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Title: A synonymous RET substitution enhances the oncogenic effect of an in-cis missense mutation by increasing constitutive splicing efficiency}
Article:
Abstract:
Synonymous mutations continue to be filtered out from most large-scale cancer genome studies, but several lines of evidence suggest they can play driver roles in neoplastic disease. We investigated a case of an aggressive, apparently sporadic medullary thyroid carcinoma( MTC) harboring a somatic RET p. Cys634Arg mutation( a known MTC driver). A germ-line RET substitution( p. Cys630=) had also been found but was considered clinically irrelevant because of its synonymous nature. Next generation sequencing( NGS) of the tumor tissues revealed that the RET mutations were in cis. There was no evidence of gene amplification. Expression analysis found an increase of RET transcript in p. Cys630=;p. Cys634Arg patient compared with that found in 7 MTCs harboring p. Cys634 mutations. Minigene expression assays demonstrated that the presence of the synonymous RET mutation was sufficient to explain the increased RET mRNA level. In silico analyses and RNA immunoprecipitation experiments showed that the p. Cys630 = variant created new exonic splicing enhancer motifs that enhanced SRp55 recruitment to the mutant allele, leading to more efficient maturation of its pre-mRNA and an increased abundance of mature mRNA encoding a constitutively active RET receptor. These findings document a novel mechanism by which synonymous mutations can contribute to cancer progression.
Introduction:
It is now clear that synonymous mutations—single-nucleotide substitutions that do not alter the amino acid encoded by the affected codon—can play functionally relevant roles in human disease[1]. Over three decades of research have shown that these mutations can affect protein synthesis and/or function by interfering with a host of cellular mechanisms ranging from pre-mRNA splicing to protein folding[2]. Despite these advances, synonymous mutations are still largely ignored in most studies of cancer, and they continue to be filtered out from large-scale analyses of cancer genomes[3]. However, a growing body of evidence indicates that these mutations might actually play driver roles in human cancer[3, 4]. Two recent studies provide evidence for the causal involvement of synonymous mutations in melanoma[5], as well as other cancer types[6]. This paper describes a series of molecular studies we conducted to explore the role played by a synonymous substitution affecting the RET proto-oncogene[MIM: 164761] in an aggressive case of medullary thyroid carcinoma( MTC[MIM: 155240]; http://www. omim. org). MTC is a relatively rare neuroendocrine calcitonin-secreting tumor derived from the parafollicular C-cells of the thyroid. Hereditary forms, including multiple endocrine neoplasia types 2A and 2B( MEN2A[MIM: 171400]; MEN2B[MIM: 162300]), in which MTC is associated with other endocrinopathies, are almost invariably associated with germ-line point mutations in RET. The membrane tyrosine kinase receptor encoded by this gene is activated by the binding of ligand—co-receptor complexes, which induces dimerization of RET proteins and autophosphorylation of intracellular tyrosine residues[7]. Gain-of-function point mutations frequently occur in RET’s cysteine-rich extracellular domain( e. g., those involving codon 634) or intracellular kinase domain( e. g., mutations at codon 918 or 804). Both result in inappropriate activation of the tyrosine kinase receptor—the former by inducing ligand-independent, disulfide-bonded homodimerization of the RET proteins, the latter by causing these proteins’ constitutive autophosphorylation[8]. Correlation has been observed between specific mutations and features of the hereditary MTC phenotype, including age at onset, tumor aggressiveness, and the presence of other endocrine tumors[9]. Somatic gain-of-function RET mutations appear to drive around half of all sporadic MTCs[10–12], but the genotype—phenotype correlations in these cases are less clear-cut[13]. The case we analyzed involved an apparently sporadic MTC harboring a somatic RET c. 1900T>C mutation[p. Cys634Arg] in a previously healthy 29-year-old man( patient ID0110M). The postoperative course was characterized by rapid disease spread. Despite multiple surgical interventions and systemic vandetanib therapy[14], the patient died approximately 3 years after diagnosis( Supplemental Note). Several clinical features of this case( e. g., age at onset, aggressive disease behavior[15]) were difficult to explain solely on the basis of the somatic RET p. Cys634Arg driver mutation identified by Sanger sequencing. Shortly before the patient’s death, we therefore began re-analyzing DNA and RNA samples from his primary and metastatic tumors using next-generation sequencing( NGS) to identify any somatic mutations in other cancer-relevant genes. No other known somatic driver mutations were found, but the results redirected our attention to the germ-line RET c. 1890C>T[p. Cys630=] substitution, which had been identified by the preoperative Sanger sequencing analysis and considered clinically inconsequential because of its synonymous nature. Our NGS data showed that the synonymous substitution was in cis with the somatic RET p. Cys634Arg driver mutation, and subsequent experiments demonstrated that it exerted appreciable effects on RET pre-mRNA splicing.
