoking and pollution, diet-related exosomes and inflammation-induced exosomes, which may synergistically increase the exosomal miR-21 burden of the melanocyte, the transformed MM cell and its tumor environment. Several therapeutic agents that suppress MM cell growth and proliferation attenuate miR-21 expression. These include miR-21 antagonists, metformin, kinase inhibitors, beta-blockers, vitamin D, and plant-derived bioactive compounds, which may represent new options for the prevention and treatment of MM.
Project description:Oncogene addiction describes how cancer cells exhibit dependence on single oncogenes to escape apoptosis and senescence. While oncogene addiction constitutes the basis for new cancer treatment strategies targeting individual kinases and pathways activated by oncogenic mutations, the biochemical basis for this addiction is largely unknown. Here we provide evidence for a metabolic rationale behind the addiction to (V600E)BRAF in two malignant melanoma cell lines. Both cell lines display a striking addiction to glycolysis due to underlying dysfunction of oxidative phosphorylation (OXPHOS). Notably, even minor reductions in glycolytic activity lead to increased OXPHOS activity (reversed Warburg effect), however the mitochondria are unable to sustain ATP production. We show that (V600E)BRAF upholds the activity of glycolysis and therefore the addiction to glycolysis de facto becomes an addiction to (V600E)BRAF. Finally, the senescence response associated with inhibition of (V600E)BRAF is rescued by overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), providing direct evidence that oncogene addiction rests on a metabolic foundation.
Project description:We previously demonstrated an inverse correlation between tyrosinase-related protein 1 (TYRP1) mRNA expression in melanoma metastases and patient survival. However, TYRP1 protein was not detected in half of tissues expressing mRNA and did not correlate with survival. Based on a study reporting that 3' untranslated region (UTR) of TYRP1 mRNA contains two miR-155-5p (named miR-155) binding sites exhibiting single-nucleotide polymorphisms (SNPs) that promote (matched miRNA-mRNA interaction) mRNA decay or not (mismatched), we aimed to investigate the role of miR-155 in the regulation of TYRP1 mRNA expression and protein translation accounting for these SNPs.The effect of miR-155 on TYRP1 mRNA/protein expression was evaluated in two melanoma cell lines harbouring matched or mismatched miR-155-TYRP1 mRNA interaction after transfection with pre-miR-155. In parallel, 192 skin and lymph node melanoma metastases were examined for TYRP1 mRNA/protein, miR-155 and SNPs and correlated with patient survival. TYRP1 mRNA, SNPs at its 3'UTR and miR-155 were analysed by RT-qPCR, whereas TYRP1 protein was evaluated by western blot in cell lines and by immunohistochemistry in metastatic tissues.The miR-155 induced a dose-dependent TYRP1 mRNA decay and hampered its translation into protein in the line with the 'match' genotype. In melanoma metastases, TYRP1 mRNA inversely correlated with miR-155 expression but not with TYRP1 protein in the 'match' group, whereas it positively correlated with protein but not with miR-155 in the 'mismatch' group. Consequently, in the latter group, TYRP1 protein inversely correlated with survival.Polymorphisms in 3'UTR of TYRP1 mRNA can affect TYRP1 mRNA regulation by miR-155 and its subsequent translation into protein. These SNPs can render TYRP1 mRNA and protein expression nonsusceptible to miR-155 activity and disclose a prognostic value for TYRP1 protein in a subgroup of melanoma patients. These data support the interest in the prognostic value of melanogenic markers and propose TYRP1 to refine prognosis in patients with advanced disease.
Project description:BRAF mutations are detected in >50% of all melanomas. These mutations impair the LKB1-AMPK signaling, an important metabolic pathway associated with cell growth, proliferation and survival. Melanoma patients with BRAF mutations are usually treated with BRAF inhibitors such as vemurafenib, but responses are short-lived as drug resistant tumors metabolically switch to mitochondrial oxidative phosphorylation (OXPHOS) to escape metabolic stress-induced BRAF inhibition. Additionally, a large subset of melanoma utilizes OXPHOS in their metabolism, which can confer de novo resistance to BRAF inhibitors. Therefore, uncoupling of OXPHOS to perturb energy homeostasis and to indirectly stimulate AMPK could be a novel treatment for melanoma and to overcome intrinsic and acquired resistance to BRAF inhibitors. Here, we investigated the effects of SR4 and niclosamide, two small molecule mitochondria uncouplers, on the growth and proliferation of treatment-naïve and vemurafenib-resistant melanomas in vitro and in vivo. SR4 and niclosamide inhibited melanoma proliferation irrespective of BRAF/NRAS status. Melanomas with greater OXPHOS phenotype (higher OCR/ECAR), with LKB1 mutation, or with acquired resistance to vemurafenib displayed greater sensitivity to both uncouplers. More importantly, SR4 and niclosamide inhibited tumor growth in both treatment-naïve and vemurafenib-resistant xenograft mice models. Mechanistic studies indicate both uncouplers induced energetic stress, modulated the AMPK-mTOR pathway, and promoted apoptosis without affecting MEK-ERK MAPK signaling. These results suggest that uncouplers such as SR4 and niclosamide may be useful as first line treatment against melanoma regardless of BRAF/NRAS status, and as an adjuvant therapy for patients failing MAPK inhibitors.
