Regulation of nucleo-cytosolic 26S proteasome translocation by aromatic amino acids via mTOR is essential for cell survival under stress.

The proteasome is responsible for removal of ubiquitinated proteins. Although several aspects of its regulation (e.g., assembly, composition, and post-translational modifications) have been unraveled, studying its adaptive compartmentalization in response to stress is just starting to emerge. We found that following amino acid starvation, the proteasome is translocated from its large nuclear pool to the cytoplasm-a response regulated by newly identified mTOR-agonistic amino acids-Tyr, Trp, and Phe (YWF). YWF relay their signal upstream of mTOR through Sestrin3 by disrupting its interaction with the GATOR2 complex. The triad activates mTOR toward its downstream substrates p62 and transcription factor EB (TFEB), affecting both proteasomal and autophagic activities. Proteasome translocation stimulates cytosolic proteolysis which replenishes amino acids, thus enabling cell survival. In contrast, nuclear sequestration of the proteasome following mTOR activation by YWF inhibits this proteolytic adaptive mechanism, leading to cell death, which establishes this newly identified pathway as a key stress-coping mechanism.

Adipocyte extracellular vesicles carry enzymes and fatty acids that stimulate mitochondrial metabolism and remodeling in tumor cells.

Extracellular vesicles are emerging key actors in adipocyte communication. Notably, small extracellular vesicles shed by adipocytes stimulate fatty acid oxidation and migration in melanoma cells and these effects are enhanced in obesity. However, the vesicular actors and cellular processes involved remain largely unknown. Here, we elucidate the mechanisms linking adipocyte extracellular vesicles to metabolic remodeling and cell migration. We show that adipocyte vesicles stimulate melanoma fatty acid oxidation by providing both enzymes and substrates. In obesity, the heightened effect of extracellular vesicles depends on increased transport of fatty acids, not fatty acid oxidation-related enzymes. These fatty acids, stored within lipid droplets in cancer cells, drive fatty acid oxidation upon being released by lipophagy. This increase in mitochondrial activity redistributes mitochondria to membrane protrusions of migrating cells, which is necessary to increase cell migration in the presence of adipocyte vesicles. Our results provide key insights into the role of extracellular vesicles in the metabolic cooperation that takes place between adipocytes and tumors with particular relevance to obesity.

Comparison of Drosophila melanogaster Embryo and Adult Proteome by SWATH-MS Reveals Differential Regulation of Protein Synthesis, Degradation Machinery, and Metabolism Modules.

An important area of modern biology consists of understanding the relationship between genotype and phenotype. However, to understand this relationship it is essential to investigate one of the principal links between them: the proteome. With the development of recent mass-spectrometry approaches, it is now possible to quantify entire proteomes and thus relate them to different phenotypes. Here, we present a comparison of the proteome of two extreme developmental states in the well-established model organism Drosophila melanogaster: adult and embryo. Protein modules such as ribosome, proteasome, tricarboxylic acid cycle, glycolysis, or oxidative phosphorylation were found differentially expressed between the two developmental stages. Analysis of post-translation modifications of the proteins identified in this study indicates that they generally follow the same trend as their corresponding protein. Comparison between changes in the proteome and the transcriptome highlighted patterns of post-transcriptional regulation for the subunits of protein complexes such as the ribosome and the proteasome, whereas protein from modules such as TCA cycle, glycolysis, and oxidative phosphorylation seem to be coregulated at the transcriptional level. Finally, the impact of the endosymbiont Wolbachia pipientis on the proteome of both developmental states was also investigated.

Adipocytes promote breast cancer resistance to chemotherapy, a process amplified by obesity: role of the major vault protein (MVP).

