MITF drives endolysosomal biogenesis and potentiates Wnt signaling in melanoma cells
ABSTRACT: Canonical Wnt signaling plays an important role in development and disease, regulating transcription of target genes and stabilizing many proteins phosphorylated by Glycogen Synthase Kinase 3 (GSK3). We observed that the MiT family of transcription factors, which includes the melanoma oncogene MITF and the lysosomal master regulator TFEB, had the highest phylogenetic conservation of three consecutive putative GSK3 phosphorylation sites in animal proteomes. This prompted us to examine the relationship between MITF, endolysosomal biogenesis and Wnt signaling. Here we report that MITF expression levels correlated with the expression of a large subset of lysosomal genes in melanoma cell lines. MITF expression in the Tetracycline-inducible C32 melanoma model caused a marked increase in vesicular structures, and increased expression of late endosomal proteins such as Rab7, LAMP1, and CD63. These late endosomes were not functional lysosomes as they were less active in proteolysis, yet were able to concentrate Axin1, phospho-LRP6, phospho-β-Catenin, and GSK3 in the presence of Wnt ligands. This relocalization significantly enhanced Wnt signaling by increasing the number of multivesicular bodies (MVBs) into which the Wnt signalosome/destruction complex becomes localized upon Wnt signaling. We also show that the MITF protein was stabilized by Wnt signaling, through the novel C-terminal GSK3 phosphorylations identified here. MITF stabilization caused an increase in MVB biosynthesis, which in turn increased Wnt signaling, generating a positive feed-back loop that may function during the proliferative stages of melanoma. The results underscore the importance of misregulated endolysosomal biogenesis in Wnt signaling and cancer. Expression of selected Lysosomal genes and CLEAR element plus MITF were compared in 51 melanoma cell lines to a mixed reference pool containing equal amounts of 47 melanoma cell lines.
Project description:About half of all melanomas harbor a constitutively active mutant BRAFV600E/K kinase that can be selectively inhibited by targeted BRAF inhibitors (BRAFi). While patients treated with BRAFi initially exhibit measurable clinical improvement, the majority of patients eventually develop drug resistance and relapse. We observe significant elevation of WNT5A in a subset of tumors from patients exhibiting disease progression on BRAFi therapy. WNT5A transcript and protein are also elevated in BRAFi-resistant melanoma cell lines generated by long-term in vitro treatment with BRAFi. RNAi-mediated reduction in levels of endogenous WNT5A in melanoma decreases cell growth, increases apoptosis in response to BRAFi challenge, and decreases the activity of pro-survival AKT signaling. Overexpression of WNT5A conversely promotes melanoma growth and tumorigenesis and activates AKT signaling. Similar to WNT5A knockdown, knockdown of the WNT receptors FZD7 and RYK inhibits growth, sensitizes melanoma cells to BRAFi, and reduces AKT activation. Together, these findings suggest that chronic BRAF inhibition elevates WNT5A expression, which then acts through FZD7 and RYK to promote AKT signaling, leading to increased growth and therapeutic resistance. Increased WNT5A expression in BRAFi-resistant melanomas also correlates with an associated transcriptional signature, which identifies potential therapeutic targets to reduce clinical resistance to BRAFi. Expression of WNT5A-correlated genes was compared in melanoma cell lines generated to be resistant to PLX4032 and the their associated naïve parental line Basal expression of the WNT5A-correlated genes was also measured in experiments comparing each naïve line to a mixed reference pool containing equal amounts of 47 melanoma cell lines.
Project description:We analyzed the transcriptional response of the human melanoma cell line MZ7 to TNF-alpha (24 hours) in a dose-dependent manner (TNF-alpha 10U/ml, 100U/ml, 1000U/ml) either transfected with control siRNA (siNT = non-targeting siRNA) or transfected with siRNAs (pool of 4 active and independent siRNAs) against the melanocytic transcription factor and lineage oncogene MITF. (Microphthalmia-associated transcription factor). The experiment was performed as biological duplicate. As MITF is critical for melanoma cell state control, we aimed to explore how MITF expression intersects with inflammation-induced plasticity pathways in melanoma. Total RNA was obtained from siRNA/TNF-treated MZ7 melanoma cell lines at various conditions and global gene expression profiling was done using the Illumina Human HT12 v4 platform.
