Steroid Hormone Regulation Of Telomerase In The Prostate

In the prostate, telomerase activity is subject to regulation by steroid hormones, particularly andro-gens. Normal prostate tissues free of cancer, as well as cultured normal prostate epithelial cells, are typically negative for telomerase activity in the presence of androgen (Table 1). However, androgen withdrawal leads to the activation of telomerase. This was first observed in the involuted residual prostate and seminal vesicles of the Copenhagen rat, in which telomerase activity becomes strongly positive after castration (199). This activity remains undiminished even at 2 years after castration, despite the near total lack of ongoing cell proliferation in the quiescent, involuted glands. These residual glands are highly enriched for prostatic stem cells, as evidenced by the rapid induction of cell division and regrowth of these glands after restoration of testosterone. During androgen-induced glandular regeneration, telomerase activity subsides (199). Based on the kinetics of the appearance and disappearance of activity as glands undergo involution and regrowth, respectively, and the results of mixing experiments between extracts of intact and involuted glands, it was concluded that telo-merase activity was negatively regulated by androgens rather than being constitutively active in a small subpopulation of normal stem cells. These studies were later replicated in a non-human primate by Ravindranath et al., who found similar results to those observed in the rodent model (200). Thus,

+ Androgen - Androgen

Normal Prostate Epithelium

Prostate

Adenocarcinoma

Fig. 4. Telomerase regulation by androgens in the prostate: healthy vs cancer. Androgens act to repress telomerase in the normal prostate and this repression is relieved after androgen withdrawal. Prostate cancer behaves in a manner opposite to that of normal prostate epithelial cells.

the overall picture for the normal prostate is that telomerase is actively repressed in an androgen-dependent manner, and that activity is upregulated after androgen withdrawal. In sharp contrast, this normal regulation of telomerase is lost in prostate cancer, because the majority of primary tumors display robust telomerase activity despite the presence of normal levels of circulating androgens. In fact, with respect to telomerase regulation, the response of prostate cancer cells to androgen seems to be exactly the opposite that of normal cells. Not only are cancer cells telomerase positive in the presence of androgen but telomerase activity is suppressed after androgen withdrawal (Fig. 4) (201). For example, Guo et al. found that hTERT mRNA levels increased in the hormone-responsive prostate cancer cell line, LNCaP, when treated with the nonmetabolizable synthetic androgen, R1881 (202). This effect was considered to be indirect, based on the kinetics of the response and also the fact that R1881 failed to activate a reporter gene construct under the control of the hTERT promoter, in keeping with the known response elements in the telomerase promoter region. Although there is an androgen response element in the hTR promoter region, none is apparent in the hTERT promoter (203-206). Guo et al. also observed that, in the hormone-responsive CWR22 prostate cancer xenograft model, both telomerase activity and hTERT mRNA decreased after castration, and this effect was reversed after re-introduction of androgen. A similar pattern was observed by Soda et al., Thelen et al., and Bosland et al., with androgen deprivation causing a decrease in telomerase activity and dihydrotestosterone treatment causing an increase in activity in LNCaP cells, but not in normal prostate epithelial cells or the hormone-insensitive prostate cancer cell line, DU-145 (207-209). In contrast, Bouchal et al. did not observe a positive effect in LNCaP with dihydrotestosterone, but did see suppression of telomerase activity after treatment with the anti-androgen, bicalutamide, in LNCaP but not DU-145 cells (210,211). Bicalutamide also decreased hTERT mRNA and c-Myc mRNA but did not change expression of a host of other telomerase modulators, including dyskerin, hsp90, p23, SIP1, Mad1, and menin. In clinical specimens, Iczkowski et al. reported that telomerase expression in cancer, as measured by antibody staining, was significantly suppressed after complete androgen ablation (212). It is worth noting that inhibition of telomerase by androgen withdrawal observed in prostate cancer, although substantial, is typically not 100% complete.

The precise details of how androgens regulate telomerase in the prostate remain to be elucidated. As mentioned previously, a potential mediator of androgen's effects on telomerase in the prostate is c-Myc. It has been shown that androgen is capable of upregulating c-Myc and that hTERT expression is positively correlated with c-Myc expression in prostate cancer (139,213). Another candidate regulator is the polycomb protein CBX7, which was recently highlighted in a screen for genes upregulated in human prostate epithelial cells that had bypassed senescence (214).

In addition to androgens, estrogen has also been implicated in telomerase regulation in the prostate. Examining normal prostate epithelial cells and BPH tissue explants in culture, Nanni et al. found

+ Androgen - Androgen

Normal Prostate Epithelium

Prostate

Adenocarcinoma

Fig. 4. Telomerase regulation by androgens in the prostate: healthy vs cancer. Androgens act to repress telomerase in the normal prostate and this repression is relieved after androgen withdrawal. Prostate cancer behaves in a manner opposite to that of normal prostate epithelial cells.

them lacking both hTERT mRNA and telomerase activity. Estrogen treatment led to induction of hTERT message and telomerase activity in these cultures, as well as increases in activity (over baseline levels) in cancer cell lines and cancer tissue explants (215). No such effects were observed in estrogen receptor-negative cells. Unlike androgens, estrogen was able to activate an hTERT promoter expression construct. Thus, in contrast to androgen action; estrogens seem to directly activate hTERT transcription, likely via interaction with the known estrogen response element located in the hTERT promoter. In support of this, chromatin immunoprecipitation experiments indicate that both estrogen receptor (ER)-a and ER-P bind to this estrogen response element (215). It was also found that 4-hydroxy tamoxifen inhibited telomerase activity in both LNCaP and DU-145 cells, perhaps via preferential recruitment of ER-P to the hTERT promoter. In addition, the selective aromatase inhibitor letrozole also decreased telomerase activity in LNCaP cells in the presence of testosterone but not the non-aromatizable synthetic androgen, R1881, thus, raising the possibility that local conversion of testosterone to estrogen may play a role in regulating telomerase in the prostate. Finally, Bouchal et al. observed a slight increase in telomerase activity in LNCaP cells after a 3-day treatment with 17-P estradiol, but not with a shorter 1-day treatment (210,211).