INTRODUCTION: Clinical studies suggest that obesity, in addition to promoting breast cancer aggressiveness, is associated with a decrease in chemotherapy efficacy, although the mechanisms involved remain elusive. As chemotherapy is one of the main treatments for aggressive or metastatic breast cancer, we investigated whether adipocytes can mediate resistance to doxorubicin (DOX), one of the main drugs used to treat breast cancer, and the mechanisms associated. METHODS: We used a coculture system to grow breast cancer cells with in vitro differentiated adipocytes as well as primary mammary adipocytes isolated from lean and obese patients. Drug cellular accumulation, distribution, and efflux were studied by immunofluorescence, flow cytometry, and analysis of extracellular vesicles. Results were validated by immunohistochemistry in a series of lean and obese patients with cancer. RESULTS: Adipocytes differentiated in vitro promote DOX resistance (with cross-resistance to paclitaxel and 5-fluorouracil) in a large panel of human and murine breast cancer cell lines independently of their subtype. Subcellular distribution of DOX was altered in cocultivated cells with decreased nuclear accumulation of the drug associated with a localized accumulation in cytoplasmic vesicles, which then are expelled into the extracellular medium. The transport-associated major vault protein (MVP), whose expression was upregulated by adipocytes, mediated both processes. Coculture with human mammary adipocytes also induced chemoresistance in breast cancer cells (as well as the related MVP-induced DOX efflux) and their effect was amplified by obesity. Finally, in a series of human breast tumors, we observed a gradient of MVP expression, which was higher at the invasive front, where tumor cells are at close proximity to adipocytes, than in the tumor center, highlighting the clinical relevance of our results. High expression of MVP in these tumor cells is of particular interest since they are more likely to disseminate to give rise to chemoresistant metastases. CONCLUSIONS: Collectively, our study shows that adipocytes induce an MVP-related multidrug-resistant phenotype in breast cancer cells, which could contribute to obesity-related chemoresistance.

A new role for extracellular vesicles: how small vesicles can feed tumors' big appetite.

Cancer cells must adapt their metabolism in order to meet the energy requirements for cell proliferation, survival in nutrient-deprived environments, and dissemination. In particular, FA metabolism is emerging as a critical process for tumors. FA metabolism can be modulated through intrinsic changes in gene expression or signaling between tumor cells and also in response to signals from the surrounding microenvironment. Among these signals, extracellular vesicles (EVs) could play an important role in FA metabolism remodeling. In this review, we will present the role of EVs in tumor progression and especially in metabolic reprogramming. Particular attention will be granted to adipocytes. These cells, which are specialized in storing and releasing FAs, are able to shift tumor metabolism toward the use of FAs and, subsequently, increase tumor aggressiveness. Recent work demonstrates the involvement of EVs in this metabolic symbiosis.

Identification of the hypoxia-inducible factor 2α nuclear interactome in melanoma cells reveals master proteins involved in melanoma development.

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors that play a key role in cellular adaptation to hypoxia. HIF proteins are composed of an α subunit regulated by oxygen pressure (essentially HIF1α or HIF2α) and a constitutively expressed ß subunit. These proteins are often overexpressed in cancer cells, and HIF overexpression frequently correlates with poor prognosis, making HIF proteins promising therapeutic targets. HIF proteins are involved in melanoma initiation and progression; however, the specific function of HIF2 in melanoma has not yet been studied comprehensively. Identifying protein complexes is a valuable way to uncover protein function, and affinity purification coupled with mass spectrometry and label-free quantification is a reliable method for this approach. We therefore applied quantitative interaction proteomics to identify exhaustively the nuclear complexes containing HIF2α in a human melanoma cell line, 501mel. We report, for the first time, a high-throughput analysis of the interactome of an HIF subunit. Seventy proteins were identified that interact with HIF2α, including some well-known HIF partners and some new interactors. The new HIF2α partners microphthalmia-associated transcription factor, SOX10, and AP2α, which are master actors of melanoma development, were confirmed via co-immunoprecipitation experiments. Their ability to bind to HIF1α was also tested: microphthalmia-associated transcription factor and SOX10 were confirmed as HIF1α partners, but the transcription factor AP2α was not. AP2α expression correlates with low invasive capacities. Interestingly, we demonstrated that when HIF2α was overexpressed, only cells expressing large amounts of AP2α exhibited decreased invasive capacities in hypoxia relative to normoxia. The simultaneous presence of both transcription factors therefore reduces cells' invasive properties. Knowledge of the HIF2α interactome is thus a useful resource for investigating the general mechanisms of HIF function and regulation, and here we reveal unexpected, distinct roles for the HIF1 and HIF2 isoforms in melanoma progression.

