MITF Activity Research Articles

Divergence of cAMP signalling pathways mediating augmented nucleotide excision repair and pigment induction in melanocytes.

Loss-of-function melanocortin 1 receptor (MC1R) polymorphisms are common in UV-sensitive fair-skinned individuals and are associated with blunted cAMP second messenger signalling and higher lifetime risk of melanoma because of diminished ability of melanocytes to cope with UV damage. cAMP signalling positions melanocytes to resist UV injury by upregulating synthesis of UV-blocking eumelanin pigment and by enhancing the repair of UV-induced DNA damage. cAMP enhances melanocyte nucleotide excision repair (NER), the genome maintenance pathway responsible for the removal of mutagenic UV photolesions, through cAMP-activated protein kinase (protein kinase A)-mediated phosphorylation of the ataxia telangiectasia-mutated and Rad3-related (ATR) protein on the S435 residue. We investigated the interdependence of cAMP-mediated melanin upregulation and cAMP-enhanced DNA repair in primary human melanocytes and a melanoma cell line. We observed that the ATR-dependent molecular pathway linking cAMP signalling to the NER pathway is independent of MITF activation. Similarly, cAMP-mediated upregulation of pigment synthesis is independent of ATR, suggesting that the key molecular events driving MC1R-mediated enhancement of genome maintenance (eg PKA-mediated phosphorylation of ATR) and MC1R-induced pigment induction (eg MITF activation) are distinct.

TFAP2 paralogs regulate melanocyte differentiation in parallel with MITF.

Mutations in the gene encoding transcription factor TFAP2A result in pigmentation anomalies in model organisms and premature hair graying in humans. However, the pleiotropic functions of TFAP2A and its redundantly-acting paralogs have made the precise contribution of TFAP2-type activity to melanocyte differentiation unclear. Defining this contribution may help to explain why TFAP2A expression is reduced in advanced-stage melanoma compared to benign nevi. To identify genes with TFAP2A-dependent expression in melanocytes, we profile zebrafish tissue and mouse melanocytes deficient in Tfap2a, and find that expression of a small subset of genes underlying pigmentation phenotypes is TFAP2A-dependent, including Dct, Mc1r, Mlph, and Pmel. We then conduct TFAP2A ChIP-seq in mouse and human melanocytes and find that a much larger subset of pigmentation genes is associated with active regulatory elements bound by TFAP2A. These elements are also frequently bound by MITF, which is considered the “master regulator” of melanocyte development. For example, the promoter of TRPM1 is bound by both TFAP2A and MITF, and we show that the activity of a minimal TRPM1 promoter is lost upon deletion of the TFAP2A binding sites. However, the expression of Trpm1 is not TFAP2A-dependent, implying that additional TFAP2 paralogs function redundantly to drive melanocyte differentiation, which is consistent with previous results from zebrafish. Paralogs Tfap2a and Tfap2b are both expressed in mouse melanocytes, and we show that mouse embryos with Wnt1-Cre-mediated deletion of Tfap2a and Tfap2b in the neural crest almost completely lack melanocytes but retain neural crest-derived sensory ganglia. These results suggest that TFAP2 paralogs, like MITF, are also necessary for induction of the melanocyte lineage. Finally, we observe a genetic interaction between tfap2a and mitfa in zebrafish, but find that artificially elevating expression of tfap2a does not increase levels of melanin in mitfa hypomorphic or loss-of-function mutants. Collectively, these results show that TFAP2 paralogs, operating alongside lineage-specific transcription factors such as MITF, directly regulate effectors of terminal differentiation in melanocytes. In addition, they suggest that TFAP2A activity, like MITF activity, has the potential to modulate the phenotype of melanoma cells.