Discussion:
Synonymous variants continue to be filtered out from pipelines used to analyze data from large-scale cancer genome/exome studies[20]. Consequently, they are under-reported, and their potential to drive cancer has been underestimated. Recent evidence shows, however, that these variants can indeed play functionally relevant roles in neoplastic disease[3, 6, 21]. Supek et al.[6] found them to be significantly over-represented in oncogenes, as compared with both cancer-unrelated genes and tumor-suppressor genes, and up to half of all oncogene-associated synonymous variants appear to be under selection. They also reported a strong association between synonymous variants in oncogenes and aberrancies involving alternative splicing, reflecting the ability of these substitutions to generate, alter, and/or eliminate exonic splicing regulatory motifs. To validate their in silico predictions, the authors used minigene expression assays to assess the effects of 12 synonymous oncogene substitutions on exon inclusion/skipping patterns in their mature transcripts. Six of the 12 were found to affect alternative splicing by creating new ESEs and/or eliminating ESSs. These changes led mainly to diminished exon skipping, which in some cases caused increased expression of full-length isoforms of the encoded protein and, consequently, enhanced oncogene activity[6]. The synonymous RET p. Cys630 = substitution found in our patient’s germ-line DNA involved a constitutive exon( exon 11), i. e., one fundamental for the functional integrity of the encoded protein. The complex multifactorial process regulating constitutive splicing has thus evolved to ensure that these essential exons are unambiguously recognized by the splicing machinery and included without fail in the mature transcript[22]. Not surprisingly then, mutations altering the sequences of cis-elements that contribute to this optimal regulatory set-up( including ESEs and ESSs) have consistently been found to cause pre-mRNA maturation that is dysfunctional[19, 20, 23, 24]. These findings prompted our original search for qualitative alterations in RET mRNA in patient ID0110M’s tumor tissues. Instead, the tissues contained correctly spliced mature RET transcript but at higher levels than those found in control tumors. Consistently, in silico analyses did not predict that the presence of RET p. Cys630 = in exon 11 would reduce or eliminate ESE motifs in the pre-mRNA transcript from the mutant allele: instead, it was expected to create de novo ESEs and enhance the binding affinity of others for SR protein splicing regulators. These findings were also in line with our in vitro demonstration, compared with its WT counterpart, immature RET p. Cys630 = minigene transcript binds more SRp55, a protein implicated in spliceosome recruitment rather than in transcription regulation[25, 26]. Therefore, instead of provoking exon-11 missplicing, the RET p. Cys630 = substitution increased levels of correctly spliced mature transcript from the mutant allele, possibly by increasing the number of molecules of pre-mRNA entering in the splicing pathway. We used RET minigenes to investigate the synonymous substitution’s effect on exon 11 splicing at both its 5′ and 3’ ends. We chose to reduce intron size to more easily incorporate RET exons 10–12 into our splicing reporter construct. This design appears to have diminished the overall splicing efficiency compared with that achieved during in vivo transcript maturation, thereby facilitating our demonstration of the synonymous substitution’s effect on RET minigene maturation. Our in vitro findings led us to exclude the possibility that the p. Cys630 = substitution affects transcription or the stability of immature or mature forms of RNA. These data strengthen our suspicion that the variant’s effects were mediated by its impact on pre-RNA splicing, although they cannot exclude the involvement of other molecular mechanisms in vivo. Patient ID0110M’s germ-line RET p. Cys630 = substitution occurred in cis with a somatic missense RET mutation( p. Cys634Arg), a known driver of MTC. It is difficult to speculate on the potential clinical impact of the p. Cys630 = substitution in the absence of p. Cys634Arg mutation( particularly in light of the substantial gaps in our patient’s family history[Supplementary Note]). It is reasonable to assume, however, that a genetic variant with no effects on the amino-acid sequence of the RET protein would not generate a constitutively activated RET receptor. In that case, RET signaling levels would remain dependent on ligand availability. In terms of its biological impact, the higher levels of mature transcript produced by the p. Cys630 = substitution-bearing allele may resemble those generated by gene amplification. Ciampi et al. reported RET amplifications in both familial and sporadic MTCs, where their presence was generally correlated with relatively poor clinical outcomes. However, with rare exceptions, these amplifications were found in tumors that also harbored a RET mutation. The authors therefore excluded the possibility that RET amplification is an alternative mechanism of RET gene activation, suggesting instead that it might exert potentiating effects during the transformation and/or progression of RET-mutated MTCs[27]. This conclusion is in line with the results of our analyses. The presence of the synonymous p. Cys630 = substitution led to more efficient maturation of transcript from the mutant RET allele, which also harbored the somatic p. Cys634Arg mutation. Expression of p. Cys630=;Cys634Arg allele was thereby increased( at both the transcript and protein levels) over levels that would be expected in the absence of p. Cys630=. The result was an excess of RET p. Cys634Arg monomers, which are capable of ligand-independent dimerization and autophosphorylation of the RET receptor. Such effects could conceivably augment the p. Cys634Arg mutation’s transforming capacity, but on the basis of currently available data, we cannot draw any meaningful conclusions on this possibility. Moreover, the tumor’s transforming potential could also be influenced by multiple other mechanisms, genetic( i. e., CNV or mutations in distant regulatory regions) and/or epigenetic, and we cannot exclude their involvement in our patient’s disease on the basis of the experiments conducted thus far. Although the mechanism we hypothesize has not been previously described, it may be more common that it currently appears, given the high frequency of synonymous substitutions in oncogenes harboring activating non-synonymous mutations and their ability in this setting to increase the number of ESE motifs[3, 6]. Further study is mandatory, but the analyses we have conducted thus far shed new and interesting light on the complex regulation of splicing.
Synonymous mutations—once considered “silent” because they do not alter the gene product’s amino-acid sequence—are now emerging as potential drivers of cancer . Our recent investigation of an aggressive medullary thyroid carcinoma ( MTC ) revealed a novel mechanism that could underlie such effects . The MTC analyzed harbored a somatic p . Cys634Arg mutation of the RET protooncogene ( a well-known MTC driver ) . A second RET substitution ( p . Cys630= ) discovered in the germline had been considered clinically irrelevant because of its synonymous nature . Our next-generation sequencing analysis of the patient’s cancer tissues revealed that the RET mutations were in cis . We also found that RET mRNA levels in patient’s MTC were significantly higher than those found in seven other MTCs with various amino-acid substitutions at position 634 in RET . Subsequent experiments demonstrated that the presence of the synonymous mutation created new exonic splicing enhancer motifs in the mutant allele , which led to more efficient maturation of its transcript and increased expression of a constitutively active RET receptor .