Tryptophanyl-Transfer RNA Synthetase Is Involved in a Negative Feedback Loop Mitigating Interferon-γ-Induced Gene Expression.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes responsible for linking a transfer RNA (tRNA) with its cognate amino acid present in all the kingdoms of life. Besides their aminoacyl-tRNA synthetase activity, it was described that many of these enzymes can carry out non-canonical functions. They were shown to be involved in important biological processes such as metabolism, immunity, development, angiogenesis and tumorigenesis. In the present work, we provide evidence that tryptophanyl-tRNA synthetase might be involved in a negative feedback loop mitigating the expression of certain interferon-γ-induced genes. Mining the available TCGA and Gtex data, we found that WARS was highly expressed in cutaneous melanoma (SKCM) compared to other cancers and is of good prognosis for this particular cancer type. WARS expression correlates with genes involved in antigen processing and presentation but also transcription factors involved in IFN-γ signaling such as STAT1. In addition, WARS was found in complex with STAT1 in A375 cells treated with IFN-γ. Finally, we showed that knocking down WARS expression during IFN-γ stimulation further increases the expression of GBP2, APOL1, ISG15, HLA-A and IDO1.

Proteolipidic composition of exosomes changes during reticulocyte maturation.

During the orchestrated process leading to mature erythrocytes, reticulocytes must synthesize large amounts of hemoglobin, while eliminating numerous cellular components. Exosomes are small secreted vesicles that play an important role in this process of specific elimination. To understand the mechanisms of proteolipidic sorting leading to their biogenesis, we have explored changes in the composition of exosomes released by reticulocytes during their differentiation, in parallel to their physical properties. By combining proteomic and lipidomic approaches, we found dramatic alterations in the composition of the exosomes retrieved over the course of a 7-day in vitro differentiation protocol. Our data support a previously proposed model, whereby in reticulocytes the biogenesis of exosomes involves several distinct mechanisms for the preferential recruitment of particular proteins and lipids and suggest that the respective prominence of those pathways changes over the course of the differentiation process.

Arginyl-tRNA-protein transferase 1 (ATE1) promotes melanoma cell growth and migration.

Arginyl-tRNA-protein transferase 1 (ATE1) catalyses N-terminal protein arginylation, a post-translational modification implicated in cell migration, invasion and the cellular stress response. Herein, we report that ATE1 is overexpressed in NRAS-mutant melanomas, while it is downregulated in BRAF-mutant melanomas. ATE1 expression was higher in metastatic tumours, compared with primary tumours. Consistent with these findings, ATE1 depletion reduced melanoma cell viability, migration and colony formation. Reduced ATE1 expression also affected cell responses to mTOR and MEK inhibitors and to serum deprivation. Among putative ATE1 substrates is the tumour suppressor AXIN1, pointing to the possibility that ATE1 may fine-tune AXIN1 function in melanoma. Our findings highlight an unexpected role for ATE1 in melanoma cell aggressiveness and suggest that ATE1 constitutes a potential new therapeutic target.

Adipocyte Extracellular Vesicles Decrease p16INK4A in Melanoma: An Additional Link between Obesity and Cancer.

Obesity is a recognized factor for increased risk and poor prognosis of many cancers, including melanoma. In this study, using genetically engineered mouse models of melanoma (NrasQ61K transgenic expression, associated or not with Cdkn2a heterozygous deletion), we show that obesity increases melanoma initiation and progression by supporting tumor growth and metastasis, thereby reducing survival. This effect is associated with a decrease in p16INK4A expression in tumors. Mechanistically, adipocytes downregulate p16INK4A in melanoma cells through ß-catenin-dependent regulation, which increases cell motility. Furthermore, ß-catenin is directly transferred from adipocytes to melanoma cells in extracellular vesicles, thus increasing its level and activity, which represses CDKN2A transcription. Adipocytes from individuals with obesity have a stronger effect than those from lean individuals, mainly owing to an increase in the number of vesicles secreted, thus increasing the amount of ß-catenin delivered to melanoma cells and, consequently, amplifying their effect. In conclusion, in this study, we reveal that adipocyte extracellular vesicles control p16INK4A expression in melanoma, which promotes tumor progression. This work expands our understanding of the cooperation between adipocytes and tumors, particularly in obesity.

