Transcription Factor Mitf Research Articles

Inonotus obliquus Extract as An Inhibitor of α-MSH-Induced Melanogenesis in B16F10 Mouse Melanoma Cells

Melanogenesis is a biosynthetic pathway that produces the pigment melanin in human skin. The catalyzation of the key enzyme tyrosinase is the first step in melanogenesis, and the downregulation of tyrosinase enzyme activity is the most reported method for inhibiting melanogenesis. Hyperpigmentation is an important issue in the cosmetic industry, and there is great demand for melanogenesis inhibitors. In the present study, we demonstrated the anti-melanogenic effect of Inonotus obliquus in alpha-melanocyte-stimulating hormone (α-MSH)-induced B16F10 mouse melanoma cells and identified it as a new melanogenesis inhibitor. Comparing the B16F10 cells treated with the control and the Inonotus obliquus extract, we identified the melanin contents, mRNA and protein expression of tyrosinase, tyrosinase activity, and microphthalmia-associated transcription factor (Mitf) activity using a constructed plasmid. Through these experiments, we confirmed that Inonotus obliquus extract inhibits melanin synthesis by downregulating the activity and expression of tyrosinase. Furthermore, we revealed that tyrosinase expression is regulated by Inonotus obliquus extract via the repression of Mitf transcriptional activity. Thus, in this study, we found that Inonotus obliquus extract has anti-melanogenic effects via the suppression of melanin synthesis. Taken together, we demonstrated that Inonotus obliquus extract is a good potential candidate for use as a natural source for the therapeutic treatment of hyperpigmentation and for applications in whitening cosmetic products.

Abstract B174: Co-expression of stimulators and inhibitors of T-cell activation in melanoma

Abstract Melanoma, one of the most aggressive skin cancers, has steadily been on the rise over the last three decades. With limited treatment options, the recent impetus in immuno-oncology drugs, especially checkpoint inhibitors, has changed the treatment landscape. While anti-CTLA4 and anti-PD1 demonstrated success in clinic, the search for additional targets is needed to enable potent comprehensive curative combination therapies. Our laboratory has characterized and screened five primary patient-derived melanoma cell lines, MEL-2, MEL-V, 3MM, KFM, and GLM-2. While immunomodulatory molecules are canonically present on antigen presenting cells and T-cells, increasing evidence suggests that they are not expressed in isolation. In an effort to identify immunomodulatory molecules expressed on our primary melanoma cells, a comprehensive RT-PCR screen of 29 co-inhibitory and co-stimulatory molecules was carried out. Several molecules including CD160, CD226, TIM1, HVEM, and BTLA were seen to be differentially expressed in melanoma cells compared to normal melanocytes at the mRNA level. Western blots and immunocytochemistry validated the differential expression of these molecules at the protein level. 50-80% of melanoma cases are positive for the BRAFV600E mutation, and are treated with a small molecule inhibitor of the mutated BRAF, vemurafenib (PLX4032). Treatment of these cells with PLX4032 led to an upregulation of transcription factors MITF and AP-1, as well as immunomodulatory molecules, CD160, CD226, TIM1, HVEM, and BTLA, a phenomenon seen only in cells positive for the BRAFV600E lesion. MITF and AP-1, owing to the binding sites present in the promoter regions of these molecules, can drive their expression upon treatment with PLX4032. These additional immune-regulatory molecules of T-cell activation and/or immune tolerance mechanisms are potential targets for a combination therapy with PLX4032 in melanoma patients positive for the BRAFV600E genetic lesion. Our future directions aim to elucidate the role of these molecules on the tumor cells and devise an effective combination with small-molecule inhibitors and immunotherapies. Citation Format: Rachana Maniyar, Robert Freund, Aryan Malhotra, Sanjukta Chakraborty, Jan Geliebter, Marc Wallack, Raj K. Tiwari. Co-expression of stimulators and inhibitors of T-cell activation in melanoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B174.

