In this study we have used DNA sequence analysis to determine whether MITF
are targets of somatic mutation during melanomagenesis. Sequencing of all MITF
coding exons in a panel of primary and metastatic melanoma samples identified sixteen de novo
mutations. In total, the primary and metastatic melanoma samples analyzed harbored non-synonymous (NS) mutations and amplifications with frequencies of 13.2% (10/76) in MITF
and 8.6% (9/105) in SOX10
(Supplementary Table 2
). Although the sample size was insufficient to document statistical significance, the mutually exclusive nature of the MITF
mutations could indicate that their effects are functionally redundant. As many of the patients from which the metastatic tissues were derived have been treated with various chemotherapeutic regimens, the cytotoxic drugs might have caused some of the observed mutations. However, the finding of similar mutations in some of the primary melanomas mitigates this concern. Taken together, this data provides the first evidence for the presence of NS mutations in both MITF and SOX10 in both primary and metastatic melanoma samples underscoring the involvement of the MITF transcriptional network in melanoma tumorigenesis (Carreira et al., 2005
; Carreira et al., 2006
; Levy et al., 2006
It is interesting to note that unlike what has been observed for metastatic melanoma, no amplifications of MITF were observed in the primary tumor samples suggesting that amplification may be a late stage alteration taking place in a context dependent genome that can tolerate increased activity of MITF. This is consistent with prior analysis using arrays CGH primary melanomas (Curtin et al., 2005
) The non-synonymous MITF mutations identified in both primary and metastatic samples were always found as heterozygous for a wild type copy of MITF. The mutations were throughout the protein, causing amino acid substitutions in conserved residues in defined functional domains (). Two of the MITF
mutations (L135V and L142F) occurred in the conserved acidic activation domain (AD1) of MITF which has been shown to be necessary for MITF transcriptional activity (Supplementary Figure 2
). Two of the five MITF mutations G244R and 4TΔ2B have corresponding alleles in mouse, Mib
(Hallsson et al., 2000
, Steingrimsson et al., 1996
), respectively. In the Mib
“brownish” mouse model the same amino acid, G244, is found mutated from a small, non-polar AA to a large, charged AA side chain (G244E). The amino acid location of G244 is at the junction of the loop and helix 2 where the occurrence of a small, non-charged or hydrophobic amino acid and is conserved in all basic helix-loop-helix, leucine-zipper proteins (Steingrimsson et al., 1996
). Functional analysis of the Mib
allele demonstrated an MITF protein with reduced DNA binding activity, either as a homodimer or as a heterodimer in combination with TFE3. The second mouse model, Mibws
, is similar to the 4TΔ2B mutation in that it ultimately results in exon skipping and deletion of exon 2B. This animal exhibits a reduction in melanocyte cell numbers, as reflected by the black and white spotted (bws
) coat phenotype (Hallsson et al., 2000
). Within this exon is Ser73, whose phosphorylation status can mediate MITF protein turnover (Wu et al., 2000
, Xu et al., 2000
). Consistent with this finding, we observed increased protein expression for the 4TΔ2B allele mutated in melanoma. Subsequent analysis will be required to determine whether the MITF mutations present in these animal models, Mib
, confer increased frequency of melanoma metastasis as measured by intercrossing with murine melanoma-prone models.
Given the mutation spectrum and the results from corresponding mouse alleles it might be surprising that most of the mutations had minimal effects on MITF's ability to transactivate the melanogenic target genes, DCT and TYR. However, MITF provides transcriptional regulation for a wide array of genes, mediating transcriptional control over both differentiation (i.e., DCT and TYR) and cell cycle progression pathways (Bismuth et al., 2005
). Our analysis suggests that these MITF mutations may lead to alterations in the capacity of MITF to regulate gene expression in a promoter specific manner, as all five of the mutations analyzed exhibited reduced transactivation activity of the p21 promoter, an MITF target gene previously shown to regulate cell cycle progression (Carreira et al., 2005
). The uncoupling of MITF's ability to regulate differentiation and proliferation is consistent with studies by Bismuth et al., (2005)
where they showed specific isoforms and mutations of MITF can have different effects on cell proliferation, DNA binding and transcriptional activity. It will be interesting to explore how these melanoma associated mutations affect different aspects of cell cycle and target gene regulation.
The mutation spectrum we observed for SOX10 suggests that SOX10 might be acting as a tumor suppressor gene. In metastatic melanoma samples two of the three mutations generated frameshifts predicted to result in protein truncations before the DNA binding domain and thus a product with inactivation of SOX10 function. Consistent with this, these mutations did not transactivate the promoter of a known SOX10 target gene, DCT. The third mutation is predicted to produce a truncation and frameshift at the c-terminus of SOX10. This product demonstrated reduced activity on the DCT promoter, and in the tumor, this mutation was associated with LOH. Data from primary melanoma also support a SOX10 tumor suppressor gene model where we found that of the five samples that harbored somatic mutations, four also exhibited LOH and only retained the NS mutated allele. The fifth sample demonstrated two NS mutations, though it is not known if these are in trans or if a wild type copy remains and is expressed. All but one (R43Q) of the NS mutations in SOX10 that were identified in primary melanoma occurred within its transactivation domain, and strikingly two different melanoma samples had mutations affecting the same residue. However, these mutations did not alter SOX10's ability to transactivate DCT activity. It is possible that the melanomagenic activity for these SOX10 alleles is due to a reduced SOX10 function that is not observable on the DCT promoter under conditions used. While these data support a hypothesis of SOX10 as a tumor suppressor, further functional studies and assessment of additional mutations (for example gene amplifications) needs to be explored before a mechanistic model can be confirmed.
Alternatively, these alleles may promote differential effects on specific target genes similar to what we have shown for the MITF melanoma alleles ability to transactivate TYR
differently. As one of the known SOX10 target genes is MITF
it is possible that the various genetic alterations identified in this study have a direct or indirect effect on altering MITF activity, selected for downregulation of MITF activity to adjust to a favorable tumorigenic outcome. As the mutations of MITF and SOX10 are mutually exclusive in the melanoma samples we analyzed, our results support the “biological rheostat” model (Carreira et al., 2006
) where tight regulation of MITF levels is essential for balancing melanocyte growth and arrest.
In summary, this is the first study demonstrating that melanoma patients harbor somatic mutations in MITF and SOX10. Our findings indicate that a substantial fraction of melanomas acquire genetic alterations in the MITF pathway, occurring at both the primary and metastatic stages and suggest that altered MITF function during melanomagenesis can be achieved by MITF amplification, MITF single base substitutions or by mutation of its regulator SOX10.