Targeting Cancer with Telomerase : Commentary re Q. Huang et al., A Novel Conditionally Replicative Adenovirus Vector Targeting Telomerase-Positive Tumor Cells. Clin. Cancer Res., 10: 1439–1445, 2004.

During the past 50 years, the development and widespread use of antibiotics, vaccines, and other antimicrobial agents has dramatically reduced the mortality caused by infectious agents. These therapeutic successes were achieved, in large part, by the identification and development of approaches that target molecules expressed exclusively by microbes. This paradigm not only continues to drive efforts to develop new antimicrobials and vaccines but has also stimulated efforts to apply this approach to the treatment of cancer.

Indeed, concerted efforts from many laboratories have identified specific molecular alterations unique to cancer cells. However, unlike microbes, cancer cells originate from normal precursors, and the majority of proteins expressed by such malignant cells are also found in normal cells. Moreover, many of the molecular changes associated with human tumors are subtle amino acid substitutions or alterations in the level of protein expression. This overall similarity of normal and malignant cells complicates the application of this paradigm that has worked so well in infectious disease to cancer treatment. In addition, because some cancer-associated molecules are not required for continued malignant potential, selection of cancer cells that have down-regulated or lost expression of the intended target represents a major mechanism of acquired resistance.

Despite these challenges, recent work indicates that this model for drug development will succeed in cancer. Approaches using small molecules or antibodies such as Gleevec, Herceptin, and Rituximab provide excellent examples of how targeting cancer-associated molecules, even those expressed by some normal cells, can lead to therapeutics that are not only effective but also less toxic than traditional chemotherapy. The challenge remains to identify and exploit other molecules critical to tumor maintenance.

Several lines of evidence now implicate telomeres and telomerase as potential anticancer therapeutic targets. Telomeres are nucleoprotein structures composed of arrays of repeated G-rich DNA (TTAGGG in mammals) and specific binding proteins. The integrity of these terminal structures protects chromosome ends from recognition as a broken fragment of DNA and thus promotes genome stability (1). In addition, recent evidence indicates that dysfunctional telomeres limit cell replicative life span and, in this manner, serve as a barrier to immortalization, a hallmark of the malignant state (2). Disruption of these two telomere functions contributes in essential ways to cancer initiation and progression.

Telomeres are maintained by the reverse transcriptase telomerase, which is composed of essential RNA (hTERC) and protein (hTERT) components. In mammalian cells, hTERC is ubiquitously expressed, whereas the expression of hTERT is tightly regulated, indicating that hTERT is the rate-limiting component of the telomerase holoenzyme (4, 5). Indeed, in normal human cells, hTERT is expressed at levels that are insufficient to maintain overall telomere length (6). As a consequence, telomere attrition occurs in such cells with successive cell divisions, eventually leading to telomere dysfunction, chromosome instability, and a proliferative arrest (7). However, if telomere shortening occurs in the setting of inactivation of the retinoblastoma (pRB) and p53 tumor suppressor pathways, rare cells will survive this period called crisis induced by dysfunctional telomeres and exhibit high levels of telomerase activity, stabilized telomere lengths, and replicative immortality (8). These observations suggest that constitutive expression of hTERT plays a critical role in malignant transformation. In consonance with this notion, most human tumors constitutively express hTERT and telomerase activity (9), and the experimental transformation of human cells requires telomerase activation (10).

Taken together, these findings suggest that strategies that target telomerase hold promise as novel anticancer agents. Indeed, genetic (11), antisense (12, 13), and pharmacological (14) strategies that inhibit telomerase activity in human cancer cell lines induce cell death by apoptosis, suggesting that clinically useful telomerase inhibitors may eventually be identified. In addition, several groups have reported that telomerase also serves as an excellent target for immunotherapeutic strategies (15), and Phase I clinical trials testing these strategies have recently been completed (15). Despite these promising developments, additional work is necessary to fully understand the usefulness and potential toxicities of these telomerase-specific approaches.

Although these pharmacological and immunotherapeutic strategies focus on targeting telomerase in cancer cells, an alternative method to use telomerase therapeutically relies on using telomerase biology to direct therapy specifically to cancer cells. This approach exploits the observation that hTERT is tightly regulated in normal cells while constitutively active in cancer cells (2, 8). Over the past several years, several groups have created gene expression vectors in which the upstream regulatory sequences for the hTERT gene are used to drive the expression of cytotoxic genes such as caspase-8 (16), diphtheria toxin A-chain (17), herpes virus thymidine kinase (18), bacterial nitroreductase (19), and Bax (20). These investigators showed that introduction of vectors encoding these hTERT promoter-driven genes in cultured human cancer cell lines rapidly induce cell death. However, implementation of these types of vectors clinically will require significant technical advances that will allow efficient insertion of these vectors into spontaneously arising human cancers while sparing normal cells and tissues.

In this issue of Clinical Cancer Research, Huang et al. (21) report on a related strategy to use telomerase biology to direct cancer gene therapy. These authors used a small portion of the upstream regulatory region of the hTERT gene to create a conditionally replicating oncolytic adenoviral vector (21). By introducing a 400-bp portion of the hTERT promoter upstream of the E1a gene, which is required for viral replication, these authors created an adenoviral vector that should only replicate in cells in which the hTERT promoter is active. Indeed, when introduced into normal and malignant human cells, this adenoviral vector showed 1000-fold more lytic activity in human tumor cells that express hTERT constitutively both in culture and in xenograph models. These observations corroborate and extend similar experiments in which different portions of the hTERT promoter were used to create other conditionally replicative adenoviruses (22, 23, 24, 25).

Although both these expression vectors and conditionally replicative, oncolytic viruses attempt to exploit the relative specificity of the hTERT promoter, hTERT-driven, replication competent adenoviruses present several potential advantages over other gene therapy strategies. Because adenoviruses infect human cells highly efficiently, this approach may permit effective introduction of such viruses into cancer cells in vivo. Moreover, once productively infected, this target cell will produce adenoviruses that can then infect and presumably kill surrounding cells through a bystander effect, potentially amplifying the cytotoxic effects.

Despite these promising findings, additional work is necessary to evaluate whether these viral vectors truly replicate in a conditional manner. Indeed, although Huang et al. (21) reported a 1000-fold difference in the replication rate measured in normal and malignant cell lines, they also observed replication in normal human cells, consistent with the observation that telomerase is transiently expressed in such cells (6). This finding raises the possibility that the use of such viruses may also replicate in normal tissues and induce unintended cell killing and toxicity. In addition, we still lack a comprehensive understanding of when telomerase is activated during malignant transformation. Only by determining which normal, premalignant, and malignant cells constitutively express hTERT will we be able to make rational decisions regarding which clinical scenarios represent the optimal situations for the testing and use of these agents.

Despite these concerns, additional work, both basic and translational, will certainly lead to a deeper understanding of telomere and telomerase biology and will define the parameters that will dictate the effectiveness of these approaches. Moreover, we can expect that advances in applied immunology and gene therapy technology will increase the efficiency of these approaches. Taken together, targeting telomerase continues to represent a promising avenue for the development of novel antineoplastic strategies, and one or more of these approaches may allow us to add a new tool to our armamentarium to treat cancer.