MITF has a central role in regulating starvation-induced autophagy in melanoma

The MITF transcription factor is a master regulator of melanocyte development and a critical factor in melanomagenesis. The related transcription factors TFEB and TFE3 regulate lysosomal activity and autophagy processes known to be important in melanoma. Here we show that MITF binds the CLEAR-box element in the promoters of lysosomal and autophagosomal genes in melanocytes and melanoma cells. The crystal structure of MITF bound to the CLEAR-box reveals how the palindromic nature of this motif induces symmetric MITF homodimer binding. In metastatic melanoma tumors and cell lines, MITF positively correlates with the expression of lysosomal and autophagosomal genes, which, interestingly, are different from the lysosomal and autophagosomal genes correlated with TFEB and TFE3. Depletion of MITF in melanoma cells and melanocytes attenuates the response to starvation-induced autophagy, whereas the overexpression of MITF in melanoma cells increases the number of autophagosomes but is not sufficient to induce autophagic flux. Our results suggest that MITF and the related factors TFEB and TFE3 have separate roles in regulating a starvation-induced autophagy response in melanoma. Understanding the normal and pathophysiological roles of MITF and related transcription factors may provide important clinical insights into melanoma therapy.

Leathesia difformis Extract Inhibits α-MSH-Induced Melanogenesis in B16F10 Cells via Down-Regulation of CREB Signaling Pathway.

Leathesia difformis (L.) Areschoug (L. difformis) is a species of littoral brown algae of the class Phaeophyceae. Only a few studies on the apoptotic, antiviral, and antioxidant properties of L. difformis have been reported, and its inhibitory effect on melanin synthesis has not been studied. The aim of this study was to investigate the anti-melanogenic effect of L. difformis extract on α-melanocyte-stimulating hormone (α-MSH)-induced B16F10 melanocytes and its mechanism of action. L. difformis was extracted using 80% ethanol (LDE) and then fractioned between ethyl acetate (LDE-EA) and water (LDE-A). Our data demonstrated that LDE-EA significantly inhibited melanin level and cellular tyrosinase activity in α-MSH-stimulated B16 cells. In addition, the expression of genes associated with melanin synthesis, such as microphthalmia-associated transcription factor (Mitf), tyrosinase (Tyr), tyrosinase-related protein-1 (Trp-1), dopachrome tautomerase (Dct), and melanocortin 1 receptor (Mc1r) was down-regulated by LDE-EA treatment. Moreover, LDE-EA decreased p-CREB levels, which suggests that the inhibition of the cAMP/PKA/CREB pathways may be involved in the anti-melanogenic effect of LDE-EA. Thus, this study revealed that LDE-EA is an effective inhibitor of hyperpigmentation through inhibition of CREB pathways and may be considered as a potential therapeutic agent for hyperpigmentation disorders.

Mutation of microphthalmia-associated transcription factor (mitf) in zebrafish sensitizes for glomerulopathy.

ABSTRACTDifferent glomerular diseases that affect podocyte homeostasis can clinically present as nephrotic syndrome with massive proteinuria, hypoalbuminemia, hyperlipidemia and edema. Up to now, no drugs that specifically target the actin cytoskeleton of podocytes are on the market and model systems for library screenings to develop anti-proteinuric drugs are of high interest. We developed a standardized proteinuria model in zebrafish using puromycin aminonucleoside (PAN) via treatment in the fish water to allow for further drug testing to develop anti-proteinuric drugs for the treatment of glomerular diseases. We noticed that fish that carry the nacre-mutation show a significantly higher susceptibility for the disruption of the glomerular filtration barrier following PAN treatment, which results in a more pronounced proteinuria phenotype. Nacre zebrafish inherit a mutation yielding a truncated version of microphthalmia-associated transcription factor/melanogenesis associated transcription factor (mitf). We hypothesized that the nacre mutation may lead to reduced formin expression and defects in cytoskeletal rearrangement. Based on the observations in zebrafish, we carried out a PAN treatment on cultured human podocytes after knockdown with MITF siRNA causing a rearrangement of the actin cytoskeleton.