15. TELOMERASE-BASED OPPORTUNITIES FOR THERAPY

There are three overall strategic paradigms for therapeutic targeting of telomeres or telomerase in cancer. The first involves taking advantage of the tumor's dependence on telomerase enzymatic activity for survival. This strategy includes approaches aimed at directly inhibiting telomerase enzymatic activity or blocking expression of either hTERT or hTR. The second approach attempts to exploit the fact that the hTERT promoter is active in most cancer cells, for example, by specifically targeting oncolytic viruses or gene therapy to tumor cells, or by directing immunotherapy against cells expressing hTERT. The third approach involves attempts at altering the telomeres themselves. Many of these approaches have undergone preclinical testing using prostate cancer cell lines and xenografts.

One concern regarding anti-telomerase therapy stems from an anticipated delay in effect after treatment. For approaches based on telomerase inhibition, it had previously been thought that after enzyme inhibition there would, of necessity, be a lag period during which telomeres would progressively shorten before any treatment effect would be observed, and that such a lag would be related to the starting telomere lengths of the cells undergoing treatment. However, several studies have observed earlier than expected negative effects on cell growth and survival, hinting that telo-merase may have other functions in the cell besides telomere length maintenance. In support of this, several studies indicate that telomerase may be playing other roles that support cell growth and survival (216-223). If true, then telomerase inhibitors may also prove useful in treating the minority of tumors possessing normal-to-long telomere lengths.

Another concern regards the question of selectivity of action against tumor cells over normal telomerase-positive cells, such as the stem cells of the hematopoietic system and those within tissues with high turnover rates. This is of particular importance for those approaches in which telomerase-positive cells are actively targeted for destruction, including immunotherapy, gene therapy, and onco-lytic viral therapies. Encouragingly, work to date describing the treatment of human tumor xenografts in mice have, in general, not produced major toxicity in normal tissues.

16. DIRECT INHIBITION OF TELOMERASE

Various small molecules have been used in attempts to find suitable inhibitors of the telomerase enzyme (224). Because telomerase is a reverse transcriptase, nucleoside analogs (e.g., AZT) were among the first agents tested. Unfortunately, these showed only weak inhibition (225,226). Non-nucleoside inhibitors have also been tried with limited success (227). Natural compounds, including genistein, silibinin, and tea catechins have also been assayed for potential anti-telomerase effects

(208,228,229). Genistein decreased hTERT levels and telomerase activity in LNCaP and DU-145 cells, and, when assessed in LNCaP, decreased c-Myc mRNA and cellular proliferation (228). Likewise, silibinin was also found to decrease hTERT mRNA and telomerase activity in LNCaP cells

Several groups have explored telomerase inhibition by use of short oligonucleotides of varying chemistries, and this general approach has been reviewed by Corey (230). This approach was first attempted with peptide nucleic acid (PNA) oligonucleotides having sequences targeted against the template RNA sequence. Here, oligonucleotide binding acts to directly block enzymatic activity (template antagonist), leading to telomere shortening, growth arrest, and apoptosis (231-234). Treatment of DU-145 prostate cancer cells with PNA oligonucleotides led to decreased cell growth but incomplete cell kill. Antisense PNAs targeted against the hTERT mRNA have also shown antiproliferative effects (235).

Herbert et al. developed a thiophosphoramidite DNA oligonucleotide template antagonist that was further optimized to give a 13-mer oligonucleotide with improved potency, stability, and bioavailability (236-238). Treatment of telomerase-positive cancer cells with the new molecule, GRN163, caused telomerase inhibition, progressive telomere shortening, and eventual senescence or apoptosis. As was predicted, growth inhibition correlated inversely with the starting telomere lengths of the cell lines used. GRN163 also showed significant antitumor effects in vivo in a DU-145 prostate cancer xenograft model without evidence of host toxicity during an 8-week treatment period.

2'-O-(2-methoxy ethyl) RNA oligonucleotides have also been studied, producing telomere shortening and eventual total cell killing in LNCaP and DU-145 cells treated in vitro (239,240). Importantly, these effects were reversible after drug withdrawal and were not observed with a control mismatched sequence oligonucleotide. The match oligonucleotide was found to have significant negative effects on cell proliferation, anchorage-independent growth, and colony formation relatively early during treatment. Thus, it should be borne in mind that a strict focus on end points such as complete cell cycle arrest or apoptosis may lead to an underestimate of potentially significant clinical effects from telomerase inhibitors. Antitumor activity was also observed in a xenograft model and combined efficacy with various chemotherapeutic agents was observed in vitro with these oligo-nucleotides, however, this effect was complex, being dependent on the particular agent and cell line used. For example, oligonucleotide treatment sensitized DU-145 cells to cisplatin and carboplatin, but not to doxorubicin, etoposide, or paclitaxel; whereas LNCaP cells were not sensitized to any of these agents.

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