Telomerase inhibition as cancer therapy

Cancer Letters 194 (2003) 209–219 www.elsevier.com/locate/canlet

Telomerase inhibition as cancer therapy Gabriele Saretzki* SCMS Gerontology, Institute of Ageing and Health, Newcastle University, General Hospital, Newcastle upon Tyne NE4 6BE, UK Received 10 September 2002; received in revised form 29 October 2002; accepted 29 October 2002

Abstract A number of different approaches have been developed to inhibit telomerase activity in human cancer cells. Different components and types of inhibitors targeting various regulatory levels have been regarded as useful for telomerase inhibition. Most methods, however, rely on successive telomere shortening. This process is very slow and causes a long time lag between the onset of inhibition and the occurrence of senescence or apoptosis as a reversal of the immortal phenotype. Many telomerase inhibitors seem to be most efficient when combined with conventional chemotherapeutics. There are some promising approaches that seem to circumvent the slow way of telomere shortening and induce fast apoptosis in treated tumor cells. It has been demonstrated that telomerase may be involved in triggering apoptosis, but the underlying molecular mechanism remains unclear. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Telomerase; hTERT; hTER; Telomerase inhibition; Telomere; Cancer; Tumor; Apoptosis; Senescence; Therapy; Treatment; Ribozyme; Antisense; G-Quartet; Telomere capping; Chemotherapeutic drug

1. Introduction Whereas mortal cells shorten their telomeres during each round of replication, cancer cells acquire indefinite growth capacity by maintaining their telomeres. The vast majority of tumors activate telomerase, and only few maintain telomeres by alternative mechanisms relying on recombination [1]. In contrast, telomerase activity is absent from most and tightly controlled in the remaining human somatic cells. Immortality can be understood as an escape from senescence and deregulation of cell cycle and proliferation programs. These observations suggest telomerase as an important target for the development of new anticancer drugs and strategies based on the reversal of * Tel.: þ44-191-256-3378; fax: þ44-191-219-5074.. E-mail address: [email protected] (G. Saretzki).

tumor growth by telomerase inhibition (summarized in Table 1). Moreover, the relatively tumor-specific expression of telomerase is used to direct cytotoxic approaches, for instance cytotoxic lymphocytes or suicide gene expression, towards tumor cells. There are excellent recent reviews covering these approaches [2,3], but they are not within the scope of the present article. Telomerase is a unique reverse transcriptase consisting of two major components, the RNA moiety (hTER) and the catalytic subunit (hTERT). A number of regulatory proteins – hsp 90, p23, TEP1 and others [4] are associated with this core. The molecular mechanism of upregulation of telomerase activity during tumorigenesis remains elusive, but a major body of knowledge regarding regulation of telomerase activity accumulated in recent years. Transcriptional regulation is of prime importance, but regulation occurs also by gene amplification, at the assembly

0304-3835/02/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi: 1 0 . 1 0 1 6 / S 0 3 0 4 - 3 8 3 5 ( 0 2 ) 0 0 7 0 8 - 5

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Table 1 Targets for telomerase inhibition Target 1. a

Telomerase core components hTER

b

hTERT

2. a

The telomere G-quartets

b

Capping function

c

Conventional Chemotherapeutics

3.

Natural compounds

4.

Small molecules

5.

Regulatory mechanisms

6.

Apoptosis induction

stage, during splicing of RNA and by posttranslational modification. Accordingly, telomerase inhibitions have been attempted at various possible levels. With the improvement in understanding of telomerase activity regulation, more sophisticated measures for optimal intervention into tumor growth are to be expected.

2. Direct targeting of core telomerase components Both components of the core telomerase enzyme, the RNA (hTER) and the catalytic subunit (hTERT), have been used as targets for telomerase inhibition. 2.1. hTer The first successful attempt was reported in 1995, using an antisense vector against the first 185 nucleotides of the hTER molecule. Telomeres of

Example

Refs.

