TET2-Dependent Hydroxymethylome Plasticity Reduces Melanoma Initiation and Progression.

Although numerous epigenetic aberrancies accumulate in melanoma, their contribution to initiation and progression remain unclear. The epigenetic mark 5-hydroxymethylcytosine (5hmC), generated through TET-mediated DNA modification, is now referred to as the sixth base of DNA and has recently been reported as a potential biomarker for multiple types of cancer. Loss of 5hmC is an epigenetic hallmark of melanoma, but whether a decrease in 5hmc levels contributes directly to pathogenesis or whether it merely results from disease progression-associated epigenetic remodeling remains to be established. Here, we show that NRAS-driven melanomagenesis in mice is accompanied by an overall decrease in 5hmC and specific 5hmC gains in selected gene bodies. Strikingly, genetic ablation of Tet2 in mice cooperated with oncogenic NRASQ61K to promote melanoma initiation while suppressing specific gains in 5hmC. We conclude that TET2 acts as a barrier to melanoma initiation and progression, partly by promoting 5hmC gains in specific gene bodies. SIGNIFICANCE: This work emphasizes the importance of epigenome plasticity in cancer development and highlights the involvement of druggable epigenetic factors in cancer.

The fragile X mental retardation protein regulates tumor invasiveness-related pathways in melanoma cells.

The fragile X mental retardation protein (FMRP) is lacking or mutated in patients with the fragile X syndrome (FXS), the most frequent form of inherited intellectual disability. FMRP affects metastasis formation in a mouse model for breast cancer. Here we show that FMRP is overexpressed in human melanoma with high Breslow thickness and high Clark level. Furthermore, meta-analysis of the TCGA melanoma data revealed that high levels of FMRP expression correlate significantly with metastatic tumor tissues, risk of relapsing and disease-free survival. Reduction of FMRP in metastatic melanoma cell lines impinges on cell migration, invasion and adhesion. Next-generation sequencing in human melanoma cells revealed that FMRP regulates a large number of mRNAs involved in relevant processes of melanoma progression. Our findings suggest an association between FMRP levels and the invasive phenotype in melanoma and might open new avenues towards the discovery of novel therapeutic targets.

Comparative oncogenomics identifies tyrosine kinase FES as a tumor suppressor in melanoma.

Identification and functional validation of oncogenic drivers are essential steps toward advancing cancer precision medicine. Here, we have presented a comprehensive analysis of the somatic genomic landscape of the widely used BRAFV600E- and NRASQ61K-driven mouse models of melanoma. By integrating the data with publically available genomic, epigenomic, and transcriptomic information from human clinical samples, we confirmed the importance of several genes and pathways previously implicated in human melanoma, including the tumor-suppressor genes phosphatase and tensin homolog (PTEN), cyclin dependent kinase inhibitor 2A (CDKN2A), LKB1, and others. Importantly, this approach also identified additional putative melanoma drivers with prognostic and therapeutic relevance. Surprisingly, one of these genes encodes the tyrosine kinase FES. Whereas FES is highly expressed in normal human melanocytes, FES expression is strongly decreased in over 30% of human melanomas. This downregulation correlates with poor overall survival. Correspondingly, engineered deletion of Fes accelerated tumor progression in a BRAFV600E-driven mouse model of melanoma. Together, these data implicate FES as a driver of melanoma progression and demonstrate the potential of cross-species oncogenomic approaches combined with mouse modeling to uncover impactful mutations and oncogenic driver alleles with clinical importance in the treatment of human cancer.

Deciphering the Role of Oncogenic MITFE318K in Senescence Delay and Melanoma Progression.

