CLEC12B Decreases Melanoma Proliferation by Repressing Signal Transducer and Activator of Transcription 3.

The potential role of CLEC12B, a gene predominantly expressed by skin melanocytes discovered through transcriptomic analysis, in melanoma is unknown. In this study, we show that CLEC12B expression is lower in melanoma and melanoma metastases than in melanocytes and benign melanocytic lesions and that its decrease correlates with poor prognosis. We further show that CLEC12B recruits SHP2 phosphatase through its immunoreceptor tyrosine-based inhibition motif domain, inactivates signal transducer and activator of transcription 1/3/5, increases p53/p21/p27 expression/activity, and modulates melanoma cell proliferation. The growth of human melanoma cells overexpressing CLEC12B in nude mice after subcutaneous injection is significantly decreased compared with that in the vehicle control group and is associated with decreased signal transducer and activator of transcription 3 phosphorylation and increased p53 levels in the tumors. Reducing the level of CLEC12B had the opposite effect. We show that CLEC12B represses the activation of the signal transducer and activator of transcription pathway and negatively regulates the cell cycle, providing a proliferative asset to melanoma cells.

Dual Covalent Inhibition of PKM and IMPDH Targets Metabolism in Cutaneous Metastatic Melanoma.

Overcoming acquired drug resistance is a primary challenge in cancer treatment. Notably, more than 50% of patients with BRAFV600E cutaneous metastatic melanoma (CMM) eventually develop resistance to BRAF inhibitors. Resistant cells undergo metabolic reprogramming that profoundly influences therapeutic response and promotes tumor progression. Uncovering metabolic vulnerabilities could help suppress CMM tumor growth and overcome drug resistance. Here we identified a drug, HA344, that concomitantly targets two distinct metabolic hubs in cancer cells. HA344 inhibited the final and rate-limiting step of glycolysis through its covalent binding to the pyruvate kinase M2 (PKM2) enzyme, and it concurrently blocked the activity of inosine monophosphate dehydrogenase, the rate-limiting enzyme of de novo guanylate synthesis. As a consequence, HA344 efficiently targeted vemurafenib-sensitive and vemurafenib-resistant CMM cells and impaired CMM xenograft tumor growth in mice. In addition, HA344 acted synergistically with BRAF inhibitors on CMM cell lines in vitro. Thus, the mechanism of action of HA344 provides potential therapeutic avenues for patients with CMM and a broad range of different cancers. SIGNIFICANCE: Glycolytic and purine synthesis pathways are often deregulated in therapy-resistant tumors and can be targeted by the covalent inhibitor described in this study, suggesting its broad application for overcoming resistance in cancer.

Germline variants in exonic regions have limited impact on immune checkpoint blockade clinical outcomes in advanced melanoma.

Immune checkpoint inhibition (ICI) treatments improve outcomes for metastatic melanoma; however, up to 60% of treated patients do not respond to ICI and/or develop immune-related adverse events (irAEs). Currently, robust and reliable biomarker to predict response and/or occurrence of irAEs to ICI are missing. Herein, we wanted to explore whether germline variants (SNPs) could predict the clinical outcomes of melanoma patients treated with ICIs. We performed a whole exome sequencing using gDNA isolated from blood, from a discovery cohort of 57 patients with metastatic melanoma. The top associations were then tested in a validation cohort of 57 patients. Our work suggests that individual germline genetic variants have no or weak impact on the response to ICIs. Only, variants in IL1RL1 have a significant impact in treatment response. The role of IL1RL1 in the immune response against melanoma and as a theranostic marker warrants further investigations.

FBXO32 links ubiquitination to epigenetic reprograming of melanoma cells.

Ubiquitination by serving as a major degradation signal of proteins, but also by controlling protein functioning and localization, plays critical roles in most key cellular processes. Here, we show that MITF, the master transcription factor in melanocytes, controls ubiquitination in melanoma cells. We identified FBXO32, a component of the SCF E3 ligase complex as a new MITF target gene. FBXO32 favors melanoma cell migration, proliferation, and tumor development in vivo. Transcriptomic analysis shows that FBXO32 knockdown induces a global change in melanoma gene expression profile. These include the inhibition of CDK6 in agreement with an inhibition of cell proliferation and invasion upon FBXO32 silencing. Furthermore, proteomic analysis identifies SMARC4, a component of the chromatin remodeling complexes BAF/PBAF, as a FBXO32 partner. FBXO32 and SMARCA4 co-localize at loci regulated by FBXO32, such as CDK6 suggesting that FBXO32 controls transcription through the regulation of chromatin remodeling complex activity. FBXO32 and SMARCA4 are the components of a molecular cascade, linking MITF to epigenetics, in melanoma cells.