Abstract 224: The role of the microphthalmia associated transcription factor in autophagy regulation in melanoma

Abstract Autophagy is considered a therapeutic target in various cancer types, including melanoma. The transcription factors TFEB and TFE3 have been shown to play a key role in autophagy regulation in various cell types by affecting gene expression required for this process. A close relative to these factors is the transcription factor MITF; often termed the master regulator of melanocyte development and function. MITF activity has also been shown to be important for various stages of melanoma formation and progression. Analysis of The Cancer Genome Atlas expression data from melanoma tumors reveals that MITF expression correlates with lysosomal and autophagy gene expression in metastatic tumors. Furthermore, analysis of expression data from 23 melanoma cell lines shows that MITF expression correlates with lysosomal and autophagy gene expression, and the analysis of available MITF ChIP sequencing data shows that MITF binds the promoters of lysosomal and autophagy genes. This binding was experimentally verified by ChIP experiments in 501Mel melanoma cells. MITF over-expression and knock down experiments show effects of MITF on lysosomal and autophagy gene expression, as well as an effect on the autophagy activity. Autophagy is high in melanoma and has been linked to tolerance against cancer treatment. We have found that MITF regulates autophagy genes and activity in melanoma cell lines. In light of both MITF and autophagy being considered a therapeutic target in melanoma, this relationship needs to be taken into consideration. Citation Format: Katrin Moller, Sara Sigurbjornsdottir, Remina Dilixiati, Solveig H. Brynjolfsdottir, Lionel Larue, Vesteinn Thorsson, Eirikur Steingrimsson, Margret H. Ogmundsdottir. The role of the microphthalmia associated transcription factor in autophagy regulation in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 224.

Control of lysosomal biogenesis and Notch-dependent tissue patterning by components of the TFEB-V-ATPase axis in Drosophila melanogaster

ABSTRACTIn vertebrates, TFEB (transcription factor EB) and MITF (microphthalmia-associated transcription factor) family of basic Helix-Loop-Helix (bHLH) transcription factors regulates both lysosomal function and organ development. However, it is not clear whether these 2 processes are interconnected. Here, we show that Mitf, the single TFEB and MITF ortholog in Drosophila, controls expression of vacuolar-type H+-ATPase pump (V-ATPase) subunits. Remarkably, we also find that expression of Vha16-1 and Vha13, encoding 2 key components of V-ATPase, is patterned in the wing imaginal disc. In particular, Vha16-1 expression follows differentiation of proneural regions of the disc. These regions, which will form sensory organs in the adult, appear to possess a distinctive endolysosomal compartment and Notch (N) localization. Modulation of Mitf activity in the disc in vivo alters endolysosomal function and disrupts proneural patterning. Similar to our findings in Drosophila, in human breast epithelial cells we observe that impairment of the Vha16-1 human ortholog ATP6V0C changes the size and function of the endolysosomal compartment and that depletion of TFEB reduces ligand-independent N signaling activity. Our data suggest that lysosomal-associated functions regulated by the TFEB-V-ATPase axis might play a conserved role in shaping cell fate.

Abstract 2682: Melanoma phenotype switching to adapt to BRAF inhibition

Abstract Melanoma can adapt to several stress factors, such as TNF treatment, by changing its phenotype from a highly differentiated (MITF and MART-1 positive) to undifferentiated (neural crest markers) states. We studied this phenomenon as a mechanism of adaptive resistance to BRAF inhibition since melanomas with both MITF high and low have been reported to result in BRAF inhibitor resistance. We treated 17 BRAFV600E mutant melanoma cell lines with the BRAF inhibitor vemurafenib for short term (3 days) or long term (17 days), and compared them to no treatment or TNF treated conditions. We profiled them phenotypically for MART1 antigen (differentiated phenotype) and nerve growth factor receptor (NGFR, neural crest phenotype) expression by flow cytometry. Results comparing median fluorescence intensity (MFI) showed a wide spectrum of adaptive responses to short term BRAF inhibition, followed by solidification or further evolution of these changes upon long term exposure and development of drug tolerance. According to each cell line distribution, we calculated a (x,y) value that organized the cell lines and conditions in four quadrants according to MART1 (x axis) or NGFR (y axis) expression. Two double negative (MART1-/NGFR-) cell lines that were unaffected by treatment and stayed in the same quadrant. Seven MART1-/NGFR+ and four MART1+/NGFR- cell lines slightly changed phenotype with vemurafenib exposure but stayed in the same quadrant. Four cell lines with MART1+/NGFR+ phenotype were greatly influenced by treatment, showing wide plasticity with cells shifting towards NGFR+/MART1- and others towards NGFR-/MART1+. Changes in position between baseline and tolerance to vemurafenib after 17days treatment were numerically quantified as an inferred number from plasticity. Cell lines with double positive (MART1+/NGFR+) phenotype had a significant change in plasticity compared to the rest of cell lines in other quadrants (p = 0.0004). This arranges cells in a four-quadrant pattern that could be likened to a U-turn toward de-differentiation. Cells that de-differentiated with BRAF inhibition mimicked the effect induced by TNF. This effect was driven by a decrease in MITF activity confirmed with a tyrosinase promoter-luciferase assay. In conclusion, baseline melanoma differentiation phenotypes guide the phenotype switch response, with those double positive for differentiation and neural crest markers being at a semi-stable state that can be prone to different plastic changes upon BRAF inhibition. Citation Format: Lidia Robert, Jennifer Tsoi, Angel Garica Diaz, Blanca Homet Moreno, Nhat Truong, Thomas Graeber, Antoni Ribas. Melanoma phenotype switching to adapt to BRAF inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2682. doi:10.1158/1538-7445.AM2015-2682