Antisense 2-5A-ODN 20 -Ome RNA DNhTERT Anti-hTERT ribozymes

[5,6] [12–16] [11] [19,20] [23–27]

Porphyrins Amidoanthraceans Pentacyclic acridine Mutant hTER template PinX1 Cisplatin Topoisomerase inhibitors Mistletoe Tea catechins Telomestatin AZT Berberine derivatives BIBR1532 Geldanamycin Transcription factors Hormones Retinoids 20 -50 ODN Anti-hTERT ribozyme

[32] [32] [33] [39] [43] [44,45] [25,48–50] [53] [55] [56] [58,59] [63] [64] [66] [71–78] [82,83] [84,85] [12–16] [25,27]

treated HeLa cells shortened over a long period of time (23 –26 population doublings) and most tumor cells of the culture eventually went into crisis [5]. Interestingly, Kondo et al. [6] using the same antisense component found a similar effect in glioma cells. After 30 doublings and presumably sufficient telomere shortening two different subpopulations were observed: one going into apoptosis, the other not. Both subpopulations showed enhanced production of the ICE caspase. The authors discuss the two pathways – apoptosis or differentiation – in the context of a differential expression of cyclin-dependent kinase inhibitors (CDKI). Those early successes showing a positive effect on tumor cell survival due to telomerase inhibition and telomere shortening encouraged the scientific community to search for other and easier applicable anti-telomerase drugs. However, a remarkable number of approaches using, e.g. antisense oligonucleotides, peptide nucleic acids or hammerhead ribozymes against the hTER

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subunit could not demonstrate attenuated tumor cell viability despite diminishing telomerase activity significantly [7 – 10]. Herbert et al. [11] developed 20 -O-methyl-RNA (20 -O-meRNA) oligomers with enhanced binding properties to hTER sequences and higher stability than the conventional DNA oligonucleotides. The oligomers efficiently inhibited telomerase activity, and that led to progressive telomere shortening in immortal breast epithelial cells. However, it took the cells over 100 days to go finally into apoptosis. Other cell lines with longer telomeres reduced their net growth upon treatment, but there was no apoptosis despite a dramatic telomere reduction even after months. The conclusion from that is that the antiproliferative effect of those oligonucleotides is telomere length-dependent and that this approach towards telomerase inhibition is not applicable to tumors with rather long telomeres. Anti-hTER oligonucleotides linked to 50 -phosphorylated, 20 -50 -linked oligoadenylates that activate the catalytic RNase L system of the cell to degrade hTER were proven to be very successful. Kondo, Cowell and others showed that this 2-5A antisense strategy could be applied to specifically degrade hTER in glioma, prostate, cervical, bladder and ovarian cancer cells within 4 –5 days and to reduce the cancer cell viability to 20– 30% 2 weeks later [6, 12 –16]. The fast reduction of cell viability was due to induction of apoptosis. Telomere shortening within this short period was not reported. The authors measured induction of caspases; however, it is unclear how telomerase inhibition could lead to caspase activation. Possibly, the treatment causes telomere uncapping by withdrawing the telomerase molecule from some or all telomeric ends, and that might trigger an apoptotic response (see Section 3.2). In addition, the authors could impressively demonstrate the successful reduction of tumor growth in a mouse xenograft model [6].

dependence of the lag phase – the time between onset of treatment and growth arrest or crisis – from the initial telomere length. Reaching sufficiently short telomeres, cells went into crisis and were characterized by increased apoptosis. Hahn et al. [20] also showed a reduced tumorigenicity in nude mice when cells were retrovirally transfected with DN-hTERT before xenotransplantation. Another possibility to target either of the two telomerase components are hammerhead ribozymes. These are small catalytically active RNA molecules cleaving their RNA substrate in a sequence-dependent manner at GUX motifs. First ribozyme approaches were against hTER and reduced telomerase activity, but had no anti-proliferative effect on tumor cells [9, 21]. Folini et al. [22] found longer doubling times but no telomere shortening after treatment of melanoma cells with an anti-hTER ribozyme. Ribozymes against the hTERT component were also designed and tested in cell studies [23 –26]. Ludwig et al. [25] demonstrated not only that cleavage of hTERT mRNA led to shortened telomeres, net growth arrest and induction of apoptosis but in addition sensitized tumor cells to inhibitors of topoisomerase II. The same group showed that adenoviral application of anti-hTERT ribozymes led to immediate induction of apoptosis in different breast and ovarian tumor cell lines. Apoptosis was independent from the initial telomere length over a wide range (4 –12 kb) and was not accompanied by telomere shortening [27]. Only few reports show that anti-telomerase ribozymes can decrease tumor size and induce apoptosis in vivo [24].

2.2. hTERT

The disruption of the telomeric structure and/or function by drugs interacting with this substrate of the telomerase enzyme has evolved into a promising way of telomerase inhibition. The telomere has been shown to be a complex structure comprised of higher order chromatin loop and specific telomere binding proteins [28]. A central role for the whole structure plays the single-stranded 30 G-rich overhang.