The ubiquitin ligase RNF5 determines acute myeloid leukemia growth and susceptibility to histone deacetylase inhibitors.

Acute myeloid leukemia (AML) remains incurable, largely due to its resistance to conventional treatments. Here, we find that increased abundance of the ubiquitin ligase RNF5 contributes to AML development and survival. High RNF5 expression in AML patient specimens correlates with poor prognosis. RNF5 inhibition decreases AML cell growth in culture, in patient-derived xenograft (PDX) samples and in vivo, and delays development of MLL-AF9-driven leukemogenesis in mice, prolonging their survival. RNF5 inhibition causes transcriptional changes that overlap with those seen upon histone deacetylase (HDAC)1 inhibition. RNF5 induces the formation of K29 ubiquitin chains on the histone-binding protein RBBP4, promoting its recruitment to and subsequent epigenetic regulation of genes involved in AML maintenance. Correspondingly, RNF5 or RBBP4 knockdown enhances AML cell sensitivity to HDAC inhibitors. Notably, low expression of both RNF5 and HDAC coincides with a favorable prognosis. Our studies identify an ERAD-independent role for RNF5, demonstrating that its control of RBBP4 constitutes an epigenetic pathway that drives AML, and highlight RNF5/RBBP4 as markers useful to stratify patients for treatment with HDAC inhibitors.

Regulation of eIF2α by RNF4 Promotes Melanoma Tumorigenesis and Therapy Resistance.

Among the hallmarks of melanoma are impaired proteostasis and rapid development of resistance to targeted therapy that represent a major clinical challenge. However, the molecular machinery that links these processes is unknown. Here we describe that by stabilizing key melanoma oncoproteins, the ubiquitin ligase RNF4 promotes tumorigenesis and confers resistance to targeted therapy in melanoma cells, xenograft mouse models, and patient samples. In patients, RNF4 protein and mRNA levels correlate with poor prognosis and with resistance to MAPK inhibitors. Remarkably, RNF4 tumorigenic properties, including therapy resistance, require the translation initiation factor initiation elongation factor alpha (eIF2α). RNF4 binds, ubiquitinates, and stabilizes the phosphorylated eIF2α (p-eIF2α) but not activating transcription factor 4 or C/EBP homologous protein that mediates the eIF2α-dependent integrated stress response. In accordance, p-eIF2α levels were significantly elevated in high-RNF4 patient-derived melanomas. Thus, RNF4 and p-eIF2α establish a positive feed-forward loop connecting oncogenic translation and ubiquitin-dependent protein stabilization in melanoma.

SPANX Control of Lamin A/C Modulates Nuclear Architecture and Promotes Melanoma Growth.

Mechanisms regulating nuclear organization control fundamental cellular processes, including the cell and chromatin organization. Their disorganization, including aberrant nuclear architecture, has been often implicated in cellular transformation. Here, we identify Lamin A, among proteins essential for nuclear architecture, as SPANX (sperm protein associated with the nucleus on the X chromosome), a cancer testis antigen previously linked to invasive tumor phenotypes, interacting protein in melanoma. SPANX interaction with Lamin A was mapped to the immunoglobulin fold-like domain, a region critical for Lamin A function, which is often mutated in laminopathies. SPANX downregulation in melanoma cell lines perturbed nuclear organization, decreased cell viability, and promoted senescence-associated phenotypes. Moreover, SPANX knockdown (KD) in melanoma cells promoted proliferation arrest, a phenotype mediated in part by IRF3/IL1A signaling. SPANX KD in melanoma cells also prompted the secretion of IL1A, which attenuated the proliferation of naïve melanoma cells. Identification of SPANX as a nuclear architecture complex component provides an unexpected insight into the regulation of Lamin A and its importance in melanoma. IMPLICATIONS: SPANX, a testis protein, interacts with LMNA and controls nuclear architecture and melanoma growth.

Obesity and melanoma: could fat be fueling malignancy?