Distribution of mast cells within the mouse heart and its dependency on Mitf

Although mast cell distribution has been described in both human and canine hearts, cardiac mast cells in mice have yet to be categorically localized. We therefore sought to describe mast cell distribution within the mouse heart and characterize their dependence on the Microphthalmia-associated transcription factor (Mitf). Cardiac mast cells were visualized using Toluidine Blue and avidin staining, and their distribution within the heart described. Cardiac mast cells were most prevalent in the epicardium (50%) or myocardium (45%). Less frequently, mast cells were noted in the endocardium (5%). Within the myocardium, 31% of the mast cells had perivascular location. By studying two different Mitf mutant strains, Mitfmi-vga9 and MitfMi-wh, we demonstrated that these mutations led to near-complete deficiency of cardiac mast cells. Accordingly, expression of the mMCP-4 and mMCP-5 genes was lost and chymase enzyme activity was severely reduced. Additionally, hearts from mice heterozygous for these Mitf mutations contained significantly fewer mast cells compared to wild-type mice. Our results demonstrated that the distribution of cardiac mast cells in mice is different from humans and dogs. Cardiac mast cells are dependent on Mitf expression, with loss-of-function mutation in the Mitf gene leading to near-complete lack of cardiac mast cells. Loss of a single Mitf allele is sufficient for relative mast cell deficiency.

Transcriptional regulation of Rab32/38, a specific marker of pigment cell formation in Ciona robusta

Through a myriad of pigments stored in different cells, animal pigmentation represents a crucial process to face disparate environmental and ecological challenges. In vertebrates, the small GTPase Rab32 and Rab38 have a conserved role in the transport of key melanogenic enzymes, as tyrosinase (tyr) and tyrosinase-related protein (tyrp), to the melanosomes in formation. We provide a survey on Rab32/38 evolution and its regulatory logics during pigment cell formation in Ciona robusta. Our phylogeny supports the existence of a single Rab32/38 gene in tunicates, which is probably the unique transporter for tyrosinase family members in this clade. Different deletions allow us to identify the minimal cis-regulatory element able to recapitulate the endogenous gene expression during pigment cell development in C. robusta. In this conserved region, we identified two putative binding sites for the transcription factor Mitf, which is known for its role as regulator of pigmentation in vertebrates. Mutational analysis revealed that both Mitf binding sites are essential for the activity of this regulatory region and we demonstrated that Mitf misexpression is able to induce ectopic activation of the Rab32/38 regulatory region in vivo.Our results strongly indicate that Mitf is involved in the regulation of Rab32/38 activity during Ciona pigment cell development.

A PAX3/BRN2 rheostat controls the dynamics of BRAF mediated MITF regulation in MITFhigh /AXLlow melanoma.

The BRAF kinase and the MAPK pathway are targets of current melanoma therapies. However, MAPK pathway inhibition results in dynamic changes of downstream targets that can counteract inhibitor‐action not only in during treatment, but also in acquired resistant tumours. One such dynamic change involves the expression of the transcription factor MITF, a crucial regulator of cell survival and proliferation in untreated as well as drug‐addicted acquired resistant melanoma. Tight control over MITF expression levels is required for optimal melanoma growth, and while it is well established that the MAPK pathway regulates MITF expression, the actual mechanism is insufficiently understood. We reveal here, how BRAF through action on the transcription factors BRN2 and PAX3 executes control over the regulation of MITF expression in a manner that allows for considerable plasticity. This plasticity provides robustness to the BRAF mediated MITF regulation and explains the dynamics in MITF expression that are observed in patients in response to MAPK inhibitor therapy.

A Targeted Quantitative Proteomic Approach Assesses the Reprogramming of Small GTPases during Melanoma Metastasis.

Small GTPases of the Ras superfamily are master regulators of intracellular trafficking and constitute essential signaling components in all eukaryotes. Aberrant small GTPase signaling is associated with a wide spectrum of human diseases, including cancer. Here, we developed a high-throughput, multiple reaction monitoring-based workflow, coupled with stable isotope labeling by amino acids in cell culture, for targeted quantification of approximately 100 small GTPases in cultured human cells. Using this method, we investigated the differential expression of small GTPases in three pairs of primary and metastatic melanoma cell lines. Bioinformatic analyses of The Cancer Genome Atlas data and other publicly available data as well as cell-based assays revealed previously unrecognized roles of RAB38 in promoting melanoma metastasis. Diminished promoter methylation and the subsequent augmented binding of transcription factor MITF contributed to elevated expression of RAB38 gene in metastatic versus primary melanoma cells. Moreover, RAB38 promoted invasion of cultured melanoma cells by modulating the expression and activities of matrix metalloproteinases-2 and -9. Together, these data establish a novel targeted proteomic method for interrogating the small GTPase proteome in human cells and identify epigenetic reactivation of RAB38 as a contributing factor to metastatic transformation in melanoma.Significance: A novel quantitative proteomic method leads to the discovery of RAB38 as a new driver of metastasis in melanoma. Cancer Res; 78(18); 5431-45. ©2018 AACR.