Background: MITF encodes an oncogenic lineage-specific transcription factor in which a germline mutation ( MITFE318K ) was identified in human patients predisposed to both nevus formation and, among other tumor types, melanoma. The molecular mechanisms underlying the oncogenic activity of MITF E318K remained uncharacterized. Methods: Here, we compared the SUMOylation status of endogenous MITF by proximity ligation assay in melanocytes isolated from wild-type (n = 3) or E318K (n = 4) MITF donors. We also used a newly generated Mitf E318K knock-in (KI) mouse model to assess the role of Mitf E318K (n = 7 to 13 mice per group) in tumor development in vivo and performed transcriptomic analysis of the tumors to identify the molecular mechanisms. Finally, using immortalized or normal melanocytes (wild-type or E318K MITF, n = 2 per group), we assessed the role of MITF E318K on the induction of senescence mediated by BRAF V600E . All statistical tests were two-sided. Results: We demonstrated a decrease in endogenous MITF SUMOylation in melanocytes from MITF E318K patients (mean of cells with hypoSUMOylated MITF, MITF E318K vs MITF WT , 94% vs 44%, difference = 50%, 95% CI = 21.8% to 67.2%, P = .004). The Mitf E318K mice were slightly hypopigmented (mean melanin content Mitf WT vs Mitf E318K/+ , 0.54 arbitrary units [AU] vs 0.36 AU, difference = -0.18, 95% CI = -0.36 to -0.007, P = .04). We provided genetic evidence that Mitf E318K enhances BRaf V600E -induced nevus formation in vivo (mean nevus number for Mitf E318K , BRaf V600E vs Mitf WT , BRaf V600E , 68 vs 44, difference = 24, 95% CI = 9.1 to 38.9, P = .006). Importantly, although Mitf E318K was not sufficient to cooperate with BRaf V600E alone in promoting metastatic melanoma, it accelerated tumor formation on a BRaf V600E , Pten-deficient background (median survival, Mitf E318K/+ = 42 days, 95% CI = 31 to 46 vs Mitf WT = 51 days, 95% CI = 50 to 55, P < .001). Transcriptome analysis suggested a decrease in senescence in tumors from Mitf E318K mice. We confirmed this hypothesis by in vitro experiments, demonstrating that Mitf E318K impaired the ability of human melanocytes to undergo BRAF V600E -induced senescence. Conclusions: We characterized the functions of melanoma-associated MITF E318K mutations. Our results demonstrate that MITF E318K reduces the program of senescence to potentially favor melanoma progression in vivo.

Regulation of Melanoma Progression through the TCF4/miR-125b/NEDD9 Cascade.

Melanoma progression from a primary lesion to a distant metastasis is a complex process associated with genetic alterations, epigenetic modifications, and phenotypic switches. Elucidation of these phenomena may indicate how to interfere with this fatal disease. The role of microRNAs as key negative regulators of gene expression, controlling all cellular processes including cell migration and invasion, is now being recognized. Here, we used in silico analysis of microRNA expression profiles of primary and metastatic melanomas and functional experiments to show that microRNA-125b (miR-125b) is a determinant candidate of melanoma progression: (i) miR-125b is more strongly expressed in aggressive metastatic than primary melanomas, (ii) there is an inverse correlation between the amount of miR-125b and overall patient survival, (iii) invasion/migration potentials in vitro are inversely correlated with the amount of miR-125b in a series of human melanoma cell lines, and (iv) inhibition of miR-125b reduces migratory and invasive potentials without affecting cell proliferation in vitro. Furthermore, we show that neural precursor cell expressed developmentally down-regulated protein 9 (i.e., NEDD9) is a direct target of miR-125b and is involved in modulating melanoma cell migration and invasion. Also, transcription factor 4, associated with epithelial-mesenchymal transition and invasion, induces the transcription of miR-125b-1. In conclusion, the transcription factor 4/miR-125b/NEDD9 cascade promotes melanoma cell migration/invasion.

Decoding the regulatory landscape of melanoma reveals TEADS as regulators of the invasive cell state.

Transcriptional reprogramming of proliferative melanoma cells into a phenotypically distinct invasive cell subpopulation is a critical event at the origin of metastatic spreading. Here we generate transcriptome, open chromatin and histone modification maps of melanoma cultures; and integrate this data with existing transcriptome and DNA methylation profiles from tumour biopsies to gain insight into the mechanisms underlying this key reprogramming event. This shows thousands of genomic regulatory regions underlying the proliferative and invasive states, identifying SOX10/MITF and AP-1/TEAD as regulators, respectively. Knockdown of TEADs shows a previously unrecognized role in the invasive gene network and establishes a causative link between these transcription factors, cell invasion and sensitivity to MAPK inhibitors. Using regulatory landscapes and in silico analysis, we show that transcriptional reprogramming underlies the distinct cellular states present in melanoma. Furthermore, it reveals an essential role for the TEADs, linking it to clinically relevant mechanisms such as invasion and resistance.

