Keith Wn

Amplification, increased dosage and in situ expression of the telomerase RNA gene in human cancer

Telomere length is maintained by the enzyme, telomerase, which has been linked to cellular immortality and tumour progression. However, the reasons for the high levels of telomerase found in human tumours are unknown. We have mapped the human telomerase RNA gene, (hTR), to chromosome 3q26.3 and show the hTR gene to be amplified in four carcinomas, (2/33 cervix, 1/31 head and neck, 1/9 lung). In addition, increased copy numbers of the hTR locus was also observed in 97% of tumours. By in situ hybridisation, the histological distribution of high levels of hTR expression could be demonstrated in a lung tumour and its metastasis with hTR amplification. These results are the first report of genetic alterations involving a known component of telomerase in human cancer. Indeed, it is also the first report of the amplification of a specific locus within the chromosome 3q region frequently subject to copy number gains in human tumours. In addition, we also show for the first time the histological distribution of the RNA component of telomerase in human tumours.

Targeting telomerase for cancer therapeutics

One of the hallmarks of advanced malignancies is continuous cell growth and this almost universally correlates with the reactivation of telomerase. Although there is still much we do not understand about the regulation of telomerase, it remains a very attractive and novel target for cancer therapeutics. Several clinical trials have been initiated, and in this review we highlight some of the most promising approaches and conclude by speculating on the role of telomerase in cancer stem cells.

Telomerase-specific suicide gene therapy vectors expressing bacterial nitroreductase sensitize human cancer cells to the pro-drug CB1954.

Telomerase activation is considered to be a critical step in cancer progression due to its role in cellular immortalization. The prevalence of telomerase expression in human cancers makes it an attractive candidate for new mechanism-based targets for cancer therapy. The selective killing of cancer cells can be achieved by gene-directed enzyme pro-drug therapy (GDEPT). In this study we have tested the feasibility of using the transcriptional regulatory sequences from the hTERT and hTR genes to regulate expression of the bacterial nitroreductase enzyme in combination with the pro-drug CB1954 in a suicide gene therapy strategy. hTERT and hTR promoter activity was compared in a panel of 10 cell lines and showed a wide distribution in activity; low activity was observed in normal cells and telomerase-negative immortal ALT cell lines, with up to 300-fold higher activity observed in telomerase positive cancer lines. Placing the nitroreductase gene under the control of the telomerase gene promoters sensitized cancer cells in tissue culture to the pro-drug CB1954 and promoter activity was predictive of sensitization to the pro-drug (2–20-fold sensitization), with cell death restricted to lines exhibiting high levels of promoter activity. The in vivo relevance of these data was tested using two xenograft models (C33a and GLC4 cells). Significant tumour reduction was seen with both telomerase promoters and the promoter-specific patterns of sensitization observed in tissue culture were retained in xenograft models. Thus, telomerase-specific suicide gene therapy vectors expressing bacterial nitroreductase sensitize human cancer cells to the pro-drug CB1954.

Hypoxic regulation of telomerase gene expression by transcriptional and post-transcriptional mechanisms

Basal telomerase activity is dependent on expression of the hTERT and hTR genes and upregulation of telomerase gene expression is associated with tumour development. It is therefore possible that signal transduction pathways involved in tumour development and features of the tumour environment itself may influence telomerase gene regulation. The majority of solid tumours contain regions of hypoxia and it has recently been demonstrated that hypoxia can increase telomerase activity by mechanisms that are still poorly defined. Here, we show that hypoxia induces the transcriptional activity of both hTR and hTERT gene promoters. While endogenous hTR expression is regulated at the transcriptional level, hTERT is subject to regulation by alternative splicing under hypoxic conditions, which involves a switch in the splice pattern in favour of the active variant. Furthermore, analysis of the chromatin landscape of the telomerase promoters reveals dynamic recruitment of a transcriptional complex involving the hypoxia-inducible factor-1 transcription factor, p300, RNA polymerase II and TFIIB, to both promoters during hypoxia, which traffics along and remains associated with the hTERT gene as transcription proceeds. These studies show that hTERT and hTR are subject to similar controls under hypoxia and highlight the rapid and dynamic regulation of the telomerase genes in vivo.