Daphnane diterpenes inhibit the metastatic potential of B16F10 murine melanoma cells in vitro and in vivo

BackgroundMelanoma is one of the most invasive and aggressive types of cancer with a very poor prognosis. Surgery remains the most efficient treatment prior melanoma invasion and metastasis formation. However, therapy becomes a challenge once the cancer cells colonized other tissues. At present, there are two main classes of therapies acting with a certain efficiency on metastatic melanoma: immune check point inhibitors (anti-PD1/PDL1) and targeted therapy such as Vemurafenib. Unfortunately, these therapies are not fully responsive, induce resistance and/or generate unwanted side effects. In this respect, it is important to continue to discover new cancer therapeutics. Here, we show that daphnane diterpenes type of compounds can prevent melanoma metastasis by inhibiting metastasis-associated matrix metalloproteinases expression without cytotoxicity.MethodsEvaluation of the anti-metastasis effect of daphnane diterpenes-rich Thymelaea hirsuta extract (TH) and its bioactive component gnidilatidin was carried out in vitro using B16 murine melanoma cells and in vivo using male C57BL/6 J mice. Global gene expression in B16 cells was done using DNA microarray, validated using real-time PCR, to further understand the effect of daphnane diterpenes, specifically daphnane diterpenoid gnidilatidin.ResultsOral administration of daphnane diterpenes-rich Thymelaea hirsuta extract (TH) suppressed MMP2 and MMP9 expression, decreasing lung tumor in mice injected with B16 murine melanoma cells. Validation of these observations in vitro showed reduced B16 cells migration, adhesion, and invasion. Results of microarray analysis of B16 cells treated with daphnane diterpenoid gnidilatidin from TH revealed an upregulation of tumor suppressor Egr1 while inhibiting metastasis-associated genes Id2 and Sytl2 expression. A downregulation of the melanoma oncogene microphthalmia-associated transcription factor (Mitf) was observed, and most likely caused by the inhibition of Id2, a gene that regulated HLH transcription factors such as MITF and also reported to promote tumor cell migration and invasion.ConclusionsDaphnane diterpenes have inhibitory effect on the metastatic potential of B16 melanoma cells, and the results of this study provided evidence for their potential for use in the prevention and inhibition of melanoma metastasis.

BRAF/MAPK and GSK3 signaling converges to control MITF nuclear export

The close integration of the MAPK, PI3K, and WNT signaling pathways underpins much of development and is deregulated in cancer. In principle, combinatorial posttranslational modification of key lineage-specific transcription factors would be an effective means to integrate critical signaling events. Understanding how this might be achieved is central to deciphering the impact of microenvironmental cues in development and disease. The microphthalmia-associated transcription factor MITF plays a crucial role in the development of melanocytes, the retinal pigment epithelium, osteoclasts, and mast cells and acts as a lineage survival oncogene in melanoma. MITF coordinates survival, differentiation, cell-cycle progression, cell migration, metabolism, and lysosome biogenesis. However, how the activity of this key transcription factor is controlled remains poorly understood. Here, we show that GSK3, downstream from both the PI3K and Wnt pathways, and BRAF/MAPK signaling converges to control MITF nuclear export. Phosphorylation of the melanocyte MITF-M isoform in response to BRAF/MAPK signaling primes for phosphorylation by GSK3, a kinase inhibited by both PI3K and Wnt signaling. Dual phosphorylation, but not monophosphorylation, then promotes MITF nuclear export by activating a previously unrecognized hydrophobic export signal. Nonmelanocyte MITF isoforms exhibit poor regulation by MAPK signaling, but instead their export is controlled by mTOR. We uncover here an unanticipated mode of MITF regulation that integrates the output of key developmental and cancer-associated signaling pathways to gate MITF flux through the import-export cycle. The results have significant implications for our understanding of melanoma progression and stem cell renewal.