Project description:PURPOSE:The purpose of this study is to determine if inhibition of mitochondrial oxidative phosphorylation (OxPhos) is an effective strategy against MAPK pathway inhibitor (MAPKi)-resistant BRAF-mutant melanomas.Experimental Design: The antimelanoma activity of IACS-010759 (OPi), a novel OxPhos complex I inhibitor, was evaluated in vitro and in vivo. Mechanistic studies and predictors of response were evaluated using molecularly and metabolically stratified melanoma cell lines. 13C-labeling and targeted metabolomics were used to evaluate the effect of OPi on cellular energy utilization. OxPhos inhibition in vivo was evaluated noninvasively by [18F]-fluoroazomycin arabinoside (FAZA) PET imaging. RESULTS:OPi potently inhibited OxPhos and the in vivo growth of multiple MAPKi-resistant BRAF-mutant melanoma models with high OxPhos at well-tolerated doses. In vivo tumor regression with single-agent OPi treatment correlated with inhibition of both MAPK and mTOR complex I activity. Unexpectedly, antitumor activity was not improved by combined treatment with MAPKi in vitro or in vivo. Signaling and growth-inhibitory effects were mediated by LKB1-AMPK axis, and proportional to AMPK activation. OPi increased glucose incorporation into glycolysis, inhibited glucose and glutamine incorporation into the mitochondrial tricarboxylic acid cycle, and decreased cellular nucleotide and amino acid pools. Early changes in [18F]-FAZA PET uptake in vivo, and the degree of mTORC1 pathway inhibition in vitro, correlated with efficacy. CONCLUSIONS:Targeting OxPhos with OPi has significant antitumor activity in MAPKi-resistant, BRAF-mutant melanomas, and merits further clinical investigation as a potential new strategy to overcome intrinsic and acquired resistance to MAPKi in patients.
Project description:Inflammation plays an important role in cardiac injuries. Here, we examined the role of miRNA in regulating inflammation and cardiac injury during myocardial infarction. We showed that mir-155 expression was increased in the mouse heart after myocardial infarction. Upregulated mir-155 was primarily presented in macrophages and cardiac fibroblasts of injured hearts, while pri-mir-155 was only expressed in macrophages. mir-155 was also presented in exosomes derived from macrophages, and it can be transferred into cardiac fibroblasts by macrophage-derived exosomes. A mir-155 mimic or mir-155 containing exosomes inhibited cardiac fibroblast proliferation by downregulating Son of Sevenless 1 expression and promoted inflammation by decreasing Suppressor of Cytokine Signaling 1 expression. These effects were reversed by the addition of a mir-155 inhibitor. In vivo, mir-155-deficient mice showed a significant reduction of the incidence of cardiac rupture and an improved cardiac function compared with wild-type mice. Moreover, transfusion of wild-type macrophage exosomes to mir-155-/- mice exacerbated cardiac rupture. Finally, the mir-155-deficient mice exhibited elevated fibroblast proliferation and collagen production, along with reduced cardiac inflammation in injured heart. Taken together, our results demonstrate that activated macrophages secrete mir-155-enriched exosomes and identify macrophage-derived mir-155 as a paracrine regulator for fibroblast proliferation and inflammation; thus, a mir-155 inhibitor (i.e., mir-155 antagomir) has the potential to be a therapeutic agent for reducing acute myocardial-infarction-related adverse events.
Project description:Loss of expression of surface antigens represents a significant problem for cancer immunotherapy. Microphthalmia-associated transcription factor (MITF-M) regulates melanocyte fate by driving expression of many differentiation genes, whose protein products can be recognized by cytolytic T lymphocytes. We previously reported that interleukin-1ß (IL-1ß) can downregulate MITF-M levels. Here we show that downregulation of MITF-M expression by IL-1ß was paralleled by an upregulation of miR-155 expression in four melanoma lines. We confirmed that miR-155 was able to target endogenous MITF-M in melanoma cells and demonstrated a role for miR-155 in the IL-1ß-induced repression of MITF-M by using an antagomiR. Notably, we also observed a strong negative correlation between MITF-M and miR-155 levels in a mouse model of melanoma. Taken together, our results indicate that MITF-M downregulation by inflammatory stimuli might be partly due to miR-155 upregulation. This could represent a novel mechanism of melanoma immune escape in an inflammatory microenvironment.
Project description:MicroRNA (miR)-155 is upregulated in breast cancer cells and in sera of patients with breast cancer, but its clinical relevance remains uncertain. The objective of the present effort was to address the transcriptional regulation of miR-155. A bioinformatics analysis of public datasets validated upregulation of miR-155 in tumor cells of patients with breast cancer, particularly those who were at early stages and had triple-negative cancers. The expression profiling and clinical relevance of miR-155 in tumor cells and blood cells were characterized by TaqMan miR assays and, in plasma and exosomes, by nest-quantitative PCR analysis. There was a positive correlation between expression of FOXP3 and miR-155 in breast cancer cell lines and primary breast cancers. In breast cancer cells, FOXP3 induced miR-155 through transcriptional repression of BRCA1. Furthermore, in an Alabama cohort, blood and plasma samples were collected from 259 participants, including patients with breast cancer or benign breast tumors, members of breast cancer families, and matched healthy female controls. For patients with early stage or localized breast cancer, there were high levels of miR-155 in both plasma and blood cells. In cultured breast cancer cells, expression of miR-155 was induced by FOXP3 but was not significantly changed in culture medium or exosomes, suggesting that circulating miR-155 originated from blood cells. These findings reveal a transcriptional axis of FOXP3-BRCA1-miR-155 in breast cancer cells and show that plasma miR-155 may serve as a non-invasive biomarker for detection of early stage breast cancer.