A Common Cancer Risk-Associated Allele in the hTERT Locus Encodes a Dominant Negative Inhibitor of Telomerase

Abstract

The TERT-CLPTM1L region of chromosome 5p15.33 is a multi-cancer susceptibility locus that encodes the reverse transcriptase subunit, hTERT, of the telomerase enzyme. Numerous cancer-associated single-nucleotide polymorphisms (SNPs), including rs10069690, have been identified within the hTERT gene. The minor allele (A) at rs10069690 creates an additional splice donor site in intron 4 of hTERT, and is associated with an elevated risk of multiple cancers including breast and ovarian carcinomas. We previously demonstrated that the presence of this allele resulted in co-production of full length (FL)-hTERT and an alternatively spliced, INS1b, transcript. INS1b does not encode the reverse transcriptase domain required for telomerase enzyme activity, but we show here that INS1b protein retains its ability to bind to the telomerase RNA subunit, hTR. We also show that INS1b expression results in decreased telomerase activity, telomere shortening, and an increased telomere-specific DNA damage response (DDR). We employed antisense oligonucleotides to manipulate endogenous transcript expression in favor of INS1b, which resulted in a decrease in telomerase activity. These data provide the first detailed mechanistic insights into a cancer risk-associated SNP in the hTERT locus, which causes cell type-specific expression of INS1b transcript from the presence of an additional alternative splice site created in intron 4 by the risk allele. We predict that INS1b expression levels cause subtle inadequacies in telomerase-mediated telomere maintenance, resulting in an increased risk of genetic instability and therefore of tumorigenesis.

Genome-Wide Association Studies in Glioma

“…In the latest glioma meta-analysis, the strongest association at 5p15.33 is with rs10069690 and GBM. Intriguingly, the rs10069690 risk allele ( T ) has been shown to create a splice-donor site leading to an alternate TERT splice isoform lacking telomerase activity (60), providing direct evidence for its functional effects on tumor development.…”

“…Notable exceptions are the 17p13.1 locus, where rs78378222 has been demonstrated to disrupt TP53 polyadenylation (27) and 5p15.33, where the risk SNP rs10069690 is reported to create a splice-donor site leading to an alternate TERT splice isoform lacking telomerase activity (60). As many of the risk SNPs are noncoding an attractive mechanism of action is through regulating expression of nearby genes.…”

Telomerase reverse transcriptase moonlights: Therapeutic targets beyond telomerase

“…One of these, rs10069690, is located in intron 4 and has a major (G) and minor (A) allele . The A allele has been studied as a candidate causal variant in a variety of malignancies . Killedar et al .…”

A splicing variant of TERT identified by GWAS interacts with menopausal estrogen therapy in risk of ovarian cancer

“…From a biological standpoint, this SNP appears to affect telomerase activity and hence, telomere maintenance, actions that could promote tumorigenesis if improperly regulated. 38 Although we cannot rule out that the observed interaction may be due to a SNP in the region that is in linkage disequilibrium with rs10069690, the fact that rs10069690 is functional with biological plausibility supporting its interaction with ET use makes it a strong candidate. The other two SNPs implicated in this analysis are intriguing as well in that they are confirmed ovarian cancer susceptibility loci.…”

“…reported rs10069690 as a likely functional SNP since its risk-associated T allele was shown to result in the co-production of full-length hTERT as well as an alternatively spliced transcript, which encodes a catalytically inactive protein that inhibits telomerase activity; this was thought to be due to a dominant negative effect of the protein since telomerase exists as a dimer and its catalytic activity requires both hTERT active sites to be functional. 38 The decreased enzymatic activity may result in shorter telomeres, which could lead to an increased risk of genetic instability and subsequent carcinogenesis. Given the evidence suggesting estrogen’s role in the transcriptional regulation of hTERT, the elevated risk of serous ovarian cancer may be attributable to the inhibition of telomerase activity from higher levels of estrogen with prolonged ET use (OR = 1.85, 95% CI 1.28–2.66 for 10+ years).…”