Over the last decade, it has become increasingly clear that adipose tissue, and particularly adipocytes, contributes to tumor progression. Obesity, an ever-increasing worldwide phenomenon, exacerbates this effect. The influence of obesity on melanoma remains poorly studied, although recent data do underline an association between the two diseases in both humans and murine models. Herein, we review the impact of obesity on melanoma incidence and progression and discuss the underlying mechanisms known to be involved. Adipose tissue favors the proliferation and aggressiveness of melanoma cells through a direct dialog, mediated by soluble factors and by exosomes, and through remodeling of the tumor microenvironment. This knowledge could, in the future, help to design new personalized therapeutic options for obese melanoma patients.

Mammary adipocytes stimulate breast cancer invasion through metabolic remodeling of tumor cells.

In breast cancer, a key feature of peritumoral adipocytes is their loss of lipid content observed both in vitro and in human tumors. The free fatty acids (FFAs), released by adipocytes after lipolysis induced by tumor secretions, are transferred and stored in tumor cells as triglycerides in lipid droplets. In tumor cell lines, we demonstrate that FFAs can be released over time from lipid droplets through an adipose triglyceride lipase-dependent (ATGL-dependent) lipolytic pathway. In vivo, ATGL is expressed in human tumors where its expression correlates with tumor aggressiveness and is upregulated by contact with adipocytes. The released FFAs are then used for fatty acid ß-oxidation (FAO), an active process in cancer but not normal breast epithelial cells, and regulated by coculture with adipocytes. However, in cocultivated cells, FAO is uncoupled from ATP production, leading to AMPK/acetyl-CoA carboxylase activation, a circle that maintains this state of metabolic remodeling. The increased invasive capacities of tumor cells induced by coculture are completely abrogated by inhibition of the coupled ATGL-dependent lipolysis/FAO pathways. These results show a complex metabolic symbiosis between tumor-surrounding adipocytes and cancer cells that stimulate their invasiveness, highlighting ATGL as a potential therapeutic target to impede breast cancer progression.

Adipocyte Exosomes Promote Melanoma Aggressiveness through Fatty Acid Oxidation: A Novel Mechanism Linking Obesity and Cancer.

Malignant progression results from a dynamic cross-talk between stromal and cancer cells. Recent evidence suggests that this cross-talk is mediated to a significant extent by exosomes, nanovesicles secreted by most cell types and which allow the transfer of proteins, lipids, and nucleic acids between cells. Adipocytes are a major component of several tumor microenvironments, including that of invasive melanoma, where cells have migrated to the adipocyte-rich hypodermic layer of the skin. We show that adipocytes secrete exosomes in abundance, which are then taken up by tumor cells, leading to increased migration and invasion. Using mass spectrometry, we analyzed the proteome of adipocyte exosomes. Interestingly, these vesicles carry proteins implicated in fatty acid oxidation (FAO), a feature highly specific to adipocyte exosomes. We further show that, in the presence of adipocyte exosomes, FAO is increased in melanoma cells. Inhibition of this metabolic pathway completely abrogates the exosome-mediated increase in migration. Moreover, in obese mice and humans, both the number of exosomes secreted by adipocytes as well as their effect on FAO-dependent cell migration are amplified. These observations might in part explain why obese melanoma patients have a poorer prognosis than their nonobese counterparts. Cancer Res; 76(14); 4051-7. ©2016 AACR.

Proteome characterization of melanoma exosomes reveals a specific signature for metastatic cell lines.

Exosomes are important mediators in cell-to-cell communication and, recently, their role in melanoma progression has been brought to light. Here, we characterized exosomes secreted by seven melanoma cell lines with varying degrees of aggressivity. Extensive proteomic analysis of their exosomes confirmed the presence of characteristic exosomal markers as well as melanoma-specific antigens and oncogenic proteins. Importantly, the protein composition differed among exosomes from different lines. Exosomes from aggressive cells contained specific proteins involved in cell motility, angiogenesis, and immune response, while these proteins were less abundant or absent in exosomes from less aggressive cells. Interestingly, when exposed to exosomes from metastatic lines, less aggressive cells increased their migratory capacities, likely due to transfer of pro-migratory exosomal proteins to recipient cells. Hence, this study shows that the specific protein composition of melanoma exosomes depends on the cells' aggressivity and suggests that exosomes influence the behavior of other tumor cells and their microenvironment.