Impairing hydrolase transport machinery prevents human melanoma metastasis.

Metastases are the major cause of cancer-related death, yet, molecular weaknesses that could be exploited to prevent tumor cells spreading are poorly known. Here, we found that perturbing hydrolase transport to lysosomes by blocking either the expression of IGF2R, the main receptor responsible for their trafficking, or GNPT, a transferase involved in the addition of the specific tag recognized by IGF2R, reduces melanoma invasiveness potential. Mechanistically, we demonstrate that the perturbation of this traffic, leads to a compensatory lysosome neo-biogenesis devoided of degradative enzymes. This regulatory loop relies on the stimulation of TFEB transcription factor expression. Interestingly, the inhibition of this transcription factor playing a key role of lysosome production, restores melanomas' invasive potential in the absence of hydrolase transport. These data implicate that targeting hydrolase transport in melanoma could serve to develop new therapies aiming to prevent metastasis by triggering a physiological response stimulating TFEB expression in melanoma.

Recycled melanoma-secreted melanosomes regulate tumor-associated macrophage diversification.

Extracellular vesicles (EVs) are important mediators of communication between cells. Here, we reveal a new mode of intercellular communication by melanosomes, large EVs secreted by melanocytes for melanin transport. Unlike small EVs, which are disintegrated within the receiver cell, melanosomes stay intact within them, gain a unique protein signature, and can then be further transferred to another cell as "second-hand" EVs. We show that melanoma-secreted melanosomes passaged through epidermal keratinocytes or dermal fibroblasts can be further engulfed by resident macrophages. This process leads to macrophage polarization into pro-tumor or pro-immune cell infiltration phenotypes. Melanosomes that are transferred through fibroblasts can carry AKT1, which induces VEGF secretion from macrophages in an mTOR-dependent manner, promoting angiogenesis and metastasis in vivo. In melanoma patients, macrophages that are co-localized with AKT1 are correlated with disease aggressiveness, and immunotherapy non-responders are enriched in macrophages containing melanosome markers. Our findings suggest that interactions mediated by second-hand extracellular vesicles contribute to the formation of the metastatic niche, and that blocking the melanosome cues of macrophage diversification could be helpful in halting melanoma progression.

An Exercise-Induced Metabolic Shield in Distant Organs Blocks Cancer Progression and Metastatic Dissemination.

Exercise prevents cancer incidence and recurrence, yet the underlying mechanism behind this relationship remains mostly unknown. Here we report that exercise induces the metabolic reprogramming of internal organs that increases nutrient demand and protects against metastatic colonization by limiting nutrient availability to the tumor, generating an exercise-induced metabolic shield. Proteomic and ex vivo metabolic capacity analyses of murine internal organs revealed that exercise induces catabolic processes, glucose uptake, mitochondrial activity, and GLUT expression. Proteomic analysis of routinely active human subject plasma demonstrated increased carbohydrate utilization following exercise. Epidemiologic data from a 20-year prospective study of a large human cohort of initially cancer-free participants revealed that exercise prior to cancer initiation had a modest impact on cancer incidence in low metastatic stages but significantly reduced the likelihood of highly metastatic cancer. In three models of melanoma in mice, exercise prior to cancer injection significantly protected against metastases in distant organs. The protective effects of exercise were dependent on mTOR activity, and inhibition of the mTOR pathway with rapamycin treatment ex vivo reversed the exercise-induced metabolic shield. Under limited glucose conditions, active stroma consumed significantly more glucose at the expense of the tumor. Collectively, these data suggest a clash between the metabolic plasticity of cancer and exercise-induced metabolic reprogramming of the stroma, raising an opportunity to block metastasis by challenging the metabolic needs of the tumor. SIGNIFICANCE: Exercise protects against cancer progression and metastasis by inducing a high nutrient demand in internal organs, indicating that reducing nutrient availability to tumor cells represents a potential strategy to prevent metastasis. See related commentary by Zerhouni and Piskounova, p. 4124.

Food-seeking behavior is triggered by skin ultraviolet exposure in males.

