The Study Of Human Telomerase Reverse Transcriptase HTERT Promoter In Tumorigenesis And Diagnosis Of Carcinomas

Telomerase is a RNA-dependent DNA reverse transcriptase, synthesizing telomeric DNA according to its RNA template. Human telomerase has two main components: human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT). A majority of experiments demonstrated that telomerase activation is a critical step in the carcinogenesis and cancer progression, in which hTERT plays a pivotal role as a rate-limiting enzyme. However, the function of telomerase and its underlying mechanism has not been well investigated. This dissertation explores the function and regulation of hTERT in tumorigenesis and diagnosis of carcinomas, mainly focusing on three fields:hTERT promoter DNA methylation, hTERT promoter mutation and hTERT transcriptional regulation.Part Ⅰ DNA methylation of hTERT promoter in the diagnosis of gastrointestinal carcinomaBackgroundGastrointestinal cancer (GIC), mainly composed of gastric cancer (GC) and colorectal cancer (CRC), is one of the most common malignant carcinomas. Endoscope examination and biospy is the main diagnostic tool of GIC, but it is invasive and expensive. Non invasive tests such as stool-based approaches are ideal for population screening and surveillance monitoring. More and more researchers are working on the fecal tumor markers in the diagnosis of GIC. DNA methylation is one of the main ways of epigenetic modification. It has been demonstrated that aberrant methylation of variety of genes such as CDH1、P16、hMLH1、MGMT、RASSF2 do exist in GC or CRC. Meanwhile, aberrant methylations have been found in the blood and stool of CRC patients, for example, SEPT9、RASSF2、SFRP2 may become the potential biomarkers in the noninvasive of CRC.It has been well established that hTERT over-expression and telomerase activation are observed in up to 90% of human malignancies including GIC.hTERT, as a catalytic subunit, is the rate-limiting component of telomerase. hTERT promoter is rich in CpG dinucleotide, which forms CpG islands. It has been pointed out that there are differential methylation statuses of hTERT promoter between normal and cancerous cells, which makes hTERT promoter methylation may serve as a potential discriminating biomarker. It is reported that hTERT promoter in GC and CRC tumor was hypermethylated compared with normal tissue adjacent to tumor, and hypermethylation of hTERT promoter is possitively associated with hTERT mRNA expression. However, no report has ever profiled the methylation status of hTERT promoter in stool.ObjectiveTo delineate hTERT promoter methylation status in GIC tumor and normal tissue adjacent to tumor; to explore whether the hTERT promoter methylation assay in stool is applicable for GIC screening and surveillance.MethodsTumor specimens of GIC and normal tissue adjacent to tumor were obtained from 34 patients who had undergone curative surgery. Feces were collected from 69 GC or CRC patients and 62 control adults without cancers or gastrointestinal diseases. Samples were immediately frozen at -80℃.Genomic DNA was extracted from the above tissues and feces, following by bisulfite conversion. Pyrosequencing was applied to quantify the methylation status of 2 CpG sites of hTERT promoter in tissues and feces. Statistical assay was applied to analyze the diffierece in the methylation between groups according age, gender, differentiation levels, lesion stages, and aggression. Sensitivity and specificity was determined for the diagnosis on GC and CRC.ResultsThe two sites of hTERT promoter were in general unmethylated or methylated at a very low level in non-malignant gastrointestinal tissues whereas significantly methylated in cancerous tissues (P=0.008 and 0.004, seperatively).The methylated sites 1 and 2 were indeed detectable in the stool while only background levels were found in non-cancer individuals (P＜0.05). Receiver operating characteristic (ROC) curves for various cutoffs by hTERT promoter methylation levels in fecal samples were made, and for overall series, the area under curve (AUC) of site1, site2 and average (combining site1 and site2) was 0.769,0.777 and 0.799, correspondingly. There was no difference in the methylation of sites 1 and 2 of fecal DNA between GC and CRC and the methylation level was independent of age and sex in all the patients. In CRC, higher levels of the methylated site 1 but not site 2 was significantly correlated with lowly differentiated tumors and advanced disease stages (duke’s stages C and D). However, in contrast, GC analyses revealed a significant correlation of the higher methylated site 2 bot not site 1 with lowly differentiated tumors, and late or metastatic stages of the disease. AUC for occult blood test (OBT) in GC was 0.537. In CRC, AUC for OBT was 0.834. When the assessment result of hTERT promoter methylation and OBT was combined together, AUCs in GC and CRC were 0.806 and 0.920, respectively.ConclusionsThe methylation levels of the two CpG sites are significantly higher in GIC tissues than that in normal tissue adjacent to