Search

Pleckstrin homology domain containing S1 (PLEKHS1) is a poorly characterized factor, although its promoter mutations were identified in human malignancies including thyroid carcinoma (TC). This study was designed to determine PLEKHS1 promoter hotspot mutations in papillary and anaplastic thyroid carcinomas (PTCs and ATCs) and to evaluate if PLEKHS1 expression influences clinical outcome. The PLEKHS1 promoter mutation was observed in 1/93 of PTCs and none of 18 ATCs in our cohort, however, PLEKHS1 expression was aberrantly up-regulated in TCs compared to adjacent non-tumorous thyroid tissues. ATC tumors, an undifferentiated TC, exhibited the highest PLEKHS1 expression. In both TCGA and present cohorts of PTCs, PLEKHS1 gene methylation density was inversely correlated with its mRNA expression and demethylation at the PLEKHS1 locus occurred at two CpGs. Higher PLEKHS1 expression was associated with lymph node and distant metastases, and shorter overall and disease-free survival in our cohort of PTC patients. Importantly, PLEKHS1 over-expression predicted shorter patient survival in PTCs lacking TERT promoter mutations. Cellular experiments showed that PLEKHS1 over-expression enhanced AKT phosphorylation and invasiveness. Collectively, the PLEKHS1 gene demethylation causes its over-expression in PTCs. PLEKHS1 promotes aggressive behavior of TCs possibly by increasing AKT activity, and its over-expression predicts poor patient outcomes.

Background: TERT promotor mutations are present in >75% of bladder tumours; these mutations are also detectable in urine. Previous studies have used urinary pellet DNA, and semi-quantitative methods unsuitable for detecting very low mutant allele frequencies. Objective: In this proof-of-principle study we use ddPCR to count the DNA molecules with wt and mutant TERT sequences in urinary cfDNA from patients whose bladder cancers harbour TERT mutations. Methods: Urinary cfDNA prepared from the urine from 104 bladder cancer patients was analysed. We determined the mutant allele frequency across stages and grades of disease, analysed concordance between cfDNA and tumour DNA, compared cfDNA with pellet DNA, and analysed the quantity and size distribution of cfDNA. Results: In 71 of 77 patients with a 228 G>A/T mutant tumour, the mutation was also detected in urinary cfDNA by ddPCR; all 6 "false negatives" were low grade pTa tumours. Overall concordance between tissue and cfDNA mutation status was 92%, and 100% was achieved for high grade disease. Median mutant allele frequencies in urinary cfDNA were 3.4, 13.4 and 32.1% in grade 1, 2 and 3 disease. The 228 G>A/T mutation was not detected in urinary cfDNA in 26 out of 27 mutation-negative patients (96% specificity). Conclusions: Concordance between tumour DNA and urinary cfDNA is high, and TERT 228 G>A/T ddPCR may prove useful for monitoring patients that harbour this mutation. Mutant allele frequencies in cfDNA are often high, but assays capable of detecting very low mutant allele frequencies will be required to achieve high sensitivity in low grade disease.

TERT (telomerase reverse transcriptase) is the catalytic component of telomerase. TERT shows little expression in normal somatic cells but is commonly re-expressed in cancers, facilitating immortalization. Recently-discovered TERT promoter mutations create binding sites for ETS-family transcription factors to upregulate TERT. ETS1 is reported to be important for TERT upregulation in melanoma. However it is unclear when in melanoma progression TERT and ETS1 proteins are expressed. To elucidate this question, ETS1 and TERT immunohistochemistry were performed on a panel of benign (n=27) and dysplastic nevi (n=34), radial growth phase (n=29), vertical growth phase (n=25) and metastatic melanomas (n=27). Lesions were scored by percentage of positive cells. ETS1 was readily detectable in all lesions, but not in normal melanocytes. TERT was located in either the nucleolus, the nucleoplasm (non-nucleolar) or both. Non-nucleolar TERT increased in prevalence with progression, from 19% of benign nevi to 78% of metastases. It did not however correlate with cell proliferation (Ki-67 immunostaining), nor differ significantly in prevalence between primary melanomas with or without a TERT promoter mutation. These results demonstrate that ETS1 is expressed very early in melanoma progression, and interestingly only non-nucleolar TERT correlates clearly in prevalence with melanoma progression. It can be acquired at various stages and by mechanisms other than promoter mutations.