Project description:The most critical stage in initiation of melanoma metastasis is the radial to vertical growth transition, yet the triggers of this transition remain elusive. We introduce a novel perspective, suggesting that the microenvironment drives melanoma metastasis independently of mutation acquisition. Here we examined the changes in microenvironment that occur during melanoma radial growth. We show that direct contact of melanoma cells with the remote epidermal layer triggers vertical invasion via Notch signaling activation, the latter serving to inhibit MITF function. Briefly, within the native Notch ligand-free microenvironment, MITF, the melanocyte lineage master regulator, binds and represses miR-222/221 promoter in an RBPJK-dependent manner. However, when radial growth brings melanoma cells into contact with distal differentiated keratinocytes that express Notch ligands, the activated Notch intracellular domain impairs MITF binding to miR-222/221 promoter. This de-repression of miR-222/221 expression triggers initiation of invasion. Our findings may direct novel prevention opportunities via targeting specific microenvironment. Two replicates of Notch-activated cells that were seeded on Delta-like-1 (DLL1) (2 ng/µl ) coated plates were compared to two replicates of cells without Notch activation. The goal of this experiment is to evaluate the changes of miRs expression in melanoma cells upon Notch signaling activation.
Project description:Ma-Mel-15 human melanoma cell cultures were transiently transfected (RNAiMax, Lipofectamin) with control siRNA, siRNA against MITF (pool of 4 siRNAs), siRNA against c-JUN (pool of 4 siRNAs) or combinations of siMITF and siJUN. Cells were then either treated with TNF-alpha (1000U/ml) for 24 hours or left untreated. The experiment was performed as biological duplicates. We aimed to determine how c-JUN cooperates with acute MITF-loss in human melanoma cells to increase inflammatory responsiveness and cell plasticity. Total RNA was obtained from siRNA/TNF-treated Ma-Mel-15 melanoma cell lines and global gene expression profiling was done using the Illumina Human HT12 v4 platform.
Project description:Melanoma cells from three surgical specimens of nodular melanoma were grown as anchorage-independent melanospheres in stem cell bFGF(+)EGF(+) medium and as adherent monolayer cultures in the presence of serum. RNA from melanospheres (DMBC2s, DMBC8s, DMBC10s) and their adherent monolayer counterparts (DMBC2a, DMBC8a, DMBC10a) were hybridized to the dual-color Agilent human genome array G4450A. A transcriptome profile was generated to explore the molecules governing phenotypes of melanospheres and monolayers. We demonstrated that melanospheres contained more pigmented cells which was consistent with higher expression of MITF and MITF-dependent genes responsible for differentiation, including TYR, TYRP1, MLANA and DCT. Expression of MITF-dependent BIRC7, BCL2 and BCL2A1 was reduced in monolayers, thus limiting the anti-apoptotic protection. The enhanced activity of MITF shown as the elevated expression of 74 MITF-dependent genes in melanospheres, identified MITF as a central regulator of this phenotype. Importantly, our study revealed that MITF and MITF-dependent genes were expressed in melanospheres at similar levels as in original tumors, much higher than in monolayers. The reduced MITF level in monolayers might be partially explained by suppression of the Wnt/beta-catenin pathway. Differential expression of Wnt/beta-catenin pathway components and target genes in melanospheres and monolayers points to strong influence of the microenvironment on the functional outcome of Wnt/beta-catenin pathway in melanoma, including differentiation, survival, proliferation and invasiveness. Silencing of the Wnt inhibitor DKK1 in monolayers increased the percentage of clonogenic cells. In summary, melanospheres more accurately mirrored the morphology, heterogeneity and gene expression profiles of original tumors than monolayers. Our study demonstrates the feasibility of utilizing melanospheres to unravel the molecular pathways sustaining melanoma heterogeneity and potentially to select compounds with anticancer activities.
