The role of telomerase reverse transcriptase in human malignancies : genetic polymorphisms and promoter mutations

Sammanfattning: Telomerase is a ribonucleoprotein with its catalytic subunit telomerase reverse transcriptase (TERT) as a key component, lengthening telomeres. In differentiated human cells, telomerase is silent due to the transcriptional repression of the TERT gene, but activated in oncogenesis. Telomerase activation/TERT induction is essential to unlimited proliferation of cancer cells via telomere lengthening, whereas recent evidence also suggests that TERT may be a master contributor of cancer hallmarks. It is thus important to define regulatory mechanisms underlying cancer-specific TERT expression, and to delineate oncogenic effects of TERT. This thesis is designed to address these issues with the following specific aims: (1) The association between single-nucleotide polymorphisms (SNPs) of the TERT gene and cancer susceptibility and (2) Biological/translational implications of cancer-specific TERT promoter mutations. The TERT SNP association with cancer risk has been extensively investigated, most studies being focused on rs2736100 and rs2736098. The rs2736100_CC genotype has been shown to be associated with higher risk for a number of cancer types. Consistently, we observed that male individuals carrying the rs2736100_CC exhibited greater susceptibility to myeloproliferative neoplasms (MPNs), clonal diseases with myeloid cell origin (PAPER I). Furthermore, a comparison between Swedish and Chinese populations revealed a significantly higher fraction of rs2736100_CC in Swedes, coupled with a higher MPN incidence (compared to that in China). In addition, we made the same genotyping in upper tract urothelial carcinoma (UTUC) and hepatocellular carcinoma (HCC). The rs2736100_AC genotype was associated with reduced UTUC risk compared to the rs2736100_AA and CC carriers (PAPER II), while there were no significant differences in the rs2736100 or rs2736098 genotype distribution between HCC patients and healthy individuals (PAPER III). Collectively, male/female and ethnical groups may harbor different germline TERT variants, thereby contributing to different incidences and susceptibility dependent on origins of malignancies. The recurrent TERT promoter mutations, recently identified in different human malignancies, stimulate TERT transcription and activate telomerase. To explore the biological and clinical implication of TERT promoter mutations, we sequenced the TERT promoter region in tumor specimens derived from patients with UTUC, bladder cancer (BC) and HCC (PAPERS III and IV), and mutations were observed in 65/220 (30%) UTUC, 41/70 (59%) BC and 57/190 (30%) of HCC patients, respectively. In UTUC, the presence of TERT promoter mutations was significantly correlated with metastases, whereas for HCC, there was a significant difference in rs2736098 and rs2736100 genotypes between wt and mutant TERT promoter-bearing tumors. The cancer risk genotype rs2736100_CC was significantly associated with a reduced incidence of TERT promoter mutations, while the rs2736098_CT genotype was significantly higher in HCCs with TERT promoter mutations. Thus, the germline TERT genetic background may substantially affect the incidence of TERT promoter mutations in HCCs. As TERT promoter mutations are absent in normal cells, we evaluated the mutant TERT promoter as a urinary biomarker for non-invasive detection of UTUC and BC. The mutant TERT promoter was indeed detectable in urine from the mutation-positive UTUC and BC patients using Sanger sequencing, but the sensitivity was only 60%. To improve it, we developed a Competitive Allele-Specific TaqMan PCR (castPCR), and achieved an overall sensitivity of 89% and specificity of 96%. Thus, castPCR assays of TERT promoter mutations may be useful tools for non-invasive, urine-based diagnostics of UTUC and BC. In summary, our findings gain new insights into the association of TERT SNPs with cancer risk and TERT promoter mutations. These results will hopefully contribute to the rational development of a TERT-based strategy for precision oncology.