Depigmenting effect of argan press-cake extract through the down-regulation of Mitf and melanogenic enzymes expression in B16 murine melanoma cells

Oil extraction from the kernels of Argania spinosa (L.) Skeels (Sapotaceae), an endemic tree of Morocco, produces argan press-cake (APC) used as a shampoo and to treat sprains, scabies, and for healing wounds. We have previously reported that argan oil has antimelanogenesis effect. Here, we determined if the by-product, APC, has melanogenesis regulatory effect using B16 murine melanoma cells. The effect of APC ethanol extract on cell proliferation and melanin content of B16 cells were measured, and to elucidate the mechanism involved, the expression level of melanogenic enzymes tyrosinase (TYR), dopachrome tautomerase (DCT), and tyrosinase-related protein 1 (TRP1) were determined and mRNA expression level of microphthalmia- associated transcription factor (Mitf) and Tyr genes were quantified. APC ethanol extract showed a significant melanin biosynthesis inhibitory effect on B16 cells in a time-dependent manner without cytotoxicity, which could be due to the decreased expression of TYR, TRP1, and DCT in a time-dependent manner. APC extract down regulated Mitf and Tyr. Decreased TRP1 and DCT levels could be due to post-translational modifications. These results suggest that APC extract may be used as a new source of natural whitening products and may be introduced as an important pharmacological agent for the treatment of hyperpigmentation disorders.

A wide variety of Mitf transcript variants are expressed in the Xenopus laevis periodic albino mutant.

The periodic albino mutant of Xenopus laevis has been used to study the development of pigment cells because both the retinal pigment epithelium (RPE) and melanophores are affected. In this mutant, "white pigment cells" containing both melanophore-specific and iridophore-specific pigment organelles appear. The present experiments were designed to investigate the structural organization and expression of microphthalmia-associated transcription factor (Mitf) in the mutant since Mitf is known to regulate the development of melanocytes and RPE. The exon structures of X.laevis mitf genes (mitf.L and mitf.S) were defined using newly obtained Mitf transcripts and X.laevis genomic data. Compared to mouse mitf, exons 3 and 6a were absent in X.laevis mitf. The four exons between exons 4 and 6b in X.laevis mitf were named 5α, 5β, 5γ, and 5δ. Exons 5α and 5δ were specific to X.laevis mitf, whereas the continuous exons 5β/γ were identical to exon 5 of mouse mitf. A wide variety of A-Mitf and M-Mitf transcript variants lacking one or more exons were found in X.laevis; however, different types of Mitf transcripts were expressed in the mutant. In addition, white pigment cells and melanophores expressed both the mitf and dopachrome tautomerase (dct) genes.

25 Strategies to overcome phenotype switch-driven therapy resistance in melanoma

ABSTRACT Introduction Phenotype switching is a key mechanism contributing to therapy resistance and metastasis in melanoma. It is marked by high expression of several receptor tyrosine kinases, most prominently AXL, and associated with resistance to the clinically oft-used BRAF and MEK inhibitors. Furthermore, it has recently been suggested that phenotype switching also contributes to resistance to immune checkpoint blockade in melanoma. Results and discussions We have shown previously that upregulation of AXL occurs concurrently with the downregulation of the melanocytic lineage transcription factor MITF and its downstream protein Melan-A1. Furthermore, we recently showed that most melanomas are highly heterogeneous for AXL expression, both untreated tumours and treatment-resistant ones. This observation provided the framework for a concept to differentially target these distinct AXL-high and -low populations. On the one hand, we targeted AXL-expressing melanoma cells by a novel antibody-drug conjugate, AXL-107-MMAE. On the other hand, the MITF-high, AXL-low melanoma cells were targeted with MAPK pathway inhibitors. Therefore, by eliminating distinct populations within heterogeneous melanoma cell pools, AXL-107-MMAE and MAPK pathway inhibitors cooperatively inhibited tumour growth in vitro and in vivo. Lastly, by inducing AXL transcription, BRAF/MEK inhibitors further potentiated the efficacy of AXL-107-MMAE2. Although the correlation between phenotype switching and resistance to targeted agents such as BRAF/MEK inhibitors has been well described, it is not well established how melanoma cells, especially in patients or in vivo, can acquire this state. Furthermore, it is as unclear if there is any functional interaction between the immune system and the phenotype switch. Investigating any such relationship may contribute to the development of therapies that aim to prevent the acquisition of an AXL-high, treatment-refractory state. We will present results of recent efforts to better understand the role that immune cells may play in the establishment of a phenotype switch and how we may re-sensitise tumour cells that have become resistant to T cell therapy as a consequence.