Single-cell RNA sequencing reveals intratumoral heterogeneity in primary uveal melanomas and identifies HES6 as a driver of the metastatic disease.

Intratumor heterogeneity has been recognized in numerous cancers as a major source of metastatic dissemination. In uveal melanomas, the existence and identity of specific subpopulations, their biological function and their contribution to metastasis remain unknown. Here, in multiscale analyses using single-cell RNA sequencing of six different primary uveal melanomas, we uncover an intratumoral heterogeneity at the genomic and transcriptomic level. We identify distinct transcriptional cell states and diverse tumor-associated populations in a subset of the samples. We also decipher a gene regulatory network underlying an invasive and poor prognosis state driven in part by the transcription factor HES6. HES6 heterogenous expression has been validated by RNAscope assays within primary human uveal melanomas, which further unveils the existence of these cells conveying a dismal prognosis in tumors diagnosed with a favorable outcome using bulk analyses. Depletion of HES6 impairs proliferation, migration and metastatic dissemination in vitro and in vivo using the chick chorioallantoic membrane assay, demonstrating the essential role of HES6 in uveal melanomas. Thus, single-cell analysis offers an unprecedented view of primary uveal melanoma heterogeneity, identifies bona fide biomarkers for metastatic cells in the primary tumor, and reveals targetable modules driving growth and metastasis formation. Significantly, our findings demonstrate that HES6 is a valid target to stop uveal melanoma progression.

ITGBL1 is a new immunomodulator that favors development of melanoma tumors by inhibiting natural killer cells cytotoxicity.

Resistances to immunotherapies remains a major hurdle towards a cure for melanoma in numerous patients. An increase in the mesenchymal phenotype and a loss of differentiation have been clearly associated with resistance to targeted therapies. Similar phenotypes have been more recently also linked to resistance to immune checkpoint therapies. We demonstrated here that the loss of MIcrophthalmia associated Transcription Factor (MITF), a pivotal player in melanocyte differentiation, favors the escape of melanoma cells from the immune system. We identified Integrin beta-like protein 1 (ITGBL1), a secreted protein, upregulated in anti-PD1 resistant patients and in MITFlow melanoma cells, as the key immunomodulator. ITGBL1 inhibited immune cell cytotoxicity against melanoma cells by inhibiting NK cells cytotoxicity and counteracting beneficial effects of anti-PD1 treatment, both in vitro and in vivo. Mechanistically, MITF inhibited RUNX2, an activator of ITGBL1 transcription. Interestingly, VitaminD3, an inhibitor of RUNX2, improved melanoma cells to death by immune cells. In conclusion, our data suggest that inhibition of ITGBL1 might improve melanoma response to immunotherapies.

Discovery of a new molecule inducing melanoma cell death: dual AMPK/MELK targeting for novel melanoma therapies.

In the search of biguanide-derived molecules against melanoma, we have discovered and developed a series of bioactive products and identified the promising new compound CRO15. This molecule exerted anti-melanoma effects on cells lines and cells isolated from patients including the ones derived from tumors resistant to BRAF inhibitors. Moreover, CRO15 was able to decrease viability of cells lines from a broad range of cancer types. This compound acts by two distinct mechanisms. First by activating the AMPK pathway induced by a mitochondrial disorder. Second by inhibition of MELK kinase activity, which induces cell cycle arrest and activation of DNA damage repair pathways by p53 and REDD1 activation. All of these mechanisms activate autophagic and apoptotic processes resulting in melanoma cell death. The strong efficacy of CRO15 to reduce the growth of melanoma xenograft sensitive or resistant to BRAF inhibitors opens interesting perspective.

Innate lymphocyte-induced CXCR3B-mediated melanocyte apoptosis is a potential initiator of T-cell autoreactivity in vitiligo.

T-cells play a crucial role in progression of autoimmunity, including vitiligo, yet the initial steps triggering their activation and tissue damage remain unknown. Here we demonstrate increased presence of type-1 innate lymphoid cells (NK and ILC1)-producing interferon gamma (IFNγ) in the blood and in non-lesional skin of vitiligo patients. Melanocytes of vitiligo patients have strong basal expression of chemokine-receptor-3 (CXCR3) isoform B which is directly regulated by IFNγ. CXCR3B activation by CXCL10 at the surface of cultured human melanocytes induces their apoptosis. The remaining melanocytes, activated by the IFNγ production, express co-stimulatory markers which trigger T-cell proliferation and subsequent anti-melanocytic immunity. Inhibiting the CXCR3B activation prevents this apoptosis and the further activation of T cells. Our results emphasize the key role of CXCR3B in apoptosis of melanocytes and identify CXCR3B as a potential target to prevent and to treat vitiligo by acting at the early stages of melanocyte destruction.