Phenotypic diversity of patient-derived melanoma populations in stem cell medium

Melanomas are highly heterogeneous tumors and there is no treatment effective at achieving long-term remission for metastatic melanoma patients. Thus, an appropriate model system for studying melanoma biology and response to drugs is necessary. It has been shown that composition of the medium is a critical factor in preserving the complexity of the tumor in in vitro settings, and melanospheres maintained in stem cell medium are a good model in this respect. In the present study, we observed that not all nodular melanoma patient-derived cell populations grown in stem cell medium were capable of forming melanospheres, and cell aggregates and anchorage-independent single-cell cultures emerged instead. Self-renewing capacity and unlimited growth potential indicated the presence of cells with stem-like properties in all patient-derived populations but immunophenotype and MITF expression exhibited variability. Enhanced MITF expression and activity was observed in melanospheres in comparison with cell aggregates and single-cell culture, and hypoxic-like conditions that increased the ability of single-cell population to form melanospheres enhanced MITF expression and cell pigmentation as well. Thus, MITF seems to be a critical transcription factor for formation of both patient-derived and hypoxia-induced melanospheres. After 2 years of continuous culturing, melanospheres progressively underwent transition into cell aggregates that was accompanied by changes in expression of several MITF-dependent genes associated with melanogenesis and survival and alterations in the composition of subpopulations but not in the frequency of ABCB5-positive cells. Several biological properties of parent tumor are well preserved in patient-derived melanospheres, but during prolonged culturing the heterogeneity is substantially lost when the melanospheres are substituted by cell aggregates. This should be considered when cell aggregates instead of melanospheres are used in the study of melanoma biology and cell response to drugs.

Deletion of histone deacetylase 7 in osteoclasts decreases bone mass in mice by interactions with MITF.

Molecular regulators of osteoclast formation and function are an important area of research due to the central role of osteoclasts in bone resorption. Transcription factors such as MITF are essential for osteoclast generation by regulating expression of the genes required for cellular differentiation and resorptive function. We recently reported that histone deacetylase 7 (HDAC7) binds to and represses the transcriptional activity of MITF in osteoclasts, and that loss of HDAC7 in vitro accelerated osteoclastogenesis. In the current study, we extend this initial observation by showing that conditional deletion of HDAC7 in osteoclasts of mice leads to an in vivo enhancement in osteoclast formation, associated with increased bone resorption and lower bone mass. Expression of multiple MITF target genes is increased in bone marrow derived osteoclast cultures from the HDAC7 knockout mice. Interestingly, multiple regions of the HDAC7 amino-terminus can bind to MITF or exert repressive activity. Moreover, mutation or deletion of the HDAC7 conserved deacetylase catalytic domain had little effect on repressive function. These observations identify HDAC7 in osteoclasts as an important molecular regulator of MITF activity and bone homeostasis, but also highlight a gap in our understanding of exactly how HDAC7 functions as a corepressor.