Altered E-Cadherin Levels and Distribution in Melanocytes Precede Clinical Manifestations of Vitiligo.

Vitiligo is the most common depigmenting disorder resulting from the loss of melanocytes from the basal epidermal layer. The pathogenesis of the disease is likely multifactorial and involves autoimmune causes, as well as oxidative and mechanical stress. It is important to identify early events in vitiligo to clarify pathogenesis, improve diagnosis, and inform therapy. Here, we show that E-cadherin (Ecad), which mediates the adhesion between melanocytes and keratinocytes in the epidermis, is absent from or discontinuously distributed across melanocyte membranes of vitiligo patients long before clinical lesions appear. This abnormality is associated with the detachment of the melanocytes from the basal to the suprabasal layers in the epidermis. Using human epidermal reconstructed skin and mouse models with normal or defective Ecad expression in melanocytes, we demonstrated that Ecad is required for melanocyte adhesiveness to the basal layer under oxidative and mechanical stress, establishing a link between silent/preclinical, cell-autonomous defects in vitiligo melanocytes and known environmental stressors accelerating disease expression. Our results implicate a primary predisposing skin defect affecting melanocyte adhesiveness that, under stress conditions, leads to disappearance of melanocytes and clinical vitiligo. Melanocyte adhesiveness is thus a potential target for therapy aiming at disease stabilization.

MDM4 is a key therapeutic target in cutaneous melanoma.

The inactivation of the p53 tumor suppressor pathway, which often occurs through mutations in TP53 (encoding tumor protein 53) is a common step in human cancer. However, in melanoma-a highly chemotherapy-resistant disease-TP53 mutations are rare, raising the possibility that this cancer uses alternative ways to overcome p53-mediated tumor suppression. Here we show that Mdm4 p53 binding protein homolog (MDM4), a negative regulator of p53, is upregulated in a substantial proportion (∼65%) of stage I-IV human melanomas and that melanocyte-specific Mdm4 overexpression enhanced tumorigenesis in a mouse model of melanoma induced by the oncogene Nras. MDM4 promotes the survival of human metastatic melanoma by antagonizing p53 proapoptotic function. Notably, inhibition of the MDM4-p53 interaction restored p53 function in melanoma cells, resulting in increased sensitivity to cytotoxic chemotherapy and to inhibitors of the BRAF (V600E) oncogene. Our results identify MDM4 as a key determinant of impaired p53 function in human melanoma and designate MDM4 as a promising target for antimelanoma combination therapy.

Biological and mathematical modeling of melanocyte development.

We aim to evaluate environmental and genetic effects on the expansion/proliferation of committed single cells during embryonic development, using melanoblasts as a paradigm to model this phenomenon. Melanoblasts are a specific type of cell that display extensive cellular proliferation during development. However, the events controlling melanoblast expansion are still poorly understood due to insufficient knowledge concerning their number and distribution in the various skin compartments. We show that melanoblast expansion is tightly controlled both spatially and temporally, with little variation between embryos. We established a mathematical model reflecting the main cellular mechanisms involved in melanoblast expansion, including proliferation and migration from the dermis to epidermis. In association with biological information, the model allows the calculation of doubling times for melanoblasts, revealing that dermal and epidermal melanoblasts have short but different doubling times. Moreover, the number of trunk founder melanoblasts at E8.5 was estimated to be 16, a population impossible to count by classical biological approaches. We also assessed the importance of the genetic background by studying gain- and loss-of-function ß-catenin mutants in the melanocyte lineage. We found that any alteration of ß-catenin activity, whether positive or negative, reduced both dermal and epidermal melanoblast proliferation. Finally, we determined that the pool of dermal melanoblasts remains constant in wild-type and mutant embryos during development, implying that specific control mechanisms associated with cell division ensure half of the cells at each cell division to migrate from the dermis to the epidermis. Modeling melanoblast expansion revealed novel links between cell division, cell localization within the embryo and appropriate feedback control through ß-catenin.