Uncovering and deciphering the pro-invasive role of HACE1 in melanoma cells.

HACE1 is an E3 ubiquitin ligase described as a tumour suppressor because HACE1-knockout mice develop multi-organ, late-onset cancers and because HACE1 expression is lost in several neoplasms, such as Wilms' tumours and colorectal cancer. However, a search of public databases indicated that HACE1 expression is maintained in melanomas. We demonstrated that HACE1 promoted melanoma cell migration and adhesion in vitro and was required for mouse lung colonisation by melanoma cells in vivo. Transcriptomic analysis of HACE1-depleted melanoma cells revealed an inhibition of ITGAV and ITGB1 as well changes in other genes involved in cell migration. We revealed that HACE1 promoted the K27 ubiquitination of fibronectin and regulated its secretion. Secreted fibronectin regulated ITGAV and ITGB1 expression, as well as melanoma cell adhesion and migration. Our findings disclose a novel molecular cascade involved in the regulation of fibronectin secretion, integrin expression and melanoma cell adhesion. By controlling this cascade, HACE1 displays pro-tumoural properties and is an important regulator of melanoma cell invasive properties.

Pivotal role of NAMPT in the switch of melanoma cells toward an invasive and drug-resistant phenotype.

In BRAFV600E melanoma cells, a global metabolomic analysis discloses a decrease in nicotinamide adenine dinucleotide (NAD+) levels upon PLX4032 treatment that is conveyed by a STAT5 inhibition and a transcriptional regulation of the nicotinamide phosphoribosyltransferase (NAMPT) gene. NAMPT inhibition decreases melanoma cell proliferation both in vitro and in vivo, while forced NAMPT expression renders melanoma cells resistant to PLX4032. NAMPT expression induces transcriptomic and epigenetic reshufflings that steer melanoma cells toward an invasive phenotype associated with resistance to targeted therapies and immunotherapies. Therefore, NAMPT, the key enzyme in the NAD+ salvage pathway, appears as a rational target in targeted therapy-resistant melanoma cells and a key player in phenotypic plasticity of melanoma cells.

Focus on cutaneous and uveal melanoma specificities.

Cutaneous melanoma (CM) and uveal melanoma (UM) derive from cutaneous and uveal melanocytes that share the same embryonic origin and display the same cellular function. However, the etiopathogenesis and biological behaviors of these melanomas are very different. CM and UM display distinct landscapes of genetic alterations and show different metastatic routes and tropisms. Hence, therapeutic improvements achieved in the last few years for the treatment of CM have failed to ameliorate the clinical outcomes of patients with UM. The scope of this review is to discuss the differences in tumorigenic processes (etiologic factors and genetic alterations) and tumor biology (gene expression and signaling pathways) between CM and UM. We develop hypotheses to explain these differences, which might provide important clues for research avenues and the identification of actionable vulnerabilities suitable for the development of new therapeutic strategies for metastatic UM.

Visfatin expression analysis in association with recruitment and activation of human and rodent brown and brite adipocytes.

Human brown adipocytes are able to burn fat and glucose and are now considered as a potential strategy to treat obesity, type 2 diabetes and metabolic disorders. Besides their thermogenic function, brown adipocytes are able to secrete adipokines. One of these is visfatin, a nicotinamide phosphoribosyltransferase involved in nicotinamide dinucleotide synthesis, which is known to participate in the synthesis of insulin by pancreatic ß cells. In a therapeutic context, it is of interest to establish whether a potential correlation exists between brown adipocyte activation and/or brite adipocyte recruitment, and adipokine expression. We analyzed visfatin expression, as a pre-requisite to its secretion, in rodent and human biopsies and cell models of brown/brite adipocytes. We found that visfatin was preferentially expressed in mature adipocytes and that this expression was higher in brown adipose tissue of rodents compared to other fat depots. However, using various rodent models we were unable to find any correlation between visfatin expression and brown or brite adipocyte activation or recruitment. Interestingly, the situation is different in humans where visfatin expression was found to be equivalent between white and brown or brite adipocytes in vivo and in vitro. In conclusion, visfatin can be considered only as a rodent brown adipocyte biomarker, independently of tissue activation.

Let-7i-5p represses brite adipocyte function in mice and humans.

