Unknown,Transcriptomics,Genomics,Proteomics

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IGR_MITF_STUDY_BBRB

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

DISEASE(S): melanoma

SUBMITTER: Philippe DESSEN

PROVIDER: E-TABM-1198 | 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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IGR_MITF_STUDY_BBRB

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