Indirect Inhibition Of Telomerase

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Antisense approaches have been used aimed at degrading either the hTERT or hTR RNAs. Hammerhead ribozymes against both of these critical telomerase components have been produced (223,241-245). 2'-5' Oligoadenylate chimeric antisense oligonucleotides targeting hTR for destruction by RNAseL have shown effect against various cancer cell types, including prostate cancer, both in vitro and in vivo (246). Schindler et al., found phosphorothioate antisense oligonucleotides targeting hTERT effective in inhibiting DU-145 cell viability (247). The use of anti-hTERT small interfering RNAs have also been reported (248).

Telomerase inhibition has also been accomplished by using dominant negative (DN) forms of the telomerase catalytic subunit; either by introduction of a catalytically dead hTERT mutant gene into cells, or by the use of oligonucleotide-mediated alternate splicing of endogenous hTERT mRNA. DN mutant hTERT was shown to inhibit telomerase activity, shorten telomeres, and lead to senescence or apoptosis in various tumor cells (249,250). Guo et al. examined the effects of expressing DN hTERT in three prostate cancer cell lines: PC3, DU-145, and LNCaP. In this study, negative effects on PC3 tumor cells, including telomere shortening, growth inhibition, decreased colony formation, and decreased tumor growth in nude mice, were dependent on the degree of DN

hTERT expression (251). Interestingly, although high level DN hTERT expression restored mortality to PC3 and LNCaP cells, this was not true for DU-145 cells. Even at high levels of expression, no growth arrest was observed in vitro, and only a slowing of tumor growth was observed in vivo in DU-145 xenografts. These findings are reminiscent of results observed in another study using antisense against hTR in DU-145 (233). On the other hand, a study using oligonucleotide-mediated alternate splicing of hTERT to generate DN hTERT in DU-145 found decreased telomerase activity and accompanying inhibition of cell growth and promotion of apoptosis (252). Such findings underscore the apparent need for thorough telomerase inhibition and imply that factors intrinsic to the specific tumor cell population may affect the ultimate response to specific approaches to inhibit telomerase. Likewise, sensitization to antitumor drugs is often observed, but only for certain agents (253-256).

Acetylation and deacetylation of histones is linked to changes in chromatin structure that can dramatically affect gene expression, and inhibitors of histone deacetylases are currently being evaluated as anticancer agents. Two such agents, sodium butyrate and trichostatin A, have been show to decrease hTERT mRNA levels and telomerase activity in LNCaP and PC3 prostate cancer cells (257). Interestingly, in one study, trichostatin A was found to activate hTERT mRNA production and telomerase activity in both normal and transformed telomerase-negative human cells (258).

Another prominent mechanism affecting gene silencing is methylation of cytosines in the CpG islands in the promoter region of genes. Guilleret et al., have reported a positive correlation between hypermethylation of the hTERT promoter and hTERT mRNA expression and telomerase activity in several cell lines, including PC3 (177). This group has also reported that treatment of telomerase-positive tumor cells with the demethylating agent 5-aza-2'-deoxycytidine led to repression of hTERT (without changes in c-Myc expression) and decreased telomerase activity (259). These results are unusual in that promoter methylation typically acts to repress gene expression, whereas demethyla-tion typically reactivates gene expression, although it is possible that the observed effects are indirect, perhaps caused by reactivation of expression of a telomerase repressor. Other groups have also observed instances in which the hTERT promoter is unmethylated in telomerase negative cells and partially or totally methylated in telomerase-positive cells, however, in at least one study, no correlation was found between methylation status of the hTERT promoter and hTERT gene expression, and, in this case, the demethylating agent 5-aza-2'-deoxycytidine induced telomerase expression in telo-merase-negative cells (260-262). More work will thus be required to clarify the apparently complex relationship between telomerase gene expression and promoter methylation status.