The gene for the catalytic subunit of telomerase was discovered in 1997 [17,18] and soon after targeted as well. Zhang et al. [19] and Hahn et al. [20] used a dominant negative mutant of the hTERT gene (DN-hTERT) for telomerase inhibition. Comparing different cell lines, they found a remarkable

3. Targeting the telomere

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3.1. G-quartets Telomeric DNA consists mainly of double stranded DNA but ends in a conserved single-stranded 30 -G-rich overhang of around 150 – 200 bases in length [29]. The 30 overhang of human telomeres is guanine rich and can form characteristic secondary structures under physiologic ionic conditions, i.e. Gquadruplex structures. It has been hypothesized that these structures might be important for telomere function [30] and this has been approved in the yeast system [31]. Moreover, this structure could hinder the telomerase from elongating the 30 overhang and inhibit the activity of the enzyme. Drugs stabilizing this confirmation are thought to be effective telomerase inhibitors.. Different compounds like porphyrins, amidoanthracens and others have been shown to act via this presumed mechanism (for review see Ref. [32]). Gowan et al. [33] described a pentacyclic acridine which inhibits telomerase, blocked growth and induced a senescence-like arrest in different cancer cell lines at a concentration below 1 mmol. Unspecific cytotoxicity was only found at concentrations about two orders of magnitude higher. Growth inhibition appeared somewhat dependent on initial telomere length, but telomere shortening was not observed [34]. Moreover, the authors also show a significant antitumor activity of the acridine compound in mouse xenografts, but only when combined with the conventional chemotherapeutic drug paclitaxel. A possible explanation could be that telomere damage induced by paclitaxel might destabilize the telomeric loop structure and promote the formation of telomeric G-quartets. 3.2. Capping Telomerase takes part in capping telomeres in tumor cells [35 – 37]. Telomeres form a higher order loop structure [28] and specific telomeric proteins are also critical for maintaining the capped situation [38]. There are different ways to uncap telomeres in human cancer cells and telomere shortening is one of them. In addition, Kim et al. [39] reported uncapping of telomeres in prostate and breast cancer cell lines without telomere shortening or loss of telomerase activity. They expressed hTER molecules with a

mutant template, which decreased cancer cell proliferation already at a low level, and which increased apoptosis rates (around 12%) in vitro and in vivo. The mutant template acted in a strong dominant negative fashion while retaining the normal wild-type telomerase activity [39]. The authors propose that uncapping of only one or a few telomeres might signal cell cycle arrest and apoptosis in human cancer cells. Cao et al. [40] showed that down-regulation of telomerase induced apoptosis in breast cancer cells, and that the cells could be rescued by expression of a hTERT mutant lacking telomerase activity, again suggesting that the anti-apoptotic role of telomerase might be different from its telomere-elongating activity. These results correspond well with suggestions of Saretzki et al. [27] who used a virally transduced anti-hTERT ribozyme for telomerase inhibition and found a fast apoptotic response (75 – 90%) within 1 week in all cell lines tested independently of their initial telomere length and without telomere shortening. In accordance with data from transgenic mice, telomerase might have an important function for tumor growth and survival, even when telomeres are long [41]. The double-strand binding telomeric proteins TRF1 and TRF2 are important for T-loop formation [28,42] and regulate telomere length (for review see Ref. [38]). Other associated proteins, like TIN2 and tankyrases, are also involved in telomere length regulation. Zhou and Lou [43] recently described the Pin2/TRF1-interacting protein PinX1 which binds to the catalytic subunit of human telomerase and inhibits its activity in vitro and in vivo. It acts as a tumor suppressor and has an additional function in rRNA processing [43]. The targeting of such proteins might become an important challenge for new anticancer strategies. Together, it appears that approaches which target the telomeric ‘capping’ function have great potential to develop into an effective and fast way to target cancer cells. 3.3. Conventional chemotherapeutics Certain classes of DNA-damaging drugs employed in conventional chemotherapy might interact preferentially with telomeric sequences, and a number of interactions between these and telomeres, with possible consequences for telomerase activity, have been