Melanoblast proliferation dynamics during mouse embryonic development. Modeling and validation.

In this paper, we are looking for mathematical modeling of mouse embryonic melanoblast proliferation dynamics, taking into account, the expression level of ß-catenin. This protein plays an important role into the whole signal pathway process. Different assumptions on some unobservable features lead to different candidate models. From real data measured, from biological experiments and from a priori biological knowledge, it was able to validate or invalidate some of the candidate models. Data assimilation and parameter identification allowed us to derive a mathematical model that is in very good agreement with biological data. As a result, the produced model can give tracks for biologists into their biological investigations and experimental evidence. Another interest is the use of this model for robust hidden parameter identification like double times or number of founder melanoblasts.

GLI2-mediated melanoma invasion and metastasis.

BACKGROUND: The transforming growth factor-beta (TGF-beta) pathway, which has both tumor suppressor and pro-oncogenic activities, is often constitutively active in melanoma and is a marker of poor prognosis. Recently, we identified GLI2, a mediator of the hedgehog pathway, as a transcriptional target of TGF-beta signaling. METHODS: We used real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to determine GLI2 expression in human melanoma cell lines and subsequently classified them as GLI2high or as GLI2low according to their relative GLI2 mRNA and protein expression levels. GLI2 expression was reduced in a GLI2high cell line with lentiviral expression of short hairpin RNA targeting GLI2. We assessed the role of GLI2 in melanoma cell invasiveness in Matrigel assays. We measured secretion of matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography and expression of E-cadherin by western blotting and RT-PCR. The role of GLI2 in development of bone metastases was determined following intracardiac injection of melanoma cells in immunocompromised mice (n = 5-13). Human melanoma samples (n = 79) at various stages of disease progression were analyzed for GLI2 and E-cadherin expression by immunohistochemistry, in situ hybridization, or RT-PCR. All statistical tests were two-sided. RESULTS: Among melanoma cell lines, increased GLI2 expression was associated with loss of E-cadherin expression and with increased capacity to invade Matrigel and to form bone metastases in mice (mean osteolytic tumor area: GLI2high vs GLI2low, 2.81 vs 0.93 mm(2), difference = 1.88 mm(2), 95% confidence interval [CI] = 1.16 to 2.60, P < .001). Reduction of GLI2 expression in melanoma cells that had expressed high levels of GLI2 substantially inhibited both basal and TGF-beta-induced cell migration, invasion (mean number of Matrigel invading cells: shGLI2 vs shCtrl (control), 52.6 vs 100, difference = 47.4, 95% CI = 37.0 to 57.8, P = .024; for shGLI2 + TGF-beta vs shCtrl + TGF-beta, 31.0 vs 161.9, difference = -130.9, 95% CI = -96.2 to -165.5, P = .002), and MMP secretion in vitro and the development of experimental bone metastases in mice. Within human melanoma lesions, GLI2 expression was heterogeneous, associated with tumor regions in which E-cadherin was lost and increased in the most aggressive tumors. CONCLUSION: GLI2 was directly involved in driving melanoma invasion and metastasis in this preclinical study.

{alpha}MSH and Cyclic AMP elevating agents control melanosome pH through a protein kinase A-independent mechanism.