There is evidence that cellular kinase and phosphates may modulate telomerase activity. Kang et al. reported that there are two potential Akt phosphorylation sites on hTERT and that the Akt inhibitor, wortmannin, decreased both Akt-dependent phosphorylation of telomerase peptides in vitro and telomerase activity in a human melanoma cell line, whereas treatment with okadaic acid, an inhibitor of protein phosphatase 2A, which counteracts Akt, produced the opposite effect (263). In another study, protein phosphatase 2A was shown to abolish telomerase activity in breast cancer cells, whereas okadaic acid treatment stimulated telomerase activity (264). Protein kinase C activity also seems to enhance telomerase activity, and some protein kinase C inhibitors have been shown to inhibit telomerase (111,265-267).

Retinoic acid and vitamin D3 have both been reported to have growth suppressive effects in cancer cells, including prostate cancer, and anti-telomerase effects have been observed with these agents (192-194). Ikeda et al. have identified a putative vitamin D receptor binding sequence within the hTERT promoter (195). Furthermore, combined treatment of retinoic acid and vitamin D3 resulted in decreased hTERT expression and telomerase activity in PC3 and LNCaP cells, and slowed tumor growth in PC3 xenografts. However, the effect of these agents on telomerase is apparently cell type dependent because combined treatment with retinoic acid and vitamin D3 did not suppress hTERT in DU-145 prostate cancer cells.

18. EXPLOITING TELOMERASE EXPRESSION IN CANCER CELLS—GENE THERAPY

Because telomerase expression is activated in most cancer cells, attempts have been made to make use of the active hTERT promoter to drive the expression of pro-apoptotic genes, suicide transgenes, or genes that facilitate selective drug uptake, in tumor cells. Pro-apoptotic and suicide genes that have been used in this approach include: caspases 6 and 8, FADD, Bax, TNF, and TRAIL and HSV-TK (268-273). Genes used for selective drug uptake include Escherichia coli nitroreductase and the noradrenaline transporter (274-278). Several of these studies have shown decreased growth and/or apoptosis in tumor cells, both in vitro and in vivo. For example, the noradrenaline transporter allows uptake of a 131I-labeled drug in transfected telomerase-positive cells. This approach is advantageous in that there is a bystander effect of the drug such that neighboring cells will also be killed, thus, 100% efficiency of transgene delivery to the tumor is not necessary. In experiments using LNCaP and DU-145 cells, the gene is expressed, cells take up the drug and exhibit dose-dependent toxicity at doses showing negligible killing of control parental cells that lack the transgene. A comparison between transporter expression constructs using the hTERT promoter or the PSA promoter showed that the telomerase promoter resulted in superior drug uptake. In addition, testing in cell spheroids confirmed that a bystander effect was operating.

19. EXPLOITING TELOMERASE EXPRESSION

IN CANCER CELLS—ONCOLYTIC VIRAL THERAPY

The hTERT promoter has been used in attempts to achieve selective replication of lytic viruses in cancer cells. The general approach was pioneered in the prostate by Rodriguez et al., who made use of the PSA promoter to restrict viral replication to prostate epithelial cells (279). For telomerase targeting, early viral genes essential for viral replication are placed under the control of the hTERT promoter, thus, limiting viral replication to hTERT-positive tumor cells while sparing the surrounding normal cells. Productive viral infection in tumor cells leads to death (lysis) of the infected cells and release of infective virions, which can then spread to other nearby susceptible cells. This allows amplification of the killing effect of the initial virus, which should increase antitumor efficacy and overcome the problem of tumor cells that were uninfected during the initial treatment phase.

The human adenovirus has been used by several groups to create selectively replicating viruses. Here, the adenovirus E1A early gene is placed under control of the hTERT promoter sequence (280282). Using this approach, Lanson et al. saw good selectivity of tumor cell killing, including DU-145 prostate cancer cells, over normal control cells (283). Likewise, Irving et al. found that viral replication and cell lysis was restricted to several telomerase-positive tumor cell types when tested in vitro (284). In vivo, activity was observed in LNCaP xenograft tumors after intratumoral viral injection, with some complete regressions noted in this study. In tests of systemic delivery in mice, no toxicity was observed for hTERT-selective virus in contrast to other adenoviral constructs driven by the CMV promoter that caused hepatotoxicity (284). Ruan et al. recently reported the addition of further restriction on viral replication by placing the adenoviral gene E4 under control of the E2F-1 transcription factor in addition to having E1 under hTERT promoter control (285). This construct demonstrated improved selectivity and decreased toxicity compared with constructs lacking this additional constraint on viral replication and was tested in LNCaP, PC3, and VCap prostate cancer cells. In LNCaP xenografts, a single intravenous injection led to complete regression in 83% of preestablished tumors (n = 12). Furthermore, when used in combination, viral infection was shown to enhance the antitumor activity of doxorubicin (285).

