Frequent mutations in the MITF pathway in melanoma

Abstract

Microphthalmia-associated transcription factor (MITF) is involved in melanocyte cell development, pigmentation and neoplasia. To determine whether MITF is somatically mutated in melanoma, we compared the sequence of MITF from primary and metastatic lesions to patient-matched normal DNA. In the 50 metastatic melanoma tumor lines analysed, we discovered four samples that had genomic amplifications of MITF and four that had MITF mutations in the regions encoding the transactivation, DNA binding or basic, helix-loop-helix domains. Sequence analysis for SOX10, a transcription factor, which both acts upstream of MITF and synergizes with MITF, identified an additional three samples with frameshift or nonsense mutations. Microphthalmia-associated transcription factor and SOX10 were found to be mutated in a mutually exclusive fashion, possibly suggesting disruption in a common genetic pathway. Taken together we found that over 20% of the metastatic melanoma cases had alterations in the MITF pathway. We show that the MITF pathway is also altered in primary melanomas: 2/26 demonstrated mutations in MITF and 6/55 demonstrated mutations in SOX10. Our findings suggest that altered MITF function during melanomagenesis can be achieved by MITF amplification, MITF single base substitutions or by mutation of its regulator SOX10.

Figure 1. MITF and SOX10 mutations in primary and metastaic melanoma

Schematic representing the protein domains of MITF and SOX10 show mutations found in primary melanoma (blue arrows) and metastatic melanoma (red arrows). Functional domains of MITF include AD1 (activation domain 1), AD2 (activation domain 2), AD3 (activation domain 3) and bHLHZip (basic helix-loop-helix leucine-zipper domain). Functional domains of SOX10 include HMG (High Mobility Group) and TAD (transcriptional activation domain).

Figure 2. Somatic mutation in tumor sample 4T results in MITF splice variant

A. RT-PCR analysis of MITF expression in tumor samples. The product of the wild type MITF band is 1260bp, the 4T sample products are 1260bp, 1193bp and 1092bp. The 1193bp transcript represents a variant with a 67bp deletion from exon 2B that did not express protein by western (data not shown). The 1092bp transcript encodes the 4TΔ2B MITF variant, reduced in size by 168bp. B. Somatic mutation of the MITF gene in sample 4T. The top sequence chromatogram was obtained from normal tissue and the lower sequence chromatogram from the 4T melanoma sample. Arrow indicates the location of the mutation. C. The MITF mutation A to G transition in nucleotide 260 is located in the splice donor and leads to a loss of exon 2B (shaded area) and an mRNA molecule that is 168 nucleotides shorter than wild-type message. D. Sequence analysis of the 1260bp cDNA.

Figure 3. Mutant forms of MITF activate HuDct and Tyrosinase luciferase but not p21 luciferase

A. HEK 293T cells were transiently transfected with the indicated constructs together with the HuDct-luciferase reporter construct. Firefly luciferase activities in samples were normalized to Renilla luciferase activities in the same samples. Ratios between firefly and Renilla luciferase activities are shown. B. and C. Immunoblots of immunoprecipitated MITF derived from transfected HEK 293T cells probed with the indicated antibodies. D. HEK 293T cells were transiently transfected with the WT or mutant MITF, or empty vector together with the Tyrosinase-luciferase reporter construct. Fold activation was calculated as in A. E. HEK 293T cells were transiently transfected with the WT or mutant MITF, or empty vector together with the p21-luciferase reporter construct. Fold activation was calculated as in A.

Figure 4. Truncated versions of SOX10 lack the ability to transactivate HuDct-Luciferase

NIH3T3 cells were transiently transfected with the indicated constructs together with the HuDct-luciferase reporter construct. Firefly luciferase activities in samples were normalized to Renilla luciferase activities in the same samples. Ratios between Firefly luciferase and Renilla luciferase activities are shown.