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reported. Cisplatin for instance is a G –G cross-linking agent and has been shown to shorten telomeres and inhibit telomerase in HeLa and hepatoma cells [44,45]. Accordingly, Kondo et al. [12,46] observed a higher sensitivity of malignant gliomas to cisplatin after telomerase inhibition by the 20 ,50 -oligoadenylate linked anti-hTER oligonucleotide in vitro and in vivo. However, such synergism was not confirmed in either human melanoma cells [47], human breast tumor cells and immortalized fibroblasts [25] or Myc/Rastransformed mouse embryonic fibroblasts [48]. Similarly, high cleavage activity of topoisomerase II at telomeres following treatment with the topoisomerase II poisons etoposide has been claimed [49], and synergism between telomerase inhibition and topoisomerase poisoning by etoposide or doxorubicin has been observed in human breast tumor cells and immortalized fibroblasts [25], but not in melanoma cells [47]. Higher sensitivity to doxorubicin in telomerase-negative mouse embryonic fibroblasts [48] and MCF-7 breast tumor cells [50] was related to telomere dysfunction, however in the latter case at least independent on telomere length. Telomeres have been shown to act as cellular sentinels for oxidative DNA damage in non-malignant human cells [51], and this function might explain part of the observed interactions with DNA damaging drugs. However, a clear distinction between specific and unspecific drug effects is seldom trivial [52], and more careful research will be necessary for a clearer picture to emerge.

4. Natural compounds and small molecules as telomerase inhibitors Many medicinal plants, known especially to Asian medicine as, for instance, Korean mistletoe, tea catechins and others have been shown to contain active substances inhibiting telomerase activity. The involvement of different pathways including downregulation of bcl-2, targeting of G-quadruplex structures leading to mitochondrially induced apoptosis [53], caspase induction [54], or telomere shortening [55,56] was demonstrated. Whereas most of these substances are effective only in micromolar ranges, one fungal derivative termed telomestatin is a natural product which potently inhibits telomerase by interacting selectively with telomeric G-quartets at a

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concentration of 5 nM [56]. The major advantage of most of these compounds is their low unspecific cytotoxicity, which makes them ideal candidates for enhancing the sensitivity to conventional anticancer drugs, especially of chemoendocrine resistant cancers [57]. Nucleoside analog inhibitors like AZT (3-azido20 ,30 -dideoxythymidine) were among the first synthetic compounds tested against telomerase. However, they exhibited only weak inhibitory activity for human telomerase and mild proliferative impairment despite telomere shortening even at rather high concentrations in the range of 10– 100 mmol [58, 59]. Chronic treatment of mammary carcinoma cells with AZT induces a senescent phenotype and reduces tumorigenicity only at very high concentrations (800 mmol) [60], possibly due to inclusion of AZT into the telomeres [61]. Novel derivatives of 7-deaza-20 deoxypurine nucleoside triphosphate are much more potent inhibitors of telomerase in vitro [62]. In a large database-screening program Naasani et al. [63] found the berberine derivative rhodacyanine FJ5002 as an potent telomerase inhibitor in human leukemia cells. If cells were long-term (, 100 population doublings) treated with 50 – 200 nM, telomere shortening, chromosomal abnormalities and senescence/crisis phenotypes were observed. However, cells in crisis acquired multidrug resistance resembling phenotype, so that the treatment concentrations had to be increased [63]. An independent screening programme resulted in the development of the non-nucleosidic, small molecule telomerase inhibitor BIBR1532 [64], which resembles effects as described for DN-hTERT [20] and antisense-oligonucleotides [11]. After long incubation periods (, 100 days, depending on the initial telomere length) the cells of four different tumor entities slowed down proliferation and obtained a senescent phenotype, independent of their p53 status. Telomere shortening was demonstrated in all four lines and the tumorigenicity of treated cells was reduced in the nude mouse model [64]. Expression array analysis showed many features of senescence in the telomerase-inhibited cells including upregulation of p21 and downregulation of BRCA 1 and 2. Telomere length at the onset of the senescence-like arrest was equal in all four cell lines, in accordance with the different time spans needed to reach this state

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provided the different starting telomere lengths. Further studies revealed that BIBR1532 interferes with the processivity of telomerase [65]. The drug is a non-competitive inhibitor, suggesting a drug binding site distinct from the sites for DNA primer and nucleotides.

5. Interference with regulatory mechanisms of telomerase Telomerase consists not only of the two major components hTERT and hTER, but is associated with several accessory proteins like TEP-1, p23, hsp90 and interacts with modifying enzymes like phosphatase A, protein kinase C, Akt-kinase and others that play important roles for assembly and function of the holoenzyme or regulate posttranslational modification. As those proteins are essential for the functioning of the enzyme, there are different strategies to influence its activity via those co-factors. For instance, the hsp90 inhibitor geldanamycin has been shown to be an attractive telomerase inhibitor [66]. The regulation of telomerase functions at different levels with multiple pathways. One important regulatory mechanism is the effect of different transcription factors on the promoters of hTER and hTERT [67 – 70]. A large number of transcription factors like SP1, c-myc, mad and others have been identified which are positively or negatively regulating the telomerase activity [71 – 77]. Especially the transcription factor c-myc, which is closely connected to the proliferation behaviour of cells, is a tempting target for the inhibition of telomerase activity. Grand et al. [78] describe a cationic porphyrin which downregulates c-myc and hTERT expression causing inhibition of tumor growth in vivo. Others discovered new binding partners for transcriptional downregulation of the hTERT expression via c-myc [79]. Splicing of the hTERT mRNA has been demonstrated as an important regulatory mechanism, and one particular splice product was found to be a dominant negative inhibitor of telomerase activity [80]. Combined regulatory options of c-myc and splicing mechanisms have been uncovered in skin [81].