tumor. The methylation levels of fecal DNA from GIC patients are much higher than that from non-cancerous patients. The methylation levels of fecal DNA are correlated with tumor differentiation, stages and aggressive. The combined assay of hTERT promoter methylation and OBT improves detection sensitivity and specificity in CRC.Part Ⅱ The study of hTERT promoter mutation in renal cell carcinomaBackgroundRenal cell carcinoma (RCC) is one of the most common urinary carcinomas, including different types, such as clear cell carcinoma (ccRCC), papillary carcinoma (pRCC) and chromophobe renal carcinoma (chRCC). Most RCCs exhibit telomerase activation, but the mechanism is still unclear.The two hTERT promoter recurrent mutations C228 and C250T were reported in melanoma, which create de novo ETS1 binding motifs, thereby facilitating the hTERT transcription. As a consequence, the activity of telomerase is enhanced 2-4 fold or the expression of hTERT mRNA is increased evidently. The mutation is thus probably the mechanism of telomerase genetic regulation or hTERT transcriptional regulation. In addition, clinical observations suggest that hTERT promoter mutations may be correlated with clinical features or the outcomes. For instance, in melanoma, hTRET promoter mutations are related with age, thickness of tumors and ulcer formation; in glioma, mutations are related with the recurrence and prognosishTERT promoter mutations were very common in bladder cancer, and were considered to be the most frequent gene alteration in bladder cancer. It is not known that whether hTERT promoter mutations occur in RCC and the relevance between the mutations and the clinical characteristics has not been reported either.ObjectiveTo delineate the hTERT promoter mutational stutus in a cohort of RCC patients and to determine its assocation with clinical and pathological characteristics, so as to understand the mechanism of telomerase activation.MethodsThe tumor specimens were derived from 109 RCC patients, and genomic DNA was extracted from tissues samples. Sanger sequencing was used to detect the C228T, C250T mutations and VHL gene alteration. Total cellular RNA was extracted from tissue samples of RCCs, and expression of hTERT mRNA was analyzed by Real-time PCR. Differences in the hTERT promoter mutaions frequency between tumors with VHL gene alteration, gender, clinical stage, metastasis and renal capsular were analyzed statistically.ResultsAmong the 109 RCC tumor specimens,10 RCC tumors were found to harbor the mutations, with 9 C228T and 1 C250T. In the ccRCC tumor specimens, a remarkable difference in the levels of hTERT mRNA between tumors with and without hTERT promoter mutations was found, and hTERT mRNA expression was significantly higher in the mutation-positive ccRCCs. Among 96 ccRCC patients,5 of 9 (55.6%) and 9 of 87 (10.3%) were at clinical stage III/IV in the mutation-positive and mutation-negative groups, respectively (P=0.003). However, no difference was found in the age, gender and tumor size between the two groups. In the 9 mutation-positive ccRCC patients,2 had metastasis and 3 had capsular invasion, while in the 87 mutation-negative ccRCC patients,4 had remote metastasis and 3 had capsular invasion. Altogether, metastasis and capsular invasion occurred in 5/9 (55.6%) and 7/87 (8.0%) of the mutation-positive and mutation-negative ccRCC groups (P=0.001), respectively. Among the 77 evaluable VHL locas of the ccRCC tumors,32(41.6%) had one or more alterations in the VHL locas, existing in 4 mutant hTERT promoter-carrying tumors and 28 wild type hTERT promoter tumors, no statistic significance was detacted (P>0.05).ConclusionThe RCC tumor specimens do harbour hTERT promoter mutations. The hTERT promoter mutations has positive correlation with hTERT mRNA expression in ccRCC tumors, and has correlations with clinical stage and aggressive. However, no difference was found in the age, gender and tumor size between the mutation-positive tumors and mutation-negative tumors. The frequency of the VHL alterations did not differ significantly between tumors with and without hTERT promoter mutations.Part Ⅲ Transcriptional regulation of hTERT in telomerase activation by seminal plasma in cervical cancerBackgroudCervical cancer is one of the most common gynecologic carcinomas, and many factors are related to its occurrence. However, the definite causes and mechanisms are still unkown. It has been demonstrated that hTERT mRNA expression and telomerase activity are aberrantly increased in cervical cancers. Furthemore, telomerase activation is very important step in the transformation of cervical intraepithelial neoplasias (CIN) to cervical cancer, however, the underlying mechanism is still elusive.It is well established that the transcription of hTERT promoter is the key step of telomerase activation, but the mechanism of hTERT transcriptional regulation is still undefined. Researches have revealed that many factors involve in the transcriptional regulation of hTERT promoter, including activation factors and suppression factors. It has been demonstrated that