The preclinical results for hTERT-selective oncolytic virus seem promising. However, there is a critical need for studies evaluating the safety of these viral constructs in humans, especially because the mouse is only a semipermissive host for adenovirus (286). Thus, although useful, human tumor xenografts in mice will not inform us fully regarding the potential toxicities we may see in patients.

There is also the potential for such viruses to replicate in normal tissue stem cells that are currently thought to express telomerase. However, these cells may be difficult to infect and typically display low levels of telomerase or intermittent activity, therefore, the desired selectivity of this approach may be maintained. A further concern specific to prostate cancer is the fact that hTERT expression is dramatically reduced after androgen withdrawal, thus, potentially reducing treatment efficacy in patients who have undergone androgen ablative therapy, but this will only be known after this approach has been refined and tested in the clinic.

20. EXPLOITING TELOMERASE EXPRESSION IN CANCER CELLS—IMMUNOTHERAPY

Because of its widespread and fairly selective expression, telomerase has been touted as a potential "universal" tumor-associated antigen for use in immunotherapy approaches. Indeed, immune effector cells recognizing hTERT do exist and can be raised experimentally (287,288). For example, peripheral blood lymphocytes from both healthy individuals and cancer patients respond to HLA-A2-restricted hTERT peptides, providing evidence for endogenous hTERT-specific cytotoxic T-Lym-phocyte (CTL) precursors. In addition, CTL obtained from three of four hormone-refractory prostate cancer patients were shown capable of lysing tumor cells in vitro, including LNCaP and PC3 (288).

In a phase I vaccination trial using ex vivo-generated telomerase peptide-pulsed autologous dendritic cells, hTERT-specific T lymphocytes were induced in more than half of patients with advanced prostate or breast cancer, without significant toxicity (289). These cells could be expanded in vitro and were competent for tumor cell killing. In addition, some partial tumor regressions were observed in association with the presence of tumor-infiltrating lymphocytes in this study. Careful choice of telomerase peptides seems necessary, because not all peptides are able to produce CTL with the ability to recognize tumor cells expressing telomerase (290).

Nair et al. have shown that a CTL response can be induced in mice immunized with dendritic cells (DC) transfected with human hTERT RNA (291). These CTL were able to lyse human tumor cells, including prostate cancer cells. In addition, it was determined that total tumor RNA was superior to hTERT RNA alone. Similarly, Heiser et al. found that the use of amplified total prostate tumor RNA to pulse DC cells induced polyclonal CTL that, although they included anti-PSA and anti-hTERT subpopulations, were more effective at tumor cell lysis than CTL induced by pulsing DC with PSA RNA or hTERT RNA alone (292).

hTERT would seem an especially attractive target for immunotherapy because, presumably, it would be difficult for tumor cells to develop resistance by antigen loss because telomerase is likely to be essential for tumor cell survival (although the telomerase-independent telomere maintenance ALT pathway has been demonstrated in certain tumors of mesenchymal origin, it does not seem to be operative in carcinomas, including prostate cancer). Of course, tumor cells may find other ways of avoiding detection by the immune system, such as loss of antigen presentation via major histocom-patibility complex or transporter for antigen presentation downregulation. Finally, although autoim-munity caused by the breaking of self-tolerance is a concern with immunotherapy approaches, thus far, there has been no evidence of this, perhaps because normal cells that express hTERT express it at only very low levels.