Cloning and characterization of human and mouse telomerase RNA gene promoter sequences.

Variation in telomerase activity is correlated with cellular senescence and tumour progression. However, although the enzymatic activity of telomerase has been well studied, very little is known about how expression of telomerase genes is regulated in mammalian cells. We have therefore cloned the promoter regions of the human (hTR), and mouse, (terc), telomerase RNA genes in order to identify the regulatory elements controlling telomerase RNA gene transcription. 1.76 kb encompassing the hTR gene promoter region was sequenced, as was 4 kb encompassing the terc promoter. No significant sequence similarity could be detected in comparisons between human and mouse 5′-regions, flanking the transcribed sequences. However, both the human and mouse telomerase RNA genes are within CpG islands and may therefore be under the regulation of DNA methylation. Transient expression of hTR-reporter gene constructs in HeLa and GM847 cells identified the elements responsible for promoter activity are contained in a 231 bp region upstream of the transcriptional start site. Transient expression of terc-reporter gene constructs in Swiss3T3 and A9 cells identified the elements responsible for promoter activity are contained in a 73 bp region upstream of the transcriptional start site. These studies have implications for novel transcription targeted cancer therapies.

Selective ablation of human cancer cells by telomerase-specific adenoviral suicide gene therapy vectors expressing bacterial nitroreductase.

Reactivation of telomerase maintains telomere function and is considered critical to immortalization in most human cancer cells. Elevation of telomerase expression in cancer cells is highly specific: transcription of both RNA (hTR) and protein (hTERT) components is strongly upregulated in cancer cells relative to normal cells. Therefore, telomerase promoters may be useful in cancer gene therapy by selectively expressing suicide genes in cancer cells and not normal cells. One example of suicide gene therapy is the bacterial nitroreductase (NTR) gene, which bioactivates the prodrug CB1954 into an active cytotoxic alkylating agent. We describe construction of adenovirus vectors harbouring the bacterial NTR gene under control of the hTR or hTERT promoters. Western blot analysis of NTR expression in normal and cancer cells infected with adenoviral vectors showed cancer cell-specific nitroreductase expression. Infection with adenoviral telomerase–NTR constructs in a panel of seven cancer cell lines resulted in up to 18-fold sensitization to the prodrug CB1954, an effect that was retained in two drug-resistant ovarian lines. Importantly, no sensitization was observed with either promoter in any of the four normal cell strains. Finally, an efficacious effect was observed in cervical and ovarian xenograft models following single intratumoural injection with low doses of vector, followed by injection with CB1954.

Tumour specific regulation of telomerase RNA gene expression visualized by in situ hybridization.

Maintenance of telomere structure by the ribonucleoprotein enzyme telomerase is considered central to the development of most human cancers. However, regulatory mechanisms governing telomerase expression during oncogenesis are largely unknown. We address potential tumour-specific regulation of telomerase RNA gene expression by RNA in situ hybridization to over 300 tumour samples of germ cell and epithelial origin. Twenty-six per cent of non-small cell lung cancers (NSCLC), expressed detectable levels of the telomerase RNA gene (hTR), and interestingly expression was almost confined to squamous carcinomas (41%), being rare in pulmonary adenocarcinomas and large-cell anaplastic carcinomas (P=0.006). Low frequency hTR expression was also associated with adenocarcinoma of the breast (13%), and ovary (17%). In comparison, hTR expression was detected in 43% of cervical cancers with no significant differences in frequency between squamous-cell carcinoma and adenocarcinoma or in transitions between intraepithelial neoplasia and invasive carcinoma. In contrast to the common epithelial cancers, the malignant cells in 73% of testicular germ-cell tumours (seminomas and teratomas), expressed hTR consistent with hTR expression in normal testicular germ cells. Differentiated tissues within ovarian germ cell tumours and in testicular teratomas lacked detectable hTR expression. These studies show that different tumour types have distinct patterns of hTR expression, which has implications for our understanding of mechanisms regulating telomerase activity and for targeting the telomerase RNA component as an anti-cancer therapy.