Telomere repeats at chromosomal ends, critical for genomic integrity, undergo age-dependent attrition and telomere length has been associated with different disorders including cancers. In this study, based on 1469 patients and 1158 healthy controls, we show a statistically significant (P =6x10(-10)) association between increased telomere length and melanoma risk. Mendelian randomization, using 5 telomere length-associated polymorphisms, ruled out confounding factors or reverse causality and showed association between increased telomere length and melanoma risk with odds ratio of 2.66 (95% confidence interval: 2.07-3.25). Age-dependent telomere attrition was faster in melanoma cases than controls (P =.01). The carriers of a highly penetrant germline -57A>C TERT promoter mutation, in a previously reported melanoma family, had longer telomeres than the noncarriers. The mutation causes increased TERT and telomerase levels through creation of a binding motif for E-twenty six (ETS) transcription factors and the carriers develop melanoma with an early age of onset and rapid progression to metastasis. In analogy, we hypothesize that increased telomere length in melanoma patients reflects stochastic increased telomerase levels due to common genetic variation. Paradoxically, we observed shorter telomeres (P =1x10(-5)) in primary tumors from unrelated melanoma patients with (121) than without (170) somatic TERT promoter mutations that similar to the germline mutation, also create binding motifs for ETS transcription factors. However, the age-dependent telomere attrition was faster in tumors with the TERT promoter mutations than in those without such mutations. Besides a robust association between increased telomere length and risk, our data show a perturbed telomere homeostasis in melanoma.

TERT (telomerase reverse transcriptase) is the catalytic component of telomerase. TERT shows little expression in normal somatic cells but is commonly re-expressed in cancers, facilitating immortalization. Recently-discovered TERT promoter mutations create binding sites for ETS-family transcription factors to upregulate TERT. ETS1 is reported to be important for TERT upregulation in melanoma. However it is unclear when in melanoma progression TERT and ETS1 proteins are expressed. To elucidate this question, ETS1 and TERT immunohistochemistry were performed on a panel of benign (n=27) and dysplastic nevi (n=34), radial growth phase (n=29), vertical growth phase (n=25) and metastatic melanomas (n=27). Lesions were scored by percentage of positive cells. ETS1 was readily detectable in all lesions, but not in normal melanocytes. TERT was located in either the nucleolus, the nucleoplasm (non-nucleolar) or both. Non-nucleolar TERT increased in prevalence with progression, from 19% of benign nevi to 78% of metastases. It did not however correlate with cell proliferation (Ki-67 immunostaining), nor differ significantly in prevalence between primary melanomas with or without a TERT promoter mutation. These results demonstrate that ETS1 is expressed very early in melanoma progression, and interestingly only non-nucleolar TERT correlates clearly in prevalence with melanoma progression. It can be acquired at various stages and by mechanisms other than promoter mutations.

TERT promoter C228T and C250T mutations occur in various malignancies including bladder cancer (BC) and may serve as urinary tumor markers. However, the mutation association with clinical variables in upper tract urothelial carcinomas (UTUCs) is unclear. There is also a lack of sensitive tools to detect the minor mutant TERT promoter in bulk urinary DNA. Here we analyzed 220 UTUC patients [98 with renal pelvic carcinoma (RPC) and 122 with ureter carcinoma (UC)] and developed a Competitive Allele-Specific TaqMan PCR (castPCR) for urinary assay. We identified C228T or C250T mutations in 42 of 98 (43%) RPC and 23 of 122 (19%) UC tumors. Distant metastases were significantly correlated with UTUC patients harboring TERT promoter mutations (P = 0.001). C228T were detected in 6/10 and 9/10 of urine samples from patients with mutation-carrying tumors using Sanger sequencing and castPCR, respectively. When urine samples from 70 BC patients were analyzed together, the sensitivity of urinary C228T assay was 89% and 50% for castPCR and Sanger sequencing, respectively (P < 0.001). Collectively, TERT promoter mutations occur in UTUCs with a high frequency in RPCs and predict distant metastasis. castPCR assays of the mutation are a useful tool for urine-based diagnostics of urological malignancies.