Abstract PR04: A melanoma transcriptional state distinction influences sensitivity to MAPK pathway inhibitors

Abstract The deployment of cancer therapeutics that exploit oncogenic dependencies has yielded remarkable advances in patient treatment. However, the therapeutic benefit of these approaches is transient and the majority of patients develop resistance within several months. BRAFV600E-mutant malignant melanoma provides an illustrative example of this phenomenon: treatment with RAF and MEK inhibitors yields clinical responses in 50-80% of patients. However, 10-20% fail to respond to treatment (intrinsic resistance) and patients that do respond become drug resistant within ∼9 months (acquired resistance), presenting a formidable and unsolved clinical challenge. It remains incompletely understood why a subset of BRAFV600-mutant melanoma patients (10-20%) fail to respond to MAPK-pathway inhibition. Here, we show that RAF inhibitor sensitive and resistant BRAFV600-mutant melanomas display distinct transcriptional profiles. RAF-inhibitor sensitive cell lines are distinguishable by expression and activity of the melanocytic lineage transcription factor MITF, whereas intrinsically drug-resistant cell lines are defined by expression of the receptor tyrosine kinase AXL and elevated levels of NF-κB signaling. In vitro, these signatures were sufficient to predict MAPK-pathway inhibitor responsiveness in independent panels of melanoma cell lines. MITF-low, AXL/NF-κB high melanomas were resistant to single-agent RAF, MEK and ERK and combined RAF/MEK inhibition. In treatment-naïve patient biopsies, markers of the drug sensitive transcriptional states were associated with improved therapeutic responses to combined RAF/MEK inhibitors in BRAFV600-mutant melanoma. Moreover, in cell lines, NF-κB activation antagonized MITF expression and induced both resistance marker genes and drug resistance. Thus, distinct cell states characterized by MITF and AXL/NF-κB activity can influence intrinsic resistance to MAPK pathway inhibitors in BRAFV600-mutant melanoma. More broadly, these data suggest that the transcriptional context in which an oncogenic event arises can have a profound impact on the establishment of oncogene-dependencies and associated drug susceptibilities. Citation Format: Cory Johannessen, David Konieczkowski, Omar Abudayyeh, Jong Wook Kim, Zachary Cooper, Adriano Piris, Dennie Frederick, Michal Barzily-Rokni, Ravid Straussman, Rizwan Haq, David Fisher, Jill Mesirov, William Hahn, Keith Flaherty, Jennifer Wargo, Pablo Tamayo, Levi Garraway. A melanoma transcriptional state distinction influences sensitivity to MAPK pathway inhibitors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr PR04.

Oncogene addiction: pathways of therapeutic response, resistance, and road maps toward a cure.

A key goal of cancer therapeutics is to selectively target the genetic lesions that initiate and maintain cancer cell proliferation and survival. While most cancers harbor multiple oncogenic mutations, a wealth of preclinical and clinical data supports that many cancers are sensitive to inhibition of single oncogenes, a concept referred to as 'oncogene addiction'. Herein, we describe the clinical evidence supporting oncogene addiction and discuss common mechanistic themes emerging from the response and acquired resistance to oncogene-targeted therapies. Finally, we suggest several opportunities toward exploiting oncogene addiction to achieve curative cancer therapies.

Abstract 2950: Regulation of the functional interface between nucleotide excision repair and transcription by MITF modulates melanoma growth