Sexual dimorphisms are responsible for profound metabolic differences in health and behavior. Whether males and females react differently to environmental cues, such as solar ultraviolet (UV) exposure, is unknown. Here we show that solar exposure induces food-seeking behavior, food intake, and food-seeking behavior and food intake in men, but not in women, through epidemiological evidence of approximately 3,000 individuals throughout the year. In mice, UVB exposure leads to increased food-seeking behavior, food intake and weight gain, with a sexual dimorphism towards males. In both mice and human males, increased appetite is correlated with elevated levels of circulating ghrelin. Specifically, UVB irradiation leads to p53 transcriptional activation of ghrelin in skin adipocytes, while a conditional p53-knockout in mice abolishes UVB-induced ghrelin expression and food-seeking behavior. In females, estrogen interferes with the p53-chromatin interaction on the ghrelin promoter, thus blocking ghrelin and food-seeking behavior in response to UVB exposure. These results identify the skin as a major mediator of energy homeostasis and may lead to therapeutic opportunities for sex-based treatments of endocrine-related diseases.

Targeting acute myeloid leukemia dependency on VCP-mediated DNA repair through a selective second-generation small-molecule inhibitor.

The development and survival of cancer cells require adaptive mechanisms to stress. Such adaptations can confer intrinsic vulnerabilities, enabling the selective targeting of cancer cells. Through a pooled in vivo short hairpin RNA (shRNA) screen, we identified the adenosine triphosphatase associated with diverse cellular activities (AAA-ATPase) valosin-containing protein (VCP) as a top stress-related vulnerability in acute myeloid leukemia (AML). We established that AML was the most responsive disease to chemical inhibition of VCP across a panel of 16 cancer types. The sensitivity to VCP inhibition of human AML cell lines, primary patient samples, and syngeneic and xenograft mouse models of AML was validated using VCP-directed shRNAs, overexpression of a dominant-negative VCP mutant, and chemical inhibition. By combining mass spectrometry-based analysis of the VCP interactome and phospho-signaling studies, we determined that VCP is important for ataxia telangiectasia mutated (ATM) kinase activation and subsequent DNA repair through homologous recombination in AML. A second-generation VCP inhibitor, CB-5339, was then developed and characterized. Efficacy and safety of CB-5339 were validated in multiple AML models, including syngeneic and patient-derived xenograft murine models. We further demonstrated that combining DNA-damaging agents, such as anthracyclines, with CB-5339 treatment synergizes to impair leukemic growth in an MLL-AF9-driven AML murine model. These studies support the clinical testing of CB-5339 as a single agent or in combination with standard-of-care DNA-damaging chemotherapy for the treatment of AML.

Tumors escape immunosurveillance by overexpressing the proteasome activator PSME3.

The success of CD8+ T cell-based cancer immunotherapy emphasizes the importance of understanding the mechanisms of generation of MHC-I peptide ligands and the possible pathways of tumor cell escape from immunosurveillance. Recently, we showed that peptides generated in the nucleus during a pioneer round of mRNA translation (pioneer translation products, or PTPs) are an important source of tumor specific peptides which correlates with the aberrant splicing and transcription events associated with oncogenesis. Here we show that up-regulation of PSME3 proteasome activator in cancer cells results in increased destruction of PTP-derived peptides in the nucleus thus enabling cancer cell to subvert immunosurveillance. These findings unveil a previously unexpected role for PSME3 in antigen processing and identify PSME3 as a druggable target to improve the efficacy of cancer immunotherapy.

Adipocytes sensitize melanoma cells to environmental TGF-ß cues by repressing the expression of miR-211.

Transforming growth factor-ß (TGF-ß) superfamily members are critical signals in tissue homeostasis and pathogenesis. Melanoma grows in the epidermis and invades the dermis before metastasizing. This disease progression is accompanied by increased sensitivity to microenvironmental TGF-ß. Here, we found that skin fat cells (adipocytes) promoted metastatic initiation by sensitizing melanoma cells to TGF-ß. Analysis of melanoma clinical samples revealed that adipocytes, usually located in the deeper hypodermis layer, were present in the upper dermis layer within proximity to in situ melanoma cells, an observation that correlated with disease aggressiveness. In a coculture system, adipocytes secreted the cytokines IL-6 and TNF-α, which induced a proliferative-to-invasive phenotypic switch in melanoma cells by repressing the expression of the microRNA miR-211. In a xenograft model, miR-211 exhibited a dual role in melanoma progression, promoting cell proliferation while inhibiting metastatic spread. Bioinformatics and molecular analyses indicated that miR-211 directly targeted and repressed the translation of TGFBR1 mRNA, which encodes the type I TGF-ß receptor. Hence, through this axis of cytokine-mediated repression of miR-211, adipocytes increased the abundance of the TGF-ß receptor in melanoma cells, thereby enhancing cellular responsiveness to TGF-ß ligands. The induction of TGF-ß signaling, in turn, resulted in a proliferative-to-invasive phenotypic switch in cultured melanoma cells. Pharmacological inhibition of TGF-ß prevented these effects. Our findings further reveal a molecular link between fat cells and metastatic progression in melanoma that might be therapeutically targeted in patients.