Telomerase activation through the induction of its catalytic component TERT is essential in carcinogenesis. The regulatory mechanism and clinical significance underlying cancer-specific TERT expression have been extensively investigated in various human malignancies, but little is known about these in Merkel cell carcinoma (MCC), an aggressive neuroendocrine skin tumor. Here we addressed these issues by determining TERT promoter mutations, gene amplification, mRNA expression and association with clinical variables in MCC. TERT mRNA was expressed in 6/6 MCC cell lines and 41 of 43 tumors derived from 35 MCC patients. Telomerase activity was detectable in all 6 cell lines and 11 tumors analyzed. TERT promoter mutations were identified in 1/6 cell lines and 4/35 (11.4%) MCC cases. The mutation exhibited UV signature and occurred in sun-exposed areas. Increased TERT gene copy numbers were observed in 1/6 cell lines and 11/14 (79%) tumors, and highly correlated with its mRNA expression (r = 0.7419, P = 0.0024). Shorter overall survival was significantly associated with higher TERT mRNA levels in MCC patients (P = 0.032). Collectively, TERT expression and telomerase activity is widespread in MCC, and may be attributable to TERT promoter mutations and gene amplification. Higher TERT expression predicts poor patient outcomes.

Glioma, the most common central nervous system cancer in adults, has poor prognosis. Here we identify a new SNP associated with glioma risk, rs1920116 (near TERC), that reached genome-wide significance (Pcombined= 8.3 × 10-9) in a meta-analysis of genome-wide association studies (GWAS) of high-grade glioma and replication data (1,644 cases and 7,736 controls). This region has previously been associated with mean leukocyte telomere length (LTL). We therefore examined the relationship between LTL and both this new risk locus and other previously established risk loci for glioma using data from a recent GWAS of LTL (n = 37,684 individuals). Alleles associated with glioma risk near TERC and TERT were strongly associated with longer LTL (P = 5.5 × 10-20and 4.4 × 10-19, respectively). In contrast, risk-associated alleles near RTEL1 were inconsistently associated with LTL, suggesting the presence of distinct causal alleles. No other risk loci for glioma were associated with LTL. The identification of risk alleles for glioma near TERC and TERT that also associate with telomere length implicates telomerase in gliomagenesis.© 2014 Nature America, Inc. All rights reserved.

Cataloged from PDF version of thesis. Includes bibliographical references (leaves 89-112). Thesis (Ph. D.): Bilkent University, Department of Molecular Biology and Genetics, İhsan Doğramacı Bilkent University, 2014. Hepatocellular Carcinoma (HCC) is one of the major causes of cancer related deaths worldwide and its incidence has been increasing drastically, especially in western countries. HCC has a heterogeneous molecular and pathological background with various underlying risk factors and survival rate of HCC patients is very low due to late diagnosis and limited curative therapies. The mechanisms involved in hepatocellular immortality gains critical importance in order to develop preventive and therapeutic options against HCC. Telomerase reactivation is a keystone for HCC cells during transformation process. TERT promoter mutations activating its promoter by creating a novel activating motif were recently identified in different cancer types. In this study; we determined TERT promoter mutation frequency in HCC cell lines and tumors which are 67% (10/15) and 34% (15/44) respectively. High frequency of TERT promoter mutations in HCC indicated a possible functional role during hepatocarcinogenesis. We performed transcriptional factor search to find a candidate TF that could bind to mutant TERT promoter and STAT1 came out of that search. To study the role of STAT1 during reactivation of TERT expression, we activated STAT1 signaling by Interferon alpha (IFN-α) treatment and down regulated STAT1 with RNA interference in several HCC cell lines. We have found that IFN-α was able to upregulate TERT expression in the HCC cell lines carrying a TERT promoter mutation and STAT1 knockdown was enough to eradicate this upregulation. In case of wild type cell lines, IFN-α treatment and STAT1 knock down had no effect on TERT expression. Our data delineates the contributions of TERT promoter mutations to hepatocellular immortality and gives insights into the potential use of TERT as a target for chemoprevention of hepatocarcinogenesis. by Dilek Çevik. Ph.D.