1198 ATP-citrate lyase contributes to melanoma growth and MAPK inhibitors resistance by epigenetically regulating MITF and oxidative phosphorylation

Melanoma is the most life-threatening skin cancer for its rapid progression and frequent resistance to BRAF/MEK inhibitors. Enhanced lipogenesis and mitochondria function are two hallmark metabolic characteristics of melanoma, but their crosstalk in regulating melanoma growth and MAPK inhibitors resistance has not been elucidated. ATP-citrate lyase (ACLY) is a critical enzyme in lipogenesis by producing acetyl-CoA, the building block for fatty acid synthesis. Herein, we first found that ACLY expression was significantly increased in melanoma and highly correlated with poor clinical outcomes. Then, the knockdown of ACLY markedly attenuated cell proliferation in vitro and suppressed tumor growth in vivo. Subsequently, through the genome-wide mRNA profile analysis, we found that ACLY specifically regulated the expression of melanocytic lineage transcription factor MITF and its downstream PGC1α. The oncogenic role of ACLY was dependent on mitochondrial oxidative phosphorylation mediated by MITF-PGC1α axis. More importantly, ACLY conferred resistance to BRAF/MEK inhibitors by potentiating MITF-PGC1α axis and mitochondria function, and the combination treatment with ACLY inhibitor sensitized BRAF mutant melanoma to MAPK inhibition. Further mechanistic study revealed that ACLY regulated acetyltransferase P300 activity, increasing the histone acetylation at the MITF locus, thus activating MITF transcription and mitochondria biogenesis. Altogether, our results demonstrate that ACLY contributes to melanoma growth and MAPK inhibitors resistance by epigenetically regulating MITF and oxidative phosphorylation, providing a novel linkage between lipid metabolism and mitochondria function in tumor biology.

Genetic background of coat colour in sheep

Abstract. The coat colour of animals is an extremely important trait that affects their behaviour and is decisive for survival in the natural environment. In farm animal breeding, as a result of the selection of a certain coat colour type, animals are characterized by a much greater variety of coat types. This makes them an appropriate model in research in this field. A very important aspect of the coat colour types of farm animals is distinguishing between breeds and varieties based on this trait. Furthermore, for the sheep breeds which are kept for skins and wool, coat/skin colour is an important economic trait. Until now the study of coat colour inheritance in sheep proved the dominance of white colour over pigmented/black coat or skin and of black over brown. Due to the current knowledge of the molecular basis of ovine coat colour inheritance, there is no molecular test to distinguish coat colour types in sheep although some are available for other species, such as cattle, dogs, and horses. Understanding the genetic background of variation in one of the most important phenotypic traits in livestock would help to identify new genes which have a great effect on the coat colour type. Considering that coat colour variation is a crucial trait for discriminating between breeds (including sheep), it is important to broaden our knowledge of the genetic background of pigmentation. The results may be used in the future to determine the genetic pattern of a breed. Until now, identified candidate genes that have a significant impact on colour type in mammals mainly code for factors located in melanocytes. The proposed candidate genes code for the melanocortin 1 receptor (MC1R), agouti signaling protein (ASIP), tyrosinase-related protein 1 (TYRP1), microphthalmia-associated transcription factor MITF, and v-kit Hardy–Zuckerman 4 feline sarcoma viral oncogene homologue (KIT). However, there is still no conclusive evidence of established polymorphisms for specific coat colour types in sheep.

Targeting CDK2 overcomes melanoma resistance against BRAF and Hsp90 inhibitors

Novel therapies are undergoing clinical trials, for example, the Hsp90 inhibitor, XL888, in combination with BRAF inhibitors for the treatment of therapy‐resistant melanomas. Unfortunately, our data show that this combination elicits a heterogeneous response in a panel of melanoma cell lines including PDX‐derived models. We sought to understand the mechanisms underlying the differential responses and suggest a patient stratification strategy. Thermal proteome profiling (TPP) identified the protein targets of XL888 in a pair of sensitive and unresponsive cell lines. Unbiased proteomics and phosphoproteomics analyses identified CDK2 as a driver of resistance to both BRAF and Hsp90 inhibitors and its expression is regulated by the transcription factor MITF upon XL888 treatment. The CDK2 inhibitor, dinaciclib, attenuated resistance to both classes of inhibitors and combinations thereof. Notably, we found that MITF expression correlates with CDK2 upregulation in patients; thus, dinaciclib would warrant consideration for treatment of patients unresponsive to BRAF‐MEK and/or Hsp90 inhibitors and/or harboring MITF amplification/overexpression.

