Ataxia Telangiectasia Mutated Signaling Delays Skin Pigmentation upon UV Exposure by Mediating MITF Function toward DNA Repair Mode.

Skin pigmentation is paused after sun exposure; however, the mechanism behind this pausing is unknown. In this study, we found that the UVB-induced DNA repair system, led by the ataxia telangiectasia mutated (ATM) protein kinase, represses MITF transcriptional activity of pigmentation genes while placing MITF in DNA repair mode, thus directly inhibiting pigment production. Phosphoproteomics analysis revealed ATM to be the most significantly enriched pathway among all UVB-induced DNA repair systems. ATM inhibition in mouse or human skin, either genetically or chemically, induces pigmentation. Upon UVB exposure, MITF transcriptional activation is blocked owing to ATM-dependent phosphorylation of MITF on S414, which modifies MITF activity and interactome toward DNA repair, including binding to TRIM28 and RBBP4. Accordingly, MITF genome occupancy is enriched in sites of high DNA damage that are likely repaired. This suggests that ATM harnesses the pigmentation key activator for the necessary rapid, efficient DNA repair, thus optimizing the chances of the cell surviving. Data are available from ProteomeXchange with the identifier PXD041121.

Primary Melanoma miRNA Trafficking Induces Lymphangiogenesis.

Melanoma, the deadliest cutaneous tumor, initiates within the epidermis; during progression, cells invade into the dermis and become metastatic through the lymphatic and blood system. Before melanoma cell invasion into the dermis, an increased density of dermal lymphatic vessels is observed, generated by a mechanism which is not fully understood. In this study, we show that, while at the primary epidermal stage (in situ), melanoma cells secrete extracellular vesicles termed melanosomes, which are uptaken by dermal lymphatic cells, leading to transcriptional and phenotypic pro-lymphangiogenic changes. Mechanistically, melanoma-derived melanosomes traffic mature let-7i to lymphatic endothelial cells, which mediate pro-lymphangiogenic phenotypic changes by the induction of type I IFN signaling. Furthermore, transcriptome analysis upon treatment with melanosomes or let-7i reveals the enhancement of IFI6 expression in lymphatic cells. Because melanoma cells metastasize primarily via lymphatic vessels, our data suggest that blocking lymphangiogenesis by repressing either melanosome release or type I IFN signaling will prevent melanoma progression to the deadly metastatic stage.

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.

Skin exposure to UVB light induces a skin-brain-gonad axis and sexual behavior.

Ultraviolet (UV) light affects endocrinological and behavioral aspects of sexuality via an unknown mechanism. Here we discover that ultraviolet B (UVB) exposure enhances the levels of sex-steroid hormones and sexual behavior, which are mediated by the skin. In female mice, UVB exposure increases hypothalamus-pituitary-gonadal axis hormone levels, resulting in larger ovaries; extends estrus days; and increases anti-Mullerian hormone (AMH) expression. UVB exposure also enhances the sexual responsiveness and attractiveness of females and male-female interactions. Conditional knockout of p53 specifically in skin keratinocytes abolishes the effects of UVB. Thus, UVB triggers a skin-brain-gonadal axis through skin p53 activation. In humans, solar exposure enhances romantic passion in both genders and aggressiveness in men, as seen in analysis of individual questionaries, and positively correlates with testosterone level. Our findings suggest opportunities for treatment of sex-steroid-related dysfunctions.

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.

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.

Negative Regulatory Loop between Microphthalmia-Associated Transcription Factor (MITF) and Notch Signaling.

Melanoma, a melanocyte-origin neoplasm, is a highly metastatic and treatment-resistance cancer. While it is well established that notch signaling activation promotes melanoma progression, little is known about the reciprocal interactions between Notch signaling and melanoma-specific pathways. Here we reveal a negative regulatory loop between Notch signaling and microphthalmia-associated transcription factor (MITF), the central regulator of melanoma progression and the driver of melanoma plasticity. We further demonstrate that Notch signaling activation, in addition to the known competition-based repression mechanism of MITF transcriptional activity, inhibits the transcription of MITF, leading to a decrease in MITF expression. We also found that MITF binds to the promoter of the gene encoding the master regulator of Notch signaling, recombination signal binding protein J kappa (RBPJK), leading to its upregulation. Our findings suggest that, once activated, Notch signaling represses MITF signaling to maintain the melanoma invasiveness and metastatic phenotype.

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.

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.

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.

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.

A widespread role of the motif environment in transcription factor binding across diverse protein families.