In response to cold or ß3-adrenoreceptor stimulation brown adipose tissue (BAT) promotes non-shivering thermogenesis, leading to energy dissipation. BAT has long been thought to be absent or scarce in adult humans. The recent discovery of thermogenic brite/beige adipocytes has opened the way to development of novel innovative strategies to combat overweight/obesity and associated diseases. Thus it is of great interest to identify regulatory factors that govern the brite adipogenic program. Here, we carried out global microRNA (miRNA) expression profiling on human adipocytes to identify miRNAs that are regulated upon the conversion from white to brite adipocytes. Among the miRNAs that were differentially expressed, we found that Let-7i-5p was down regulated in brite adipocytes. A detailed analysis of the Let-7i-5p levels showed an inverse expression of UCP1 in murine and human brite adipocytes both in vivo and in vitro. Functional studies with Let-7i-5p mimic in human brite adipocytes in vitro revealed a decrease in the expression of UCP1 and in the oxygen consumption rate. Moreover, the Let-7i-5p mimic when injected into murine sub-cutaneous white adipose tissue inhibited partially ß3-adrenergic activation of the browning process. These results suggest that the miRNAs Let-7i-5p participates in the recruitment and the function of brite adipocytes.

The K+ channel TASK1 modulates ß-adrenergic response in brown adipose tissue through the mineralocorticoid receptor pathway.

Brown adipose tissue (BAT) is essential for adaptive thermogenesis and dissipation of caloric excess through the activity of uncoupling protein (UCP)-1. BAT in humans is of great interest for the treatment of obesity and related diseases. In this study, the expression of Twik-related acid-sensitive K(+) channel (TASK)-1 [a pH-sensitive potassium channel encoded by the potassium channel, 2-pore domain, subfamily K, member 3 (Kcnk3) gene] correlated highly with Ucp1 expression in obese and cold-exposed mice. In addition, Task1-null mice, compared with their controls, became overweight, mainly because of an increase in white adipose tissue mass and BAT whitening. Task1(-/-)-mouse-derived brown adipocytes, compared with wild-type mouse-derived brown adipocytes, displayed an impaired ß3-adrenergic receptor response that was characterized by a decrease in oxygen consumption, Ucp1 expression, and lipolysis. This phenotype was thought to be caused by an exacerbation of mineralocorticoid receptor (MR) signaling, given that it was mimicked by corticoids and reversed by an MR inhibitor. We concluded that the K(+) channel TASK1 controls the thermogenic activity in brown adipocytes through modulation of ß-adrenergic receptor signaling.

Oxytocin reverses osteoporosis in a sex-dependent manner.

The increase of life expectancy has led to the increase of age-related diseases such as osteoporosis. Osteoporosis is characterized by bone weakening promoting the occurrence of fractures with defective bone regeneration. Men aged over 50 have a prevalence for osteoporosis of 20%, which is related to a decline in sex hormones occurring during andropause or surgical orchidectomy. As we previously demonstrated in a mouse model for menopause in women that treatment with the neurohypophyseal peptide hormone oxytocin (OT) normalizes body weight and prevents the development of osteoporosis, herein we addressed the effects of OT in male osteoporosis. Thus, we treated orchidectomized mice, an animal model suitable for the study of male osteoporosis, for 8 weeks with OT and then analyzed trabecular and cortical bone parameters as well as fat mass using micro-computed tomography. Orchidectomized mice displayed severe bone loss, muscle atrophy accompanied by fat mass gain as expected in andropause. Interestingly, OT treatment in male mice normalized fat mass as it did in female mice. However, although OT treatment led to a normalization of bone parameters in ovariectomized mice, this did not happen in orchidectomized mice. Moreover, loss of muscle mass was not reversed in orchidectomized mice upon OT treatment. All of these observations indicate that OT acts on fat physiology in both sexes, but in a sex specific manner with regard to bone physiology.

The ω6-fatty acid, arachidonic acid, regulates the conversion of white to brite adipocyte through a prostaglandin/calcium mediated pathway.

OBJECTIVE: Brite adipocytes are inducible energy-dissipating cells expressing UCP1 which appear within white adipose tissue of healthy adult individuals. Recruitment of these cells represents a potential strategy to fight obesity and associated diseases. METHODS/RESULTS: Using human Multipotent Adipose-Derived Stem cells, able to convert into brite adipocytes, we show that arachidonic acid strongly inhibits brite adipocyte formation via a cyclooxygenase pathway leading to secretion of PGE2 and PGF2α. Both prostaglandins induce an oscillatory Ca(++) signaling coupled to ERK pathway and trigger a decrease in UCP1 expression and in oxygen consumption without altering mitochondriogenesis. In mice fed a standard diet supplemented with ω6 arachidonic acid, PGF2α and PGE2 amounts are increased in subcutaneous white adipose tissue and associated with a decrease in the recruitment of brite adipocytes. CONCLUSION: Our results suggest that dietary excess of ω6 polyunsaturated fatty acids present in Western diets, may also favor obesity by preventing the "browning" process to take place.