Mutated MITF-E87R in Melanoma Enhances Tumor Progression via S100A4.

Melanoma, a melanocyte origin neoplasm, is the most lethal type of skin cancer, and incidence is increasing. Several familial and somatic mutations have been identified in the gene encoding the melanocyte lineage master regulator, MITF; however, the neoplastic mechanisms of these mutant MITF variants are mostly unknown. Here, by performing unbiased analysis of the transcriptomes in cells expressing mutant MITF, we identified calcium-binding protein S100A4 as a downstream target of MITF-E87R. By using wild-type and mutant MITF melanoma lines, we found that both endogenous wild-type and MITF-E87R variants occupy the S100A4 promoter. Remarkably, whereas wild-type MITF represses S100A4 expression, MITF-E87R activates its transcription. The opposite effects of wild-type and mutant MITF result in opposing cellular phenotypes, because MITF-E87R via S100A4 enhanced invasion and reduced adhesion in contrast to wild-type MITF activity. Finally, we found that melanoma patients with altered S100A4 expression have poor prognosis. These data show that a change in MITF transcriptional activity from repression to activation of S100A4 that results from a point mutation in MITF alters melanoma invasive ability. These data suggest new opportunities for diagnosis and treatment of metastatic melanoma.

OCT4 impedes cell fate redirection by the melanocyte lineage master regulator MITF in mouse ESCs.

Ectopic expression of lineage master regulators induces transdifferentiation. Whether cell fate transitions can be induced during various developmental stages has not been systemically examined. Here we discover that amongst different developmental stages, mouse embryonic stem cells (mESCs) are resistant to cell fate conversion induced by the melanocyte lineage master regulator MITF. By generating a transgenic system we exhibit that in mESCs, the pluripotency master regulator Oct4, counteracts pro-differentiation induced by Mitf by physical interference with MITF transcriptional activity. We further demonstrate that mESCs must be released from Oct4-maintained pluripotency prior to ectopically induced differentiation. Moreover, Oct4 induction in various differentiated cells represses their lineage identity in vivo. Alongside, chromatin architecture combined with ChIP-seq analysis suggest that Oct4 competes with various lineage master regulators for binding promoters and enhancers. Our analysis reveals pluripotency and transdifferentiation regulatory principles and could open new opportunities in the field of regenerative medicine.

ETO2-GLIS2 Hijacks Transcriptional Complexes to Drive Cellular Identity and Self-Renewal in Pediatric Acute Megakaryoblastic Leukemia.

Chimeric transcription factors are a hallmark of human leukemia, but the molecular mechanisms by which they block differentiation and promote aberrant self-renewal remain unclear. Here, we demonstrate that the ETO2-GLIS2 fusion oncoprotein, which is found in aggressive acute megakaryoblastic leukemia, confers megakaryocytic identity via the GLIS2 moiety while both ETO2 and GLIS2 domains are required to drive increased self-renewal properties. ETO2-GLIS2 directly binds DNA to control transcription of associated genes by upregulation of expression and interaction with the ETS-related ERG protein at enhancer elements. Importantly, specific interference with ETO2-GLIS2 oligomerization reverses the transcriptional activation at enhancers and promotes megakaryocytic differentiation, providing a relevant interface to target in this poor-prognosis pediatric leukemia.

Cutting Edge: NANOG Activates Autophagy under Hypoxic Stress by Binding to BNIP3L Promoter.

Hypoxia upregulates the core pluripotency factors NANOG, SOX2, and OCT4, associated with tumor aggressiveness and resistance to conventional anticancer treatments. We have previously reported that hypoxia-induced NANOG contributed in vitro to tumor cell resistance to autologous-specific CTL and in vivo to the in situ recruitment of immune-suppressive cells. In this study, we investigated the mechanisms underlying NANOG-mediated tumor cell resistance to specific lysis under hypoxia. We demonstrated the tumor-promoting effect of hypoxia on tumor initiation into immunodeficient mice using human non-small lung carcinoma cells. We next showed a link between NANOG and autophagy activation under hypoxia because inhibition of NANOG decreased autophagy in tumor cells. Chromatin immunoprecipitation and luciferase reporter assays revealed a direct binding of NANOG to a transcriptionally active site in a BNIP3L enhancer sequence. These data establish a new link between the pluripotency factor NANOG and autophagy involved in resistance to CTL under hypoxia.

