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.

Slow Transcription of the 99a/let-7c/125b-2 Cluster Results in Differential MiRNA Expression and Promotes Melanoma Phenotypic Plasticity.

Almost half of the human microRNAs (miRNAs) are encoded in clusters. Although transcribed as a single unit, the levels of individual mature miRNAs often differ. The mechanisms underlying differential biogenesis of clustered miRNAs and the resulting physiological implications are mostly unknown. In this study, we report that the melanoma master transcription regulator MITF regulates the differential expression of the 99a/let-7c/125b-2 cluster by altering the distribution of RNA polymerase II along the cluster. We discovered that MITF interacts with TRIM28, a known inhibitor of RNA polymerase II transcription elongation, at the mIR-let-7c region, resulting in the pausing of RNA polymerase II activity and causing an elevation in mIR-let-7c expression; low levels of RNA polymerase II occupation over miR-99a and miR-125b-2 regions decreases their biogenesis. Furthermore, we showed that this differential expression affects the phenotypic state of melanoma cells. RNA-sequencing analysis of proliferative melanoma cells that express miR-99a and miR-125b mimics revealed a transcriptomic shift toward an invasive phenotype. Conversely, expression of a mIR-let-7c mimic in invasive melanoma cells induced a shift to a more proliferative state. We confirmed direct target genes of these miRNAs, including FGFR3, BAP1, Bcl2, TGFBR1, and CDKN1A. Our study demonstrates an MITF-governed biogenesis mechanism that results in differential expression of clustered 99a/let-7c/125b-2 miRNAs that control melanoma progression.

DNA methylation directs microRNA biogenesis in mammalian cells.

MicroRNA (miRNA) biogenesis initiates co-transcriptionally, but how the Microprocessor machinery pinpoints the locations of short precursor miRNA sequences within long flanking regions of the transcript is not known. Here we show that miRNA biogenesis depends on DNA methylation. When the regions flanking the miRNA coding sequence are highly methylated, the miRNAs are more highly expressed, have greater sequence conservation, and are more likely to drive cancer-related phenotypes than miRNAs encoded by unmethylated loci. We show that the removal of DNA methylation from miRNA loci leads to their downregulation. Further, we found that MeCP2 binding to methylated miRNA loci halts RNA polymerase II elongation, leading to enhanced processing of the primary miRNA by Drosha. Taken together, our data reveal that DNA methylation directly affects miRNA biogenesis.

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.

UV-Protection Timer Controls Linkage between Stress and Pigmentation Skin Protection Systems.

Skin sun exposure induces two protection programs: stress responses and pigmentation, the former within minutes and the latter only hours afterward. Although serving the same physiological purpose, it is not known whether and how these programs are coordinated. Here, we report that UVB exposure every other day induces significantly more skin pigmentation than the higher frequency of daily exposure, without an associated increase in stress responses. Using mathematical modeling and empirical studies, we show that the melanocyte master regulator, MITF, serves to synchronize stress responses and pigmentation and, furthermore, functions as a UV-protection timer via damped oscillatory dynamics, thereby conferring a trade-off between the two programs. MITF oscillations are controlled by multiple negative regulatory loops, one at the transcriptional level involving HIF1α and another post-transcriptional loop involving microRNA-148a. These findings support trait linkage between the two skin protection programs, which, we speculate, arose during furless skin evolution to minimize skin damage.

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.

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.

Enhancer methylation dynamics contribute to cancer plasticity and patient mortality.

During development, enhancers play pivotal roles in regulating gene expression programs; however, their involvement in cancer progression has not been fully characterized. We performed an integrative analysis of DNA methylation, RNA-seq, and small RNA-seq profiles from thousands of patients, including 25 diverse primary malignances and seven body sites of metastatic melanoma. We found that enhancers are consistently the most differentially methylated regions (DMR) as cancer progresses from normal to primary tumors and then to metastases, compared to other genomic features. Remarkably, identification of enhancer DMRs (eDMRs) enabled classification of primary tumors according to physiological organ systems, and in metastasis eDMRs are the most correlated with patient outcome. To further understand the eDMR role in cancer progression, we developed a model to predict genes and microRNAs that are regulated by enhancer and not promotor methylation, which shows high accuracy with chromatin architecture methods and was experimentally validated. Interestingly, among all metastatic melanoma eDMRs, the most correlated with patient survival were eDMRs that "switched" their methylation patterns back and forth between normal, primary, and metastases and target cancer drivers, e.g., KIT We further demonstrated that eDMR target genes were modulated in melanoma by the bone metastasis microenvironment, suggesting that eDMRs respond to microenvironmental cues in metastatic niches. Our findings that aberrant methylation in cancer cells mostly affects enhancers, which contribute to tumor progression and cancer cell plasticity, will facilitate development of epigenetic anticancer approaches.