Abstract Nucleotide excision repair (NER) is an essential instrument to maintain genomic stability in eukaryotes especially towards ultraviolet radiation (UVR). There is profound knowledge of the mechanistic execution of NER, however its regulation is not well understood. Here we provide evidence that the lineage-dependent oncogene MITF regulates the functional interface between NER and transcription through transactivation of XPG and GTF2H1, which constitute core elements of the NER and general transcription factor machineries, respectively. For this purpose, flow cytometry and microscopy fluorescence assays were applied to evaluate the temporal repair kinetics of DNA lesions after UVR in the presence and upon depletion of MITF in human primary melanocytes and several melanoma cell lines. Moreover, ChIP and luciferase reporter assays were performed to show the in vivo occupancy and a MITF dependent transactivation of the XPG and GTF2H1 promoters. Transcriptome as well as tissue microarrays (TMA) analysis were used to demonstrate the relationship between MITF and TFIIH. Finally, in vivo experiments were performed utilizing a subcutaneous xenograft tumor model in SCID mice to confirm our hypothesis. Our results showed that the NER/TFIIH complex is controlled by MITF in the presence and absence of UVR mediated genotoxic attack resulting in nucleotide repair deficiency and breakdown of global transcription upon MITF depletion. Importantly, in primary human melanoma the TFIIH subunit GTF2H1, which is involved in NER and RNA transcription, is linked to MITF abundance and prognostic melanoma stage. Moreover, RNA-interference mediated repression of GTF2H1 led to a significant reduction in tumor formation in a melanoma xenograft model. Collectively, these results describe an unanticipated role of MITF in the regulation of intimately linked NER and transcription machineries in the melanocytic lineage, which is preserved upon transformation into melanoma. Through its control of the NER/TFIIH complex MITF might coordinately regulate repair and transcription processes, thereby optimizing the rapid resumption of transcriptional activity after completion of strand repair, which is vitally important for cellular survival. The very same mechanism may drive the genesis of melanoma and its progression in the context of aberrant transcriptional activity of MITF due to SUMOylation defective germline mutation or genomic amplification. Citation Format: Marcos Seoane, Laia Pagerols Raluy, Karoline Kaufmann, Julia Strauss, Kevin Dierck, Jüergen Thomale, Johanna M. Brandner, Udo Schumacher, Peter Nollau, Peter J. Wild, Martin Zimmermann, Jinyan Du, David E. Fisher, Martin A. Horstmann. Regulation of the functional interface between nucleotide excision repair and transcription by MITF modulates melanoma growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2950. doi:10.1158/1538-7445.AM2014-2950

Next-generation sequencing of translocation renal cell carcinoma reveals novel RNA splicing partners and frequent mutations of chromatin-remodeling genes.

MITF/TFE translocation renal cell carcinoma (TRCC) is a rare subtype of kidney cancer. Its incidence and the genome-wide characterization of its genetic origin have not been fully elucidated. We performed RNA and exome sequencing on an exploratory set of TRCC (n = 7), and validated our findings using The Cancer Genome Atlas (TCGA) clear-cell RCC (ccRCC) dataset (n = 460). Using the TCGA dataset, we identified seven TRCC (1.5%) cases and determined their genomic profile. We discovered three novel partners of MITF/TFE (LUC7L3, KHSRP, and KHDRBS2) that are involved in RNA splicing. TRCC displayed a unique gene expression signature as compared with other RCC types, and showed activation of MITF, the transforming growth factor β1 and the PI3K complex targets. Genes differentially spliced between TRCC and other RCC types were enriched for MITF and ID2 targets. Exome sequencing of TRCC revealed a distinct mutational spectrum as compared with ccRCC, with frequent mutations in chromatin-remodeling genes (six of eight cases, three of which were from the TCGA). In two cases, we identified mutations in INO80D, an ATP-dependent chromatin-remodeling gene, previously shown to control the amplitude of the S phase. Knockdown of INO80D decreased cell proliferation in a novel cell line bearing LUC7L3-TFE3 translocation. This genome-wide study defines the incidence of TRCC within a ccRCC-directed project and expands the genomic spectrum of TRCC by identifying novel MITF/TFE partners involved in RNA splicing and frequent mutations in chromatin-remodeling genes.

A melanoma cell state distinction influences sensitivity to MAPK pathway inhibitors.