Melanoma miRNA trafficking controls tumour primary niche formation.

Melanoma originates in the epidermis and becomes metastatic after invasion into the dermis. Prior interactions between melanoma cells and dermis are poorly studied. Here, we show that melanoma cells directly affect the formation of the dermal tumour niche by microRNA trafficking before invasion. Melanocytes, cells of melanoma origin, are specialized in releasing pigment vesicles, termed melanosomes. In melanoma in situ, we found melanosome markers in distal fibroblasts before melanoma invasion. The melanosomes carry microRNAs into primary fibroblasts triggering changes, including increased proliferation, migration and pro-inflammatory gene expression, all known features of cancer-associated fibroblasts (CAFs). Specifically, melanosomal microRNA-211 directly targets IGF2R and leads to MAPK signalling activation, which reciprocally encourages melanoma growth. Melanosome release inhibitor prevented CAF formation. Since the first interaction of melanoma cells with blood vessels occurs in the dermis, our data suggest an opportunity to block melanoma invasion by preventing the formation of the dermal tumour niche.

Parkin Somatic Mutations Link Melanoma and Parkinson's Disease.

Epidemiological studies suggest a direct link between melanoma and Parkinson's disease (PD); however, the underlying molecular basis is unknown. Since mutations in Parkin are the major driver of early-onset PD and Parkin was recently reported to play a role in cancer development, we hypothesized that Parkin links melanoma and PD. By analyzing whole exome/genome sequencing of Parkin from 246 melanoma patients, we identified five non-synonymous mutations, three synonymous mutations, and one splice region variant in Parkin in 3.6% of the samples. In vitro analysis showed that wild-type Parkin plays a tumor suppressive role in melanoma development resulting in cell-cycle arrest, reduction of metabolic activity, and apoptosis. Using a mass spectrometry-based analysis, we identified potential Parkin substrates in melanoma and generated a functional protein association network. The activity of mutated Parkin was assessed by protein structure modeling and examination of Parkin E3 ligase activity. The Parkin-E28K mutation impairs Parkin ubiquitination activity and abolishes its tumor suppressive effect. Taken together, our analysis of genomic sequence and in vitro data indicate that Parkin is a potential link between melanoma and Parkinson's disease. Our findings suggest new approaches for early diagnosis and treatment against both diseases.

Interactions of Melanoma Cells with Distal Keratinocytes Trigger Metastasis via Notch Signaling Inhibition of MITF.

The most critical stage in initiation of melanoma metastasis is the radial to vertical growth transition, yet the triggers of this transition remain elusive. We suggest that the microenvironment drives melanoma metastasis independently of mutation acquisition. Here we examined the changes in microenvironment that occur during melanoma radial growth. We show that direct contact of melanoma cells with the remote epidermal layer triggers vertical invasion via Notch signaling activation, the latter serving to inhibit MITF function. Briefly, within the native Notch ligand-free microenvironment, MITF, the melanocyte lineage master regulator, binds and represses miR-222/221 promoter in an RBPJK-dependent manner. However, when radial growth brings melanoma cells into contact with distal differentiated keratinocytes that express Notch ligands, the activated Notch intracellular domain impairs MITF binding to miR-222/221 promoter. This de-repression of miR-222/221 expression triggers initiation of invasion. Our findings may direct melanoma prevention opportunities via targeting specific microenvironments.

Transcription factor/microRNA axis blocks melanoma invasion program by miR-211 targeting NUAK1.

Melanoma is one of the deadliest human cancers, responsible for approximately 80% of skin cancer mortalities. The aggressiveness of melanoma is due to its capacity to proliferate and rapidly invade surrounding tissues, leading to metastases. A recent model suggests melanoma progresses by reversibly switching between proliferation and invasion transcriptional signatures. Recent studies show that cancer cells are more sensitive to microRNA (miRNA) perturbation than are non-cancer cells; however, the roles of miRNAs in melanoma plasticity remain unexplored. Here, we use the gene expression profiles of melanoma and normal melanocytes to characterize the transcription factor-miRNA relationship that modulates the proliferative and invasive programs of melanoma. We identified two sets of miRNAs that likely regulate these programs. Interestingly, one of the miRNAs involved in melanoma invasion is miR-211, a known target of the master regulator microphthalmia-associated transcription factor (MITF). We demonstrate that miR-211 contributes to melanoma adhesion by directly targeting a gene, NUAK1. Inhibition of miR-211 increases NUAK1 expression and decreases melanoma adhesion, whereas upregulation of miR-211 restores adhesion through NUAK1 repression. This study defines the MITF/miR-211 axis that inhibits the invasive program by blocking adhesion. Furthermore, we have identified NUAK1 as a potential target for the treatment of metastatic melanoma.