Several hormones have been identified to play a role in the upstream signalling of the hTERT transcriptional activation, e.g. estrogen and progesterone [72,82,83] and retinoids [84,85]. Moreover, histone acetylation plays a role for the transcriptional regulation of hTERT as well [86]. However, hormone-dependent regulation of telomerase activity is cell- and tissue-dependent. For instance, the antiestrogenic agent tamoxifen blocked hTERT transcription and cell growth in estrogen-receptor positive breast tumor cells, but stimulated transcription of hTERT in endometrial cancer cells [52,87,88].

6. Telomerase inhibition and fast induction of apoptosis To date, most attempts for telomerase inhibition still rely on telomere shortening. This can be a slow process, as cells reduce their telomere length under normal circumstances only by 50 –100 base pairs per cell division [89] and can lead to long lag phases. In fact, treatment times of 3 months and more were often necessary even in experimental cell culture settings, to obtain complete growth arrest and/or apoptosis even for tumor cells with relatively short telomeres [11,55,63,64]. This lag phase needed for subsequent telomere shortening is evidently a serious obstacle for the application of telomerase inhibition as an antitumor strategy, especially in tumors with long telomeres. One way to improve this situation, suggested after the preferential sensitivity of telomeres to oxidative stress-induced single strand breaks was discovered [90,91], is the combination of telomerase inhibition with DNA-damaging chemotherapeutic drugs. Success so far, however, has been limited with this approach (see above). Another, independent possibility is the use of the hTERT promoter to target toxic or apoptosis-enhancing agents into tumor cells ([92, 93]; for review see Ref. [2]). The most promising approach towards tumor killing within a reasonable time scale is the fast induction of apoptosis as observed by using oligoadenylated anti-hTER antisense oligonucleotides [6, 15,94], hammerhead ribozymes against the hTERT Tmotif [25,27] or antisense techniques [40]. More and more data show that telomerase acts as a pro-survival,

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anti-apoptotic enzyme in neurons [95 – 97] by suppressing DNA damage and apoptotic signals [98], as well as in muscle cells [99,100] and cancer cells [40]. Telomerase has been proposed to take part in telomere capping [35,36,39,41]. How can telomerase cap telomeres? Previous experiments have shown that single-stranded telomeric oligonucleotides can block cell growth [101, 102]. One explanation is that accumulation of G-rich oligonucleotides in the nucleus resembles the opening of the telomeric loop and the unscheduled exposure of the telomeric overhang which signals a p53 dependent growth arrest. However, telomerase-positive cells were able to upregulate telomerase activity and to overcome the arrest [102]. Our interpretation of those results is, that telomerase presumably binds the free 30 overhang and by this capping abolishes a signal to emanate from telomeres. Thus, at least three major factors, namely the presence of sufficiently long telomeric repeats, of telomere-binding, loop-stabilizing proteins and of telomerase, together appear to be involved in telomere capping and maintenance of functional telomeres. Conceivably, each of these components can to some extent compensate for the other. This is in accord with reports underlining the importance of both telomerase and telomere length for the sensitivity to apoptosis [48,103]. Thus, a better understanding of functions of telomerase distinct from its telomere elongating role should spawn a completely new generation of telomerase inhibition approaches.

7. Conclusion Over the last years there has been a burst of approaches aiming to inhibit telomerase activity as a new treatment strategy for human cancer. Most approaches still relay on telomere shortening in tumor cells under the treatment. Unfortunately, these approaches generally require long treatments times in the order of 100 days to achieve sufficient reduction of telomere length and are strongly dependent on the initial telomere length in the tumor. Different groups found an enhanced sensitivity of telomerase-inhibited cancer cells to common chemotherapeutic drugs which favours approaches combining telomerase inhibition with conventional

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chemotherapeutics. However, the most promising strategies of telomerase inhibition seem to be those that directly disrupt capping of telomeres by telomerase. These approaches achieve onset of apoptosis within days and might even work in tumors with long telomeres. However, their efficiency in vivo still needs to be tested comprehensively.

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