Most melanomas harbor oncogenic BRAF(V600) mutations, which constitutively activate the MAPK pathway. Although MAPK pathway inhibitors show clinical benefit in BRAF(V600)-mutant melanoma, it remains incompletely understood why 10% to 20% of patients fail to respond. Here, we show that RAF inhibitor-sensitive and inhibitor-resistant BRAF(V600)-mutant melanomas display distinct transcriptional profiles. Whereas most drug-sensitive cell lines and patient biopsies showed high expression and activity of the melanocytic lineage transcription factor MITF, intrinsically resistant cell lines and biopsies displayed low MITF expression but higher levels of NF-κB signaling and the receptor tyrosine kinase AXL. In vitro, these MITF-low/NF-κB-high melanomas were resistant to inhibition of RAF and MEK, singly or in combination, and ERK. Moreover, in cell lines, NF-κB activation antagonized MITF expression and induced both resistance marker genes and drug resistance. Thus, distinct cell states characterized by MITF or NF-κB activity may influence intrinsic resistance to MAPK pathway inhibitors in BRAF(V600)-mutant melanoma. Although most BRAF(V600)-mutant melanomas are sensitive to RAF and/or MEK inhibitors, a subset fails to respond to such treatment. This study characterizes a transcriptional cell state distinction linked to MITF and NF-κB that may modulate intrinsic sensitivity of melanomas to MAPK pathway inhibitors.

Fishful thinking: the rise and fall of MITF in melanoma

Fishful thinking: the rise and fall of <scp>MITF</scp> in melanoma

Abstract 2980: Integrated transcriptome and methylome sequencing of Xp11 translocation renal cell carcinomas reveal uncoupling of DNA methylation and transcriptional profiles.

Abstract Background: Xp11 translocation renal cell carcinoma (RCC) is a rare subtype of RCC characterized by the fusion of TFE3 gene to multiple partners. Despite its genetic homogeneity, there is considerable heterogeneity in the clinical course of this disease, probably due to epigenetic events that follow the initial translocation. Material and methods: We performed whole-transcriptome next-gen paired end sequencing of 7 Xp11 RCC samples, 10 clear-cell RCC and 19 papillary RCC. In addition, we performed genome-wide analysis of DNA methylation of 9 Xp11 samples by Digital Restriction Enzyme analysis of Methylation (DREAM), based on next-generation sequencing. Results: Using a robust bioinformatics pipelines, we confirmed 6 previously known TFE3 translocation partners, and discovered a novel partner involving the splicing gene LUC7L3 that we validated by RT-PCR. Unsupervised clustering of gene expression profiling revealed genetic homogeneity of Xp11 cases that clustered together, independently from clear-cell and papillary RCC subgroups. Ingenuity analysis confirmed MITF activation. Gene Set Enrichment Analysis (GSEA) showed mTOR pathway activation. In contrast with genetic homogeneity of gene expression, unsupervised clustering analysis of CpG sites located within CpG islands (CGI) revealed: (1) a CpG islands methylator phenotype (CIMP+) subgroup related to patients with metastatic disease (n=3), and (2) a CIMP negative (CIMP-) subgroup related to patients with local disease (n=6). Integrative analysis showed that genes with differentially methylated levels between CIMP+ and CIMP- tumors (SD: +/-20%) and which were down-regulated in CIMP+ subtype as compared to normal kidney, were also down-regulated in CIMP- subgroup . Of note, FZD10 and CCDC8 were found to gain promoter methylation in three cases of Xp11 RCC, but their baseline expression was already low in normal kidney. In contrast, small gain of promoter DNA methylation in cancer samples was associated with gene silencing of putative tumor suppressor genes. Interestingly, GSEA for gene body methylation identified enrichment of MAPK signaling pathway in CIMP+ versus CIMP- cases. Exome-sequencing of 2 out of 3 cases with CIMP+ phenotype identified non-synonymous IDH1 (p.V71I) mutation present in COSMIC database for one case and a frameshift mutation of the chromatin remodeling enzyme INO80D in the second case. Conclusion: Our data reveal uncoupling of DNA methylation and gene expression profiling in Xp11 RCC. Patients with metastatic disease showed higher methylation level, with CIMP+ phenotype possibly related to IDH1 mutation in one case. These results suggest that additional epigenetic alterations following the initial Xp11 translocation may explain the aggressive biology of some Xp11 RCCs. Citation Format: Gabriel G. Malouf, Xiaoping Su, Hui Yao, Christopher G. Wood, Yue Lu, Shoudan Liang, Erika J. Thompson, Denaha J. Doss, Jaroslav Jelinek, Jean-pierre Issa, Nizar M. Tannir. Integrated transcriptome and methylome sequencing of Xp11 translocation renal cell carcinomas reveal uncoupling of DNA methylation and transcriptional profiles. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2980. doi:10.1158/1538-7445.AM2013-2980

The masters talk: the PGC-1α-MITF axis as a melanoma energizer.