Feed-forward microprocessing and splicing activities at a microRNA-containing intron.

The majority of mammalian microRNA (miRNA) genes reside within introns of protein-encoding and non-coding genes, yet the mechanisms coordinating primary transcript processing into both mature miRNA and spliced mRNA are poorly understood. Analysis of melanoma invasion suppressor miR-211 expressed from intron 6 of melastatin revealed that microprocessing of miR-211 promotes splicing of the exon 6-exon 7 junction of melastatin by a mechanism requiring the RNase III activity of Drosha. Additionally, mutations in the 5' splice site (5'SS), but not in the 3'SS, branch point, or polypyrimidine tract of intron 6 reduced miR-211 biogenesis and Drosha recruitment to intron 6, indicating that 5'SS recognition by the spliceosome promotes microprocessing of miR-211. Globally, knockdown of U1 splicing factors reduced intronic miRNA expression. Our data demonstrate novel mutually-cooperative microprocessing and splicing activities at an intronic miRNA locus and suggest that the initiation of spliceosome assembly may promote microprocessing of intronic miRNAs.

Hypoxia-induced transcriptional repression of the melanoma-associated oncogene MITF.

Microphthalmia-associated transcription factor (MITF) regulates normal melanocyte development and is also a lineage-selective oncogene implicated in melanoma and clear-cell sarcoma (i.e., melanoma of soft parts). We have observed that MITF expression is potently reduced under hypoxic conditions in primary melanocytes and melanoma and clear cell sarcoma cells through hypoxia inducible factor 1 (HIF1)-mediated induction of the transcriptional repressor differentially expressed in chondrocytes protein 1 (DEC1) (BHLHE40), which subsequently binds and suppresses the promoter of M-MITF (melanocyte-restricted MITF isoform). Correspondingly, hypoxic conditions or HIF1α stabilization achieved by using small-molecule prolyl-hydroxylase inhibitors reduced M-MITF expression, leading to melanoma cell growth arrest that was rescued by ectopic expression of M-MITF in vitro. Prolyl hydroxylase inhibition also potently suppressed melanoma growth in a mouse xenograft model. These studies illuminate a physiologic hypoxia response in pigment cells leading to M-MITF suppression, one that suggests a potential survival advantage mechanism for MITF amplification in metastatic melanoma and offers a small-molecule strategy for suppression of the MITF oncogene in vivo.

Intronic miR-211 assumes the tumor suppressive function of its host gene in melanoma.

When it escapes early detection, malignant melanoma becomes a highly lethal and treatment-refractory cancer. Melastatin is greatly downregulated in metastatic melanomas and is widely believed to function as a melanoma tumor suppressor. Here we report that tumor suppressive activity is not mediated by melastatin but instead by a microRNA (miR-211) hosted within an intron of melastatin. Increasing expression of miR-211 but not melastatin reduced migration and invasion of malignant and highly invasive human melanomas characterized by low levels of melastatin and miR-211. An unbiased network analysis of melanoma-expressed genes filtered for their roles in metastasis identified three central node genes: IGF2R, TGFBR2, and NFAT5. Expression of these genes was reduced by miR-211, and knockdown of each gene phenocopied the effects of increased miR-211 on melanoma invasiveness. These data implicate miR-211 as a suppressor of melanoma invasion whose expression is silenced or selected against via suppression of the entire melastatin locus during human melanoma progression.

Control of melanocyte differentiation by a MITF-PDE4D3 homeostatic circuit.

Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates a variety of biological processes. The magnitude and duration of cAMP expression are regulated by both production and hydrolysis. Melanocyte-stimulating hormone (MSH) plays a crucial role in pigment cell differentiation via cAMP-regulated expression of the master transcription factor MITF. We report the identification of phosphodiesterase 4D3 as a direct target of the MSH/cAMP/MITF pathway. This creates a negative feedback loop that induces refractoriness to chronic stimulation of the cAMP pathway in melanocytes. This homeostatic pathway highlights a potent mechanism controlling melanocyte differentiation that may be amenable to pharmacologic manipulation for skin cancer prevention.