c-MYC, Sp1, estrogen are hTERT transcriptional activators, while P53, Transcriptioanl Activation Protein 1, Madl act as suppressive factors of hTERT transcription.As the important component of semen, seminal plasma contains multiple substances, such as prostaglandins, estrogen, progestogen, vascular growth factor, cytokines, protease, and protein kinase. It has been reported that seminal plasma can promote the development and progression of cervical cancer. For instance, in the HeLa cells exposed to seminal plasma or prostaglandin E2 (PGE2), the espression of receptor EP1, EP4 and epidermal growth factor receptor (EGFR) were induced, leading tumorgenesis and cancer progression. The substances in the seminal plasma can also stimulate the mRNA expression of interleukin 1-β (IL1-β), interleukin 6 (IL-6), tumor necrosis factor a (TNF-a), and cyclooxygenase-2 (COX-2) in the endometrial epithelial cells, regulating the physiological function of these cells.ObjectiveTo delineate the function of hTERT and its transcriptional regulation mechanisms during the process of cervical transformation stimulated by seminal plasma.Methods(1) Assessment of hTERT mRNA expression:Three cervical cancer cell lines including HeLa, Caski and SiHa, and normal cervical epithelial cells isolated from normal cervical tissue, were separately incubated in FCS-free medium for 48h and then exposed to seminal plasma at various concentrations. And 24h later, cells were harvested for total RNA isolation. Quantitive real-time PCR was performed to determine the abundance of hTERT mRNA. (2) Assessment of telomerase activity: HeLa, Caski, SiHa and cervical epithelial cells were incubated and harvested (same as above). Telomerase activity was assayed with the TeloTAGGG Telomerase PCR ELISA kit. (3) Western blotting was used to determine the abundance of COX-2 and Spl at protein level. (4) hTERT promoter activity assessment:hTERT promoter activity reporter and Luciferase activity duo-reporting system were used. (5) siRNA: COX-2 expression in HeLa cells were knocked down using siRNA specifically targeting COX-2.(6) Chromatin immunoprecipitation assay (ChIP):HeLa cells were crosslinked by incubating them in formaldehyde and then sonicated to make soluble chromatin with DNA fragments of 200-1000 bp. Antibodies against Sp1 and acetylated histone H3 and H4 were used to precipitate DNA fragments bound by their corresponding elements. The protein-DNA complex was collected with protein A/G Sepharose beads, eluted and reverse-crosslinked. The recovered DNA was resuspended in TE buffer and used for the PCR amplification. (7) Xenographt transplanting experiments:Six week-old female BALB/c nude mice were injected with serum starved HeLa cells (pretreated with or without seminal plasma). After 3 weeks, the mice were sacrificed and the tumours were removed and measured.Results(1) In the three cervical cell lines HeLa, Caski, and SiHa, when treated with seminal plasma, hTERT mRNA abundance was strongly increased (P＜0.01). And in HeLa and Caski cells, telomerase activity was increased separately for the seminal plasma exposure group (P＜0.05 and P＜0.01, respectively). Control cervical epithelial cells without seminal plasma expressed negligible amounts of hTERT mRNA, while seminal plasma treatment at 1:10 induced significantly higher levels of hTERT mRNA (P＜0.05), as well as the telomerase activity (P<0.05). (2) After COX-2 depletion by siRNA, hTERT mRNA expression between the seminal plasma treatment group and the control group did not show a significant difference. However, seminal plasma exposure only enhanced hTERT mRNA expression in HeLa cells treated with control siRNA. (3) When HeLa cells were transfected with a hTERT promoter reporter construct p181wt and then exposed to seminal plasma, luciferase activity was significantly increased (P＜0.05). However, when COX-2 was knocked down, the stimulation effect of seminal plasma on luciferase activity was abolished. (4) The levels of Spl protein increased 5.6-fold and 6.5-fold, when HeLa cells exposed to seminal plasma and PGE2, respectively. (5) By ChIP assay, we found that the hTERT promoter fragment was significantly enriched in Spl antibody-precipitated DNA derived from seminal plasma-treated or PGE2-treated HeLa cells. Meanwhile, the increased Spl occupancy on the hTERT promoter was coupled with more acetylated histone H3 and H4 locally in HeLa cells treated with seminal plasma or PGE2.(6) Mouse xenophagft transplanting experiments showed that significantly larger tumors were observed in mice receiving seminal plasma-treated cells than the untreated cells.ConclusionsSenimal plasma can stimulate hTERT mRNA expression and telomerase activation in both cervical cancer cell lines and cervical epithelial cells. The transcriptional activity of hTERT promoter was increased in seminal plasma-treated HeLa cells and this effect was closely correlated with increased Spl expression and binding to the hTERT promoter. COX-2 plays an important role in the seminal plasma induced hTERT expression and telomerase activation via COX-2-PGE2-Spl axis.