The masters talk: the PGC-1α-MITF axis as a melanoma energizer.

Activation of MITF by Argan Oil Leads to the Inhibition of the Tyrosinase and Dopachrome Tautomerase Expressions in B16 Murine Melanoma Cells

Argan (Argania spinosa L.) oil has been used for centuries in Morocco as cosmetic oil to maintain a fair complexion and to cure skin pimples and chicken pox pustules scars. Although it is popular, the scientific basis for its effect on the skin has not yet been established. Here, the melanogenesis regulatory effect of argan oil was evaluated using B16 murine melanoma cells. Results of melanin assay using B16 cells treated with different concentrations of argan oil showed a dose-dependent decrease in melanin content. Western blot results showed that the expression levels of tyrosinase (TYR), tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase (DCT) proteins were decreased. In addition, there was an increase in the activation of MITF and ERK1/2. Real-time PCR results revealed a downregulation of Tyr, Trp1, Dct, and Mitf mRNA expressions. Argan oil treatment causes MITF phosphorylation which subsequently inhibited the transcription of melanogenic enzymes, TYR and DCT. The inhibitory effect of argan oil on melanin biosynthesis may be attributed to tocopherols as well as the synergistic effect of its components. The results of this study provide the scientific basis for the traditionally established benefits of argan oil and present its therapeutic potential against hyperpigmentation disorders.

Otx but Not Mitf Transcription Factors Are Required for Zebrafish Retinal Pigment Epithelium Development

Otx and Mitf transcription factors have been implicated in the development of the retinal pigmented epithelium (RPE), but the relationship between these factors and their specific roles in the development of the RPE have not been fully defined. The role of the three Otx transcription factors (Otx1a, Otx1b, and Otx2) and two Mitf transcription factors (Mitfa and Mitfb) in the development of the zebrafish RPE was explored in these experiments. The loss of Otx activity through morpholino knockdown produced variable eye defects, ranging from delayed RPE pigmentation to severe coloboma, depending on the combination of Otx factors that were targeted. Expression analysis through in situ hybridization demonstrates that otx transcription factors are necessary for the proper expression of mitfa and mitfb while Mitf transcription factors are not required for the expression of otx genes. Surprisingly, the loss of Mitf activity in mitfa, mitfb, or double mitf mutant zebrafish had no effect on RPE pigmentation or development. Moreover, histological analysis revealed that retinal lamination is unaffected in mitf mutants, as well as in otx morphants, even in regions lacking RPE. Otx and Mitf combined loss of function experiments suggest that mitfa and mitfb may still influence zebrafish RPE development. This is further supported by the ability of mitfa to induce pigmentation in the zebrafish retina when misexpressed. These findings suggest that one or more Otx targets in addition to mitfa and mitfb, possibly another mitf family member, are necessary for development of the RPE in zebrafish.