Telomerase-directed molecular therapeutics

The management of malignant disease remains one of the most challenging areas of modern medicine. The lifetime risk of developing cancer in the western world is estimated to be as high as 1 in 3. Traditionally, surgery, chemotherapy and radiotherapy have been the primary choice of treatment for patients with malignant tumours. Despite advances in the use and development of conventional cytotoxic agents, the cure rate remains disappointing in most patients with advanced disease of the common solid tumours. Consequently, the development of novel anti-cancer therapies is a high priority in cancer medicine. In recent years, a new generation of cancer therapies has emerged, based on a growing understanding of the molecular events that contribute to malignant transformation. A major difference between normal and cancer cells is the ability of cancer cells to multiply in an unrestricted and ungoverned fashion. In this context, there is considerable interest in elucidating the mechanisms that allow this unrestricted proliferation and that ultimately result in immortal cancer cells. It is now clear that the enzyme telomerase confers immortality on cells in most types of cancer. With the cancer cell reliant on telomerase for its survival, telomerase represents an extremely attractive mechanism-based target for the development of new cancer therapeutics.

Dysregulated expression of the major telomerase components in leukaemic stem cells

Telomere loss is rapid during the progression of chronic myeloid leukaemia (CML) and correlates with prognosis. We therefore sought to measure expression of the major telomerase components (hTR and hTERT) in CD34+ cells from CML patients and normal controls, to determine if their altered expression may contribute to telomere attrition in vivo. High-purity (median 94.1%) BCR-ABL+ CD34+ cells from CML (n=16) and non-CML (n=14) patients were used. CML samples had a small increase in telomerase activity (TA) compared to normal samples (approximately 1.5-fold, P=0.004), which was inversely correlated with the percentage of G0 cells (P=0.02) suggesting TA may not be elevated on a cell-to-cell basis in CML. Consistent with this, hTERT mRNA expression was not significantly elevated; however, altered mRNA splicing appeared to play a significant role in determining overall full length, functional hTERT levels. Interestingly, Q-RT-PCR for hTR demonstrated a mean five-fold reduction in levels in the chronic phase (CP) CML samples (P=0.002), raising the possibility that telomere homeostasis is disrupted in CML. In summary, the molecular events regulating telomerase gene expression and telomere maintenance during the CP of CML may influence the disease progression observed in these patients.

A gene expression signature classifying telomerase and ALT immortalization reveals an hTERT regulatory network and suggests a mesenchymal stem cell origin for ALT.

Telomere length is maintained by two known mechanisms, the activation of telomerase or alternative lengthening of telomeres (ALT). The molecular mechanisms regulating the ALT phenotype are poorly understood and it is unknown how the decision of which pathway to activate is made at the cellular level. We have shown earlier that active repression of telomerase gene expression by chromatin remodelling of the promoters is one mechanism of regulation; however, other genes and signalling networks are likely to be required to regulate telomerase and maintain the ALT phenotype. Using gene expression profiling, we have uncovered a signature of 1305 genes to distinguish telomerase-positive and ALT cell lines. By combining this with the gene expression profiles of liposarcoma tissue samples, we refined this signature to 297 genes. A network analysis of known interactions between genes within this signature revealed a regulatory signalling network consistent with a model of human telomerase reverse transcriptase (hTERT) repression in ALT cell lines and liposarcomas. This network expands on our existing knowledge of hTERT regulation and provides a platform to understand differential regulation of hTERT in different tumour types and normal tissues. We also show evidence to suggest a novel mesenchymal stem cell origin for ALT immortalization in cell lines and mesenchymal tissues.