21. TELOMERE-TARGETED THERAPIES

Targeting the telomeres themselves has also been explored as a potential anticancer approach. G-rich DNA single-stranded overhangs present at the telomeres are able to adopt a structural motif known as a G-quartet or G-quadruplex. G-quartets are folded in such a way that sets of four guanine nucleotide bases associate with one another via non-Watson and Crick hydrogen bonds. This peculiar structure restricts the access of telomerase, thereby inhibiting telomere elongation. Several compounds have been developed with the aid of computer modeling designed to interact with and stabi lize G-quartets (293-297). It is thought that such agents will act to inhibit telomerase, thereby leading to senescence or apoptosis of cancer cells. Testing G-quartet interactive cationic porphyrins on DU-145, PC3, and LNCaP cells, Izbicka et al. noted relatively rapid effects on cell growth and proposed that structural changes at the telomere, in addition to telomerase inhibition, likely contributed to the observed effects (298). Likewise, Incles et al. noted senescence induction in DU-145 at rates faster than predicted based on the inhibition of telomerase alone (299). The observation of extensive end-to-end chromosome fusions in this study strongly implied that widespread telomere uncapping had occurred, thus, the effects observed were likely the result of structural changes induced at the telom-eres by the agent. Such results highlight the need for inclusion of appropriate normal cell controls in studies of G-quartet-promoting agents to ensure that any effects observed are selective for cancer cells.

One other approach based on targeting the telomere itself involves the introduction of a construct coding for a mutated version of the telomerase template RNA (248,300). Mutations in the template region of hTR lead to incorporation of mutant telomere repeats by telomerase in telomerase-positive cells. These, in turn, disrupt the interaction between the telomere and telomere-specific binding proteins, causing uncapping of one or more telomeres. Expression of mutant hTR in LNCaP and MCF-7 cancer cells causes decreased growth and colony formation and increased apoptosis, as well as decreased tumor growth in an MCF-7 xenograft model, even if expression of the mutated hTR was low. This is in sharp contrast to telomerase inhibition strategies, which seem to require high levels of enzyme inhibition to produce significant effects.

22. CONCLUSION—INTEGRATING TELOMERE BIOLOGY INTO THE PROCESS OF PROSTATE CARCINOGENESIS

Telomere shortening can initiate CIN and occurs early during prostate tumorigenesis; it is, therefore, a likely contributor to the complex genetic changes underlying the phenotypic diversity of prostate cancer. If true, this helps resolve the puzzle of why prostate cancers seem to lack prevalent genetic changes in specific tumor suppressors, oncogenes, or genome stability genes. Instead, the source of CIN may stem from defects in fundamental structural components of the chromosomes themselves, namely, the telomeres.

Early observations of abnormally short telomeres plus the nearly universal activation of telomerase in primary prostate cancers strongly suggested that the majority of prostate cancers undergo critical telomere shortening during their development. Recently, it was found that this occurs early in the disease process, by the pre-invasive PIN stage, in which telomeres are abnormally short and telomerase activity first becomes evident. Notably, basal epithelial cells, which express genome protective genes, such as GSTP1, retain normal telomere lengths in PIN and, thus, may not be the ultimate target cells for prostate carcinogenesis. Other cells with phenotypes intermediate between basal and luminal epithelial cells have been proposed as potential transformation targets in the prostate (301-303). These putative transit amplifying/transient proliferating (TA/TP) cells, which are thought to lie along the differentiation pathway between basal stem cells and luminal secretory cells, possess features of the basal stem cell compartment, such as protection from apoptosis and proliferative competence—features also typically found in prostate cancer cells (304).

The root cause of the telomere shortening that occurs during malignant transformation of the prostate is unknown. Because telomeres shorten during cell division, one possibility is that telomeres are progressively lost through normal division as cells are replaced during aging. This would help explain the strong dependence of prostate cancer on age (6). On the other hand, the normal rate of cell division in the prostate is relatively low, thus, it may be that other processes serve to accelerate cell turnover. One such process may be inflammation, a common histological finding in the prostate that also increases with age (9,305). Locally, the inflammatory response generates genotoxic chemical species, such as oxy-radicals as part of the host defense mechanism against pathogens. Exposure to

Dieses Resulting Process Mitos

Fig. 5. Changes in telomere length, telomerase activity, and chromosomal instability (CIN) during prostate tumorigenesis and prostate cancer progression. Telomeres (solid line) shorten early in the disease process, driving CIN and the development of prostatic intraepithelial neoplasia (PIN). At this stage, telomerase becomes active, partially stabilizing the shortened telomeres and reducing CIN. Telomerase activity allows unlimited replication, thus, fostering the progression from PIN to invasive cancer and, ultimately, metastatic disease. Androgen ablative therapy (arrow) results in suppression of telomerase activity reigniting CIN in surviving prostate cancer cells, thus, promoting the development of the hormone-refractory lethal cancer phenotype.