Abstract 3093: Elucidation of c-KIT- dependent signaling to MITF

Abstract MITF, the master regulator of melanocytes, is a basic basic- helix- loop-helix- leucine zipper transcription factor that is essential for the development of melanocytes. MITF is also a lineage- survival oncogene in melanoma and recently, it has been shown that germline mutations in MITF predispose to this malignant disease. In melanoma, MITF has been proposed to act as a rheostat where high levels of MITF contribute to a proliferative phenotype whereas low levels result in a more invasive phenotype. The biological output generated by high or low levels of MITF is well characterized. However, how the activity of MITF is regulated to mediate these effects is less well known. It has been proposed that MITF is phosphorylated upon ligand induced activation of the receptor tyrosine kinase c- KIT. C-KIT engages the MAP kinase signaling cascade where ERK2 directly phosphorylates Ser73 of MITF and P90RSK phosphorylates the Ser409 residue of MITF. As a consequence, MITF activity increases and the protein is subjected to ubiquitin dependent degradation.Here, we show for the first time that c-KIT induces phosphorylation of MITF through the survival PI3 kinase- AKT pathway, the P38 stress pathway as well as the well-known MAP kinase-ERK signaling pathway. In addition, we show that the SRC kinase upstream of ERK is essential for c-KIT induced MITF activation. Moreover, we used several c-KIT phosphorylation site mutants to map the specific sites that are involved in the mediating transcription activation potential of MITF. These mutants also show less effects on MITF-mediated proliferation.We have generated phosphorylation specific antibodies against Ser73 and Ser409 of MITF and characterized the phosphorylation pattern of MITF using these reagents. Our results suggest that there is a particular relationship between Ser73 and Ser409 with subsequent effects on function. Based on our findings, we propose that phosphorylation of MITF might act as negative feedback mechanism that keeps MITF activity in check. Our model challenges the current knowledge of MITF modulation and provides insights that may have a profound impact on our understanding of the role of MITF in melanocyte and melanoma biology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3093. doi:1538-7445.AM2012-3093

Sox2 and Mitf cross-regulatory interactions consolidate progenitor and melanocyte lineages in the cranial neural crest

The cellular origin and molecular mechanisms regulating pigmentation of head and neck are largely unknown. Melanocyte specification is controlled by the transcriptional activity of Mitf, but no general logic has emerged to explain how Mitf and progenitor transcriptional activities consolidate melanocyte and progenitor cell fates. We show that cranial melanocytes arise from at least two different cellular sources: initially from nerve-associated Schwann cell precursors (SCPs) and later from a cellular source that is independent of nerves. Unlike the midbrain-hindbrain cluster from which melanoblasts arise independently of nerves, a large center of melanocytes in and around cranial nerves IX-X is derived from SCPs, as shown by genetic cell-lineage tracing and analysis of ErbB3-null mutant mice. Conditional gain- and loss-of-function experiments show genetically that cell fates in the neural crest involve both the SRY transcription factor Sox2 and Mitf, which consolidate an SCP progenitor or melanocyte fate by cross-regulatory interactions. A gradual downregulation of Sox2 in progenitors during development permits the differentiation of both neural crest- and SCP-derived progenitors into melanocytes, and an initial small pool of nerve-associated melanoblasts expands in number and disperses under the control of endothelin receptor B (Ednrb) and Wnt5a signaling.

C-KIT Signaling Depends on Microphthalmia-Associated Transcription Factor for Effects on Cell Proliferation

The development of melanocytes is regulated by the tyrosine kinase receptor c-KIT and the basic-helix-loop-helix-leucine zipper transcription factor Mitf. These essential melanocyte survival regulators are also well known oncogenic factors in malignant melanoma. Despite their importance, not much is known about the regulatory mechanisms and signaling pathways involved. In this study, we therefore sought to identify the signaling pathways and mechanisms involved in c-KIT mediated regulation of Mitf. We report that c-KIT stimulation leads to the activation of Mitf specifically through the c-KIT phosphorylation sites Y721 (PI3 kinase binding site), Y568 and Y570 (Src binding site). Our study not only confirms the involvement of Ras-Erk signaling pathway in the activation of Mitf, but also establishes that Src kinase binding to Y568 and Y570 of c-KIT is required. Using specific inhibitors we observe and verify that c-KIT induced activation of Mitf is dependent on PI3-, Akt-, Src-, p38- or Mek kinases. Moreover, the proliferative effect of c-KIT is dependent on Mitf in HEK293T cells. In contrast, c-KIT Y568F and Y721F mutants are less effective in driving cell proliferation, compared to wild type c-KIT. Our results reveal novel mechanisms by which c-KIT signaling regulates Mitf, with implications for understanding both melanocyte development and melanoma.