Transcriptional regulation requires the binding of transcription factors (TFs) to short sequence-specific DNA motifs, usually located at the gene regulatory regions. Interestingly, based on a vast amount of data accumulated from genomic assays, it has been shown that only a small fraction of all potential binding sites containing the consensus motif of a given TF actually bind the protein. Recent in vitro binding assays, which exclude the effects of the cellular environment, also demonstrate selective TF binding. An intriguing conjecture is that the surroundings of cognate binding sites have unique characteristics that distinguish them from other sequences containing a similar motif that are not bound by the TF. To test this hypothesis, we conducted a comprehensive analysis of the sequence and DNA shape features surrounding the core-binding sites of 239 and 56 TFs extracted from in vitro HT-SELEX binding assays and in vivo ChIP-seq data, respectively. Comparing the nucleotide content of the regions around the TF-bound sites to the counterpart unbound regions containing the same consensus motifs revealed significant differences that extend far beyond the core-binding site. Specifically, the environment of the bound motifs demonstrated unique sequence compositions, DNA shape features, and overall high similarity to the core-binding motif. Notably, the regions around the binding sites of TFs that belong to the same TF families exhibited similar features, with high agreement between the in vitro and in vivo data sets. We propose that these unique features assist in guiding TFs to their cognate binding sites.

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.

Human disease locus discovery and mapping to molecular pathways through phylogenetic profiling.

Genes with common profiles of the presence and absence in disparate genomes tend to function in the same pathway. By mapping all human genes into about 1000 clusters of genes with similar patterns of conservation across eukaryotic phylogeny, we determined that sets of genes associated with particular diseases have similar phylogenetic profiles. By focusing on those human phylogenetic gene clusters that significantly overlap some of the thousands of human gene sets defined by their coexpression or annotation to pathways or other molecular attributes, we reveal the evolutionary map that connects molecular pathways and human diseases. The other genes in the phylogenetic clusters enriched for particular known disease genes or molecular pathways identify candidate genes for roles in those same disorders and pathways. Focusing on proteins coevolved with the microphthalmia-associated transcription factor (MITF), we identified the Notch pathway suppressor of hairless (RBP-Jk/SuH) transcription factor, and showed that RBP-Jk functions as an MITF cofactor.

Predictive value of TP53 fluorescence in situ hybridization in cytogenetic subgroups of acute myeloid leukemia.

Acute myeloid leukemia (AML) with a complex karyotype (CK) has frequent alterations in TP53 and a very poor prognosis. We examined whether a prompt and simple fluorescence in situ hybridization (FISH) analysis for 17p13 deletion at diagnosis has a predictive value for response to therapy and overall survival in subgroups of AML. In 15 patients with a normal karyotype the TP53 FISH analysis was normal, whereas in 16 patients with CK 75% had only one copy of the TP53 allele. The deletion was also detected in 33% of six patients with monosomy or partial monosomy of chromosome 5, 7, 9, or 12. This loss of TP53 correlated significantly with a poor response to chemotherapy, and the median survival time of these patients was shorter. TP53 FISH analysis carried out at diagnosis has a predictive value with respect to chemotherapy response and can therefore facilitate a rapid decision on treatment strategies.

The human Frizzled 6 (HFz6) acts as a negative regulator of the canonical Wnt. beta-catenin signaling cascade.

Previously we have cloned the human Frizzled 1 (HFz1) and shown that it transmits the Wnt-3a-induced canonical pathway. We also cloned the human Frizzled 6 (HFz6) and show in the present study that, as opposed to HFz1, HFz6 did not activate the canonical Wnt pathway following exposure to various Wnts, whether belonging to the Wnt-1 or to the Wnt-5a group. Moreover we show that HFz6 repressed Wnt-3a-induced canonical signaling when co-expressed with HFz1. HFz6 repressed the canonical Wnt cascade activated also by various Wnt signaling intracellular mediators such as Dishevelled-1, a stabilized beta-catenin(S33Y) mutant, and LiCl-mediated repression of glycogen synthase kinase-3beta activity. Removal of HFz6 N'- or C'-terminal sequences abolished HFz6 repressive activity. As the HFz6 repressive effect was not associated with a decrease in the level of beta-catenin, it is suggested that HFz6 does not affect beta-catenin stabilization, implying that HFz6 transmits a repressive signaling that cross-talks with and inhibits the canonical Wnt pathway downstream of beta-catenin destruction complex. HFz6 did not affect the level of nuclear T-cell factor 4 (TCF4) nor did it affect beta-catenin.TCF4 complex formation. However, electrophoretic mobility shift assays indicated that HFz6 repressed the binding of TCF/lymphoid enhancer factor transcription factors to target DNA. Moreover we present data suggesting that HFz6 activates the transforming growth factor-beta-activated kinase-NEMO-like kinase pathway that blocks TCF/lymphoid enhancer factor binding to target promoters, thereby inhibiting the ability of beta-catenin to activate transcription of Wnt target genes.