MITF-M regulates melanogenesis in mouse melanocytes

BackgroundAlthough the impact of the microphthalamia-associated transcription factor (Mitf) signaling pathway on melanocytes progression has been extensively studied, the specific molecular mechanisms behind MITF-M-enhanced melanin production in melanocytes still need to be clarified. MethodsIn this study, we analyzed the levels of Mitf-M in skin tissues of different coat mice in order to further reveal the relationship between Mitf-M and skin pigmentation. To address the function of Mitf-M on melanogenesis, we have used an overexpression system and combined morphological and biochemical methods to investigate its localization in different coat color mice and pigmentation-related genes’ expression in mouse melanocytes. ResultsThe qRT-PCR assay and Western blotting analysis revealed that Mitf-M mRNA and protein were synthesized in all tested mice skin samples, with the highest expression level in brown skin, a moderate expression level in grey skin and the lowest expression level in black skin. Simultaneously, immunofluorescence staining revealed that MITF-M was mainly expressed in the hair follicle matrix and inner and outer root sheath in the skin tissues with different coat colors. Furthermore, overexpression of MITF-M led to increased melanin content and variable pigmentation-related gene expression. ConclusionThese results directly demonstrate that MITF-M not only influences melanogenesis, but also determines the progression of melanosomal protein in mouse melanocytes.

Multi-color RGB marking enables clonality assessment of liver tumors in a murine xenograft model.

We recently introduced red-green-blue (RGB) marking for clonal cell tracking based on individual color-coding. Here, we applied RGB marking to study clonal development of liver tumors. Immortalized, non-tumorigenic human fetal hepatocytes expressing the human telomerase reverse transcriptase (FH-hTERT) were RGB-marked by simultaneous transduction with lentiviral vectors encoding mCherry, Venus, and Cerulean. Multi-color fluorescence microscopy was used to analyze growth characteristics of RGB-marked FH-hTERT in vitro and in vivo after transplantation into livers of immunodeficient mice with endogenous liver damage (uPA/SCID). After initially polyclonal engraftment we observed oligoclonal regenerative nodules derived from transplanted RGB-marked FH-hTERT. Some mice developed monochromatic invasive liver tumors; their clonal origin was confirmed both on the molecular level, based on specific lentiviral-vector insertion sites, and by serial transplantation of one tumor. Vector insertions in proximity to the proto-oncogene MCF2 and the transcription factor MITF resulted in strong upregulation of mRNA expression in the respective tumors. Notably, upregulated MCF2 and MITF expression was also observed in 21% and 33% of 24 human hepatocellular carcinomas analyzed. In conclusion, liver repopulation with RGB-marked FH-hTERT is a useful tool to study clonal progression of liver tumors caused by insertional mutagenesis in vivo and will help identifying genes involved in liver cancer.

SHARPIN-mediated regulation of protein arginine methyltransferase 5 controls melanoma growth.

SHARPIN, an adaptor for the linear ubiquitin chain assembly complex (LUBAC), plays important roles in NF-κB signaling and inflammation. Here, we have demonstrated a LUBAC-independent role for SHARPIN in regulating melanoma growth. We observed that SHARPIN interacted with PRMT5, a type II protein arginine methyltransferase, and increased its multiprotein complex and methyltransferase activity. Activated PRMT5 controlled the expression of the transcription factors SOX10 and MITF by SHARPIN-dependent arginine dimethylation and inhibition of the transcriptional corepressor SKI. Activation of PRMT5 by SHARPIN counteracted PRMT5 inhibition by methylthioadenosine, a substrate of methylthioadenosine phosphorylase, which is codeleted with cyclin-dependent kinase inhibitor 2A (CDKN2A) in approximately 15% of human cancers. Collectively, we identified a LUBAC-independent role for SHARPIN in enhancing PRMT5 activity that contributes to melanomagenesis through the SKI/SOX10 regulatory axis.