Team finds amino acid change in MITF prevents sumoylation and increases transcription of protein’s target genes.
Researchers have identified a genetic variant that appears to double the background risk of familial or sporadic melanoma. The mutation leads to a single amino acid change in microphthalmia-associated transcription factor (MITF), a protein that has previously been found to be overexpressed or mutated in melanoma tumors.
The work, reported by a consortium of researchers in the U.S., U.K., and Australia, showed that the amino acid change effected by the germline MITF mutation blocks sumoylation at one of two sumoylation sites on MITF, which results in the increased transcription of a number of genes regulated by the protein.
The researchers, led by corresponding author Kevin M. Brown, M.D., at the Phoenix-based Translational Genomics Research Institute and the National Cancer Institute, say the data provide insights into the potential mechanism by which MITF mutation could cause cancer risk. Their studies are reported in Nature in a paper titled “A novel recurrent mutation in MITF predisposes to familial and sporadic melanoma.”
Candidate-gene and genome-wide association studies of melanoma have identified a number of variants linked with increased risk of skin cancer in the general population, but to date only two genes, CDKN2A and CDK4, have been linked with familial cases of melanoma, the researchers report. Prior research has indicated that mutations in CDKN2A account for approximately 40% of familial cases, while predisposing mutations in CDK4 have been reported in a very small number of melanoma kindreds.
The team’s hunt for additional genes that might be involved in familial melanoma started by sequencing the genome of a melanoma patient from an eight-case melanoma family that was negative for mutations in both CDKN2A and CDK4. This initial work identified some 410 novel variants that were predicted to affect protein structure, but the researchers homed in one germline SNP that resulted in a glutamic acid to lysine substitution in microphthalmia-associated transcription factor (MITF). When the UCSD team genotyped the remaining available affected individuals in the family, they found the same MITF E318K mutation in three out of seven cases, “consistent with it being a medium-penetrance melanoma risk variant.”
They point out that while linkage and GWAS studies haven’t previously found MITF to be associated with predisposition to melanoma or pigmentation-related disorders, the gene is somatically amplified, mutated, or overexpressed in some melanomas and is known to regulate genes involved in melanocyte development, function, and survival.
To see how often the MITF E318K mutation occurred in other melanoma patient cohorts and in the general population, the researchers genotyped two large Australian melanoma case-control samples. The MITF E318K variant was found in 14 out of 1,953 controls (a carrier frequency of 0.0072), which makes it rare in the population as a whole, they note. In contrast, it was present in 34 of the 2,059 cases, a carrier frequency of 0.0165.
This indicated that the E318K variant actually has a larger effect size than previously reported variants identified in melanoma GWAS, and a similar effect size to that of red-hair-color-associated variants of the melanocortin 1 receptor (MCIR) gene. Interestingly, the team points out, the MITF E318K variant was particularly enriched in cases displaying multiple primary melanomas and in those with family history of melanoma, but not in cases with where diagnosis occurred before the age of 40.
The investigators subsequently replicated their findings in two independent population-based case-control samples from the U.K. Combined analyses from these two cohorts confirmed that the MITF E318K variant occurred much more commonly in cases than control. The SNP was similarly associated with melanoma in the pooled U.K. and Australian data, with a calculated odds ratio of 2.19.
An association between MITF E318K and familial melanoma specifically was found through genetic screening of a cohort of 182 U.K. families with at least two melanoma cases, and another 88 Australian families with at least three cases. All the cases from these cohorts were negative for mutations in CDKN2A and CDK4. While the results showed that six families (2.2%) overall carried the mutation, in the U.K. cohort E318K was particularly enriched in those families with at least three melanoma cases. 7.4% of three-case families (4/54) carried the variant, but only 0.8% (1/128) of the families with two melanoma cases. Significantly, the MITF variant allele was also found to be strongly associated with nevus count in both the Australian and U.K. populations, and also, perhaps more surprisingly, with non-blue eye color. E318K wasn’t, however, linked with skin color, hair color, or freckling.
The E318K variant is sited within one of two IKXE consensus sites on MITF that have previously shown to be post-translationally modified by the addition of the small ubiquitin-like modifier (SUMO). Mutating the residue to which SUMO covalently binds has in addition been shown to block MITF sumoylation and increase MITF transcriptional activity, the team adds. They therefore hypothesized that the E318K variant might similarly block sumoylation and increase MITF transcription.
To look at this more closely they constructed a cDNA encoding His-tagged MITF carrying the E318K variation, and evaluated the effects of E318K on sumoylation, in comparison with both wild-type MITF and two previously characterized synthetic mutations, K316R and K182R, which are sited on the known MITF sumoylation sites. When these constructs were co-transfected into COS-7 cells along with hemagglutinin (HA)-tagged SUMO1, the wild-type MITF showed two SUMO1-modified forms, whereas the K316R and K182R variants showed only one modified form each. Similarly, the E318K variant resulted in complete loss of the doubly sumoylated form of MITF, and reduced the level of monosumoylated MITF. “When the second site is mutated (K182R) simultaneously with E318K, MITF sumoylation is completely abolished,” the team adds.
The next stage was to see what effects abrogation of sumoylation had on MITF target gene transcription. To this end the investigators developed a tetracycline-inducible system for the expression of wild-type MITF or the E318K variant in melanoma cell lines with constitutively low or undetectable levels of endogenous MITF. Initial studies showed that induction of wild-type or E318K MITF led to increased cell proliferation compared to uninduced controls, for each of the cell lines, although there was no significant difference in growth rate. At the genetic level, however, 17 of the 37 genes commonly regulated by both wild-type and E318K MITF were differentially expressed (at least a 1.25-fold difference) in the two cell lines. “We also identified two gene products that were uniquely differentially regulated compared to uninduced cells by the induction of wild-type MITF but not MITF E318K in both parental cell lines, and 16 gene products after induction of MITF E318K but not wild-type MITF,” they note. Of these, 61% had previously been identified as MITF targets.
Of particular note was the observed transcriptional differences in gene products known to be involved in pigmentation, such as DCT and MLANA, and the 22% increase in melanin content observed in HT-144 melanoma cells after induction of MITF E318K, compared with induction of wild-type MITF.
The increased expression of pigmentation genes associated with MITF E318K was consistent with an additional observation by the team that carriers were more likely to have darker (that is, non-blue) eye color, but conflicts somewhat with previously reported melanoma risk variants that are associated with fair skin, they point out. Also it just isn’t known yet whether the enhanced expression of pigment genes associated with increased E318K transcriptional activity is a contributor of melanomagenesis, or whether it simply reflects increased MITF activity.
“The new MITF variant reported here shows reasonably strong linkage to melanoma (lod score 2.7) but crucially not a high enough signal to be clearly visible in previous genome-wide linkage scans,” the UCSD team concludes. “Although the individual changes in transcription induced by the mutant E318K MITF in comparison to wild-type MITF are modest, the orchestrated change in the levels of multiple MITF target genes is likely to be biologically important, especially over the lifetime of a person.”
The doubling of background melanoma risk associated with the MITF variant is about the same as that conferred by severe sunburn, comments co-senior author Hensin Tsao, M.D., at the MGH Department of Dermatology and the Wellman Center for Photomedicine. “However, an important aspect of this study is the elegant demonstration of how the E318K change affects MITF function.”