Melanins are synthesized in melanocytes within specialized organelles called melanosomes. Numerous studies have shown that the pH of melanosome plays a key role in the regulation of melanin synthesis. However, until now, acute regulation of melanosome pH by a physiological stimulus has never been demonstrated. In the present study, we show that the activation of the cAMP pathway by alphaMSH or forskolin leads to an alkalinization of melanosomes and a concomitant regulation of vacuolar ATPases and ion transporters of the solute carrier family. The solute carrier family members include SLC45A2, which is mutated in oculocutaneous albinism type IV, SLC24A4 and SLC24A5, proteins implicated in the control of eye, hair, and skin pigmentation, and the P protein, encoded by the oculocutaneous albinism type II locus. Interestingly, H89, a pharmacological inhibitor of protein kinase A (PKA), prevents the cAMP-induced pigmentation and induces acidification of melanosomes. The drastic depigmenting effect of H89 is not due to an inhibition of tyrosinase expression. Indeed, H89 blocks the induction of melanogenesis induced by LY294002, a potent inhibitor of the PI 3-kinase pathway, without any effect on tyrosinase expression. Furthermore, PKA is not involved in the inhibition of pigmentation promoted by H89 because LY294002 induces pigmentation independently of PKA. Also, other PKA inhibitors do not affect pigmentation. Taken together, our results strengthen the support for a key role of melanosome pH in the regulation of melanin synthesis and, for the first time, demonstrate that melanosome pH is regulated by cAMP and alphaMSH. Notably, these are both mediators of the response to solar UV radiation, the main physiological stimulus of skin pigmentation.

Beta-catenin induces immortalization of melanocytes by suppressing p16INK4a expression and cooperates with N-Ras in melanoma development.

Tumor progression is a multistep process in which proproliferation mutations must be accompanied by suppression of senescence. In melanoma, proproliferative signals are provided by activating mutations in NRAS and BRAF, whereas senescence is bypassed by inactivation of the p16(Ink4a) gene. Melanomas also frequently exhibit constitutive activation of the Wnt/beta-catenin pathway that is presumed to induce proliferation, as it does in carcinomas. We show here that, contrary to expectations, stabilized beta-catenin reduces the number of melanoblasts in vivo and immortalizes primary skin melanocytes by silencing the p16(Ink4a) promoter. Significantly, in a novel mouse model for melanoma, stabilized beta-catenin bypasses the requirement for p16(Ink4a) mutations and, together with an activated N-Ras oncogene, leads to melanoma with high penetrance and short latency. The results reveal that synergy between the Wnt and mitogen-activated protein (MAP) kinase pathways may represent an important mechanism underpinning the genesis of melanoma, a highly aggressive and increasingly common disease.

Stable overexpression of Smad7 in human melanoma cells impairs bone metastasis.

Melanoma has a propensity to metastasize to bone, where it is exposed to high concentrations of transforming growth factor-beta (TGF-beta). Because TGF-beta promotes bone metastases from other solid tumors, such as breast cancer, we tested the role of TGF-beta in melanoma metastases to bone. 1205Lu melanoma cells, stably transfected to overexpress the natural TGF-beta/Smad signaling inhibitor Smad7, were studied in an experimental model of bone metastasis whereby tumor cells are inoculated into the left cardiac ventricle of nude mice. All mice bearing parental and mock-transfected 1205Lu cells developed osteolytic bone metastases 5 weeks post-tumor inoculation. Mice bearing 1205Lu-Smad7 tumors had significantly less osteolysis on radiographs and longer survival compared with parental and mock-transfected 1205Lu mice. To determine if the reduced bone metastases observed in mice bearing 1205Lu-Smad7 clones was due to reduced expression of TGF-beta target genes known to enhance metastases to bone from breast cancer cells, we analyzed gene expression of osteolytic factors, parathyroid hormone-related protein (PTHrP) and interleukin-11 (IL-11), the chemotactic receptor CXCR4, and osteopontin in 1205Lu cells. Quantitative reverse transcription-PCR analysis indicated that PTHrP, IL-11, CXCR4, and osteopontin mRNA steady-state levels were robustly increased in response to TGF-beta and that Smad7 and the TbetaRI small-molecule inhibitor, SB431542, prevented such induction. In addition, 1205Lu-Smad7 bone metastases expressed significantly lower levels of IL-11, connective tissue growth factor, and PTHrP. These data suggest that TGF-beta promotes osteolytic bone metastases due to melanoma by stimulating the expression of prometastatic factors via the Smad pathway. Blockade of TGF-beta signaling may be an effective treatment for melanoma metastasis to bone.