Fig. 5. Changes in telomere length, telomerase activity, and chromosomal instability (CIN) during prostate tumorigenesis and prostate cancer progression. Telomeres (solid line) shorten early in the disease process, driving CIN and the development of prostatic intraepithelial neoplasia (PIN). At this stage, telomerase becomes active, partially stabilizing the shortened telomeres and reducing CIN. Telomerase activity allows unlimited replication, thus, fostering the progression from PIN to invasive cancer and, ultimately, metastatic disease. Androgen ablative therapy (arrow) results in suppression of telomerase activity reigniting CIN in surviving prostate cancer cells, thus, promoting the development of the hormone-refractory lethal cancer phenotype.

Telomeres Cancer
Fig. 6. Model of telomere length changes and telomerase activity in prostate carcinogenesis. PIN, prostatic intraepithelial neoplasia. TA/TP, transit amplifying/transient proliferating.

these chemicals causes co-lateral host cell killing, leading to proliferation to replace the lost cells, and oxidative stress itself is able to cause rapid telomere loss (306). Chronic inflammation not only increases cell turnover and telomere loss, but the accompanying genotoxic stress may impair telo-mere-sensitive senescence/apoptosis checkpoints, as well as DNA-damage sensitive checkpoints, perhaps in cells whose genomic protection is impaired, for instance, because of epigenetic downregula-tion of glutathione-S-transferase-n (17,307).

Figure 5 outlines a model of the potential involvement of telomeres and telomerase in prostate carcinogenesis. After puberty, proliferation of telomerase negative transit amplifying/transient proliferating cells during normal aging, or driven by chronic inflammation, leads to moderate telomere shortening that engages tumor suppressive cell cycle checkpoints. If, however, there is genetic or epigenetic inactivation of cell senescence checkpoints (such as the p16/pRB checkpoint), proliferating telomerase-negative cells continue to divide, despite their shortened telomeres (308,309). With time, rare intermediate cells that also have experienced mutations in key growth regulatory pathways, such as the Hedgehog signaling pathway, undergo abnormal clonal expansion (310). During this time, telomeres continue to shorten until one or more telomeres become dysfunctional, thereby initiating CIN, culminating in the emergence of PIN. CIN involves DNA damage, therefore DNA damage checkpoints should act to limit a PIN lesion from progressing further. A subset of PIN lesions may bypass this second checkpoint (DNA damage checkpoint), leading to further oncogenic mutations, resulting in invasive cancer. Finally, immortalization by telomere length stabilization, most commonly through telomerase activation, allows unlimited tumor expansion (although all chromosomes in all cells may not achieve complete stabilization).

Presumably, the chance that initiated cells and premalignant lesions will abrogate both telomere length checkpoints and DNA damage checkpoints is small, explaining why most initiated cells and lesions apparently fail to fully progress to cancer. Even cells that have overcome these tumor-sup-pressive barriers must still eventually re-stabilize their telomeres to prevent runaway lethal genetic instability (postmitotic cell death) (311). Thus, the acquisition of telomerase activity likely represents one of the final hurdles on the road to cancer.

Lastly, telomeres may play an important role in the development of advanced hormone-refractory prostate cancer. Because telomerase activity is significantly suppressed in prostate cancer cells after androgen withdrawal, androgen ablative therapies may reignite CIN in the surviving prostate cancer cells (Fig. 6). Such cells represent a source of cancer cell variation from which a more aggressive, hormone-refractory lethal tumor may evolve.

In summary, short dysfunctional telomeres may be critically involved in the initiation and progression of prostate cancer, as well as the emergence of hormone-refractory cancer after androgen ablation. If so, a thorough understanding of telomere biology as it relates to prostate cancer should provide new opportunities for effective disease prevention and treatment.

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