Antagonistic functions of LMNA isoforms in energy expenditure and lifespan.

Alternative RNA processing of LMNA pre-mRNA produces three main protein isoforms, that is, lamin A, progerin, and lamin C. De novo mutations that favor the expression of progerin over lamin A lead to Hutchinson-Gilford progeria syndrome (HGPS), providing support for the involvement of LMNA processing in pathological aging. Lamin C expression is mutually exclusive with the splicing of lamin A and progerin isoforms and occurs by alternative polyadenylation. Here, we investigate the function of lamin C in aging and metabolism using mice that express only this isoform. Intriguingly, these mice live longer, have decreased energy metabolism, increased weight gain, and reduced respiration. In contrast, progerin-expressing mice show increased energy metabolism and are lipodystrophic. Increased mitochondrial biogenesis is found in adipose tissue from HGPS-like mice, whereas lamin C-only mice have fewer mitochondria. Consistently, transcriptome analyses of adipose tissues from HGPS and lamin C-only mice reveal inversely correlated expression of key regulators of energy expenditure, including Pgc1a and Sfrp5. Our results demonstrate that LMNA encodes functionally distinct isoforms that have opposing effects on energy metabolism and lifespan in mammals.

Oxytocin reverses ovariectomy-induced osteopenia and body fat gain.

Osteoporosis and overweight/obesity constitute major worldwide public health burdens that are associated with aging. A high proportion of women develop osteoporosis and increased intraabdominal adiposity after menopause. which leads to bone fractures and metabolic disorders. There is no efficient treatment without major side effects for these 2 diseases. We previously showed that the administration of oxytocin (OT) normalizes ovariectomy-induced osteopenia and bone marrow adiposity in mice. Ovariectomized mice, used as an animal model mimicking menopause, were treated with OT or vehicle. Trabecular bone parameters and fat mass were analyzed using micro-computed tomography. Herein, we show that this effect on trabecular bone parameters was mediated through the restoration of osteoblast/osteoclast cross talk via the receptor activator of nuclear factor-κB ligand /osteoprotegerin axis. Moreover, the daily administration of OT normalized body weight and intraabdominal fat depots in ovariectomized mice. Intraabdominal fat mass is more sensitive to OT that sc fat depots, and this inhibitory effect is mediated through inhibition of adipocyte precursor's differentiation with a tendency to lower adipocyte size. OT treatment did not affect food intake, locomotors activity, or energy expenditure, but it did promote a shift in fuel utilization favoring lipid oxidation. In addition, the decrease in fat mass resulted from the inhibition of the adipose precursor's differentiation. Thus, OT constitutes an effective strategy for targeting osteopenia, overweight, and fat mass redistribution without any detrimental effects in a mouse model mimicking the menopause.

Chondrogenic potential of stem cells derived from adipose tissue: a powerful pharmacological tool.

Chondrogenesis has been widely investigated in vitro using bone marrow-derived mesenchymal stromal cells (BM-MSCs) or primary chondrocytes. However, their use raises some issues partially circumvented by the availability of Adipose tissue-derived MSCs. Herein; we characterized the chondrogenic potential of human Multipotent Adipose-Derived Stem (hMADS) cells, and their potential use as pharmacological tool. hMADS cells are able to synthesize matrix proteins including COMP, Aggrecan and type II Collagen. Furthermore, hMADS cells express BMP receptors in a similar manner to BM-MSC, and BMP6 treatment of differentiated cells prevents expression of the hypertrophic marker type X Collagen. We tested whether IL-1ß and nicotine could impact chondrocyte differentiation. As expected, IL-1ß induced ADAMTS-4 gene expression and modulated negatively chondrogenesis while these effects were reverted in the presence of the IL-1 receptor antagonist. Nicotine, at concentrations similar to those observed in blood of smokers, exhibited a dose dependent increase of Aggrecan expression, suggesting an unexpected protective effect of the drug under these conditions. Therefore, hMADS cells represent a valuable tool for the analysis of in vitro chondrocyte differentiation and to screen for potentially interesting pharmacological drugs.