Unknown,Transcriptomics,Genomics,Proteomics

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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.

ORGANISM(S): Homo sapiens

SUBMITTER: HSIAOWANG CHEN

PROVIDER: E-GEOD-64497 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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MITF drives endolysosomal biogenesis and potentiates Wnt signaling in melanoma cells

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