Project description:We analyzed the transcriptional response of the human melanoma cell line Ma-Mel-15 either transfected with control siRNA (siNT = non-targeting siRNA) or transfected with siRNAs (pool of 4 active and independent siRNAs) directed against the melanocytic transcription factor and lineage oncogene MITF (Microphthalmia-associated transcription factor). The experiment was performed as biological duplicates and RNA was isolated 48 hours after siRNA transfection. We aimed to determine novel markers and pathways of melanoma cell plasticity. Total RNA was obtained from siRNA-treated Ma-Mel-15 melanoma cell lines and global gene expression profiling was done using the Illumina Human HT12 v4 platform.
Project description:A recent study of the characteristics of coexisting melanoma and renal cell carcinoma RCC in the same patients supports a genetic predisposition underlying the association between these two cancers. The microphthalmia associated transcription factor (MITF) was proposed to act as a melanoma oncogene; it also stimulates the transcription of hypoxia inducible factor (HIF1A), whose pathway is targeted by kidney cancer susceptibility genes. By sequencing the MITF gene, we detected a germ line missense substitution (p.E318K) in 5 of 62 patients affected with melanoma and RCC. When compared with 1,659 controls, this MITF substitution occurred at a significantly higher frequency in melanoma + RCC patients (p = 1.3x10-4), melanoma -only patients (p = 7.8 x10-5), and RCC-only patients (p = 0.008). Overall, p.E318K substitution carriers had a higher than fivefold increased risk of developing melanoma, RCC or both cancers (odds-ratio = 5.55, 95% confidence interval = 2.59 to 12.91). Codon 318 falls in the second MITF-conserved, small-ubiquitin like modifier (SUMO)-1 consensus binding site (YKXE) and the p.E318K substitution severely impairs SUMOylation of MITF. MITF p.E318K binds more efficiently to the HIF1A promoter and increases its activation compared to the wild type MITF. In RCC cell line, transcriptomic approach identifies a MITF p.E318K signature related to cell growth, proliferation and inflammation. MITF p.E318K is more potent than wild type MITF in promoting melanocytic and renal cell clonogenicity, migration and invasion, consistent with a gain-of-function role in tumorigenesis. Our data provide novel insights on the link between transcription, sumoylation and cancer, possibly mediated by oxidative stress. This set of 26 arrays presents expression data for 3 conditions (reference cell line, with addition of wt MITF or e318K MITF for two different cell lines (melanoma A375 and renal RCC4), in dye swap and 3 independant biological replicates.
Project description:Investigation of expression differences between skin and melanomas from a transgenic BRAFV600E zebrafish model of melanoma The embryos described in this study are further analyzed in a manuscript submitted for publication by White, et al. A 15 chip study using RNA extracted from either WT zebrafish skin, mitf-BRAFV600E;p53-/- skin or mitf-BRAFV600E;p53-/- melanoma
Project description:We aimed to analyze the effects of Wnt-1 overexpression on the mRNA expression profile of human melanoma in a mouse xenograft model and correlated the results with then presence or absence of lymphangiogenesis and metastasis. Affymetrix gene expression analysis revealed activation of canonical and non-canonical targets genes in response to Wnt-1 as compared with controls. In regard to lymphangiogenic factors, the amount of VEGF-C was the single best marker to correlate with the amount of lymph-angiogenesis. mRNA expression array of human melanoma orthotopically grown in SCID mice. Comparison includes mRNA expression profile of two melanoma cell-lines (A375 and M24met) stably overexpressing control vector or Wnt-1 treated with or without CsA. Comparison #1 comprised Wnt-1 versus control in A375 and M24met melanoma, respectively. Comparison #2 comprised Wnt-1 + Cyclosporine A (CsA) versus Wnt-1 without CsA.