Telomerase and Cancer Kirk A

Telomerase and Cancer Kirk A. Landon - AACR Prize for Basic Cancer Research Lecture

Elizabeth H. Blackburn

Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California

Telomerase is commonly expressed in human cancer cells. round of replication. Thus, incomplete replication of the Increased telomerase expression produces vulnerability of chromosome ends occurs, which, if left unchecked by any cancer cells, distinguishing them from normal cells in the compensatory mechanism, leads to a problem of maintaining body, although normal cells do also have some active the full-length of chromosomal DNA molecules. The telomerase. Recent studies also suggest that telomerase is chromosome ends become progressively shorter, which was implicated in tumor progression in unexpected ways. These predicted eventually to lead, by some unknown mechanism, to observations lead us to investigate the most efficient means of cells ceasing to divide. The solution to this theoretical inhibiting telomerase in cancer cells. problem was the enzyme telomerase (1). Carol Greider and I identified the telomerase enzyme first in the ciliated A Brief Review of Telomeres and Telomerase protozoan Tetrahymena. We used this organism because it Telomeres, the ends of the chromosomes, are required to has a large number of telomeres and is a relatively rich source protect chromosomal ends. A complex set of biological of telomerase. Telomerase contains an RNA component. The functions is encompassed in the word ‘‘capping’’ of the ends RNA has a short template sequence that is copied into DNA, of the chromosomes. The telomere, after all, represents a DNA which extends, and thus lengthens, the chromosomal DNA. end, which is at the end of the linear chromosomal DNA, but it We were able to show that this also occurs in vivo. It is this has to be treated in a different way from an accidental DNA addition of telomeric DNA in increments to the ends of break in a chromosome. Primarily, capping is accomplished chromosomes that offsets and counterbalances the shortening through formation of a specific DNA-protein complex at the of chromosome ends. chromosomal terminus. Telomerase is by nature a reverse transcriptase by virtue of A popular metaphor for what is involved in capping is that its action of copying the short RNA template sequence within the telomere acts in a way comparable to an aglet, which is the telomerase RNA into DNA; an enzyme that copies RNA the end of a shoelace. Like an aglet, the telomere prevents that into DNA is by definition a reverse transcriptase. Unlike viral end from fraying away. What this means at the molecular or retroviral reverse transcriptases, such as that of HIV-1, the level is that the telomeric DNA contains binding sites for cellular enzyme telomerase specializes in making the multiple proteins that protect the end of the chromosome. This was first short tandem repeats that are at the ends of chromosomes. The shown in a model organism, the ciliate Tetrahymena, and protein component of telomerase, telomerase reverse transcrip- subsequently in many other organisms. The telomeres consist, tase (TERT), is indeed a protein enzyme and its amino acid first, of a DNA scaffold composed of a tract of very simple sequence includes reverse transcriptase motifs. However, the repeated DNA sequences. In the case of humans, the repeat RNA is also critically important to the enzyme action, and not only because it provides the template. The template is only a sequence T2AG3 is repeated, in tandem, thousands of times at the end of every chromosome. That sequence tract provides a minor part of the entire telomerase RNA molecule. The molecular scaffold on which binds a set of proteins, about telomerase RNA is built into the structure of the core which much is known. These proteins include DNA ribonucleoprotein complex of telomerase, which contains the sequence–specific binding proteins and other proteins that protein TERT and the telomerase RNA component. After much together, in effect, sheathe the chromosome end. The resulting study, it has emerged that the RNAs from evolutionarily diverse structure is very dynamic. The proteins come on and off the eukaryotes share a common core structure (2). The core telomere with surprising speed. Hence, the telomeric structure structure includes a conserved pseudoknot, which is one of the is continually being assembled and disassembled. Further- parts of RNA that interact tightly and specifically with the more, the underlying DNA is also dynamic, for two reasons. protein TERT in the enzyme complex. The pseudoknot is also One reason is that, as was predicted in the 1970s, the known critically important for the action of telomerase. Thus, the RNA mechanism of DNA polymerase prevents complete DNA and the protein of telomerase collaborate in the enzymatic replication all the way to the ends of the chromosome at every action. That property also makes telomerase a very unusual reverse transcriptase. The ciliated protozoan Tetrahymena thermophila normally has constitutive telomerase and can continue to proliferate

Received 8/23/05; accepted 8/23/05. indefinitely like a cancer cell. We were originally able to show Note: This lecture was presented at the AACR 96th Annual Meeting, April 16-20, that if we interfered with the action of telomerase in this 2005, Anaheim, CA organism, the telomeres gradually became shorter and cells Requests for reprints: Elizabeth H. Blackburn, E-mail: [email protected] Copyright D 2005 American Association for Cancer Research. eventually ceased to divide. Thus, we converted an effectively doi:10.1158/1541-7786.MCR-05-0147 immortal, perpetually growing organism into a mortal one by

the simple action of making its telomerase nonfunctional, Intervening in the Action of Telomerase in thereby showing that telomerase was important for the Human Cancer Cells continued maintenance of the telomeres and of cell proli- As mentioned previously, if telomerase is present in feration (3). sufficient amounts, it permits cells to keep multiplying. And, Humans also have telomerase. With its different RNA indeed, many labs and many groups have now shown that template sequence, it makes the sequence at the ends of our the apparent increase in telomerase is a common feature of chromosomes, T2AG3 repeats, which is very much like that of human tumor cells. This increased expression of telomerase some ciliated protozoans, T2G4 repeats. Those T2AG3 repeats makes it an attractive target for intervention. We investigated are the critical binding sites for sequence-specific telomeric the effect of telomerase inhibition on cancer cells using two protective proteins. Telomerase levels are very highly regulated different approaches. in normal human cells. As you might expect, telomerase is active in human stem cells and in germ line cells. At various levels, it is also active in other adult human cells, but the levels Forcing Telomerase to Make Mutant Telomeric are often very low and not enough to sustain telomere length DNA in Cancer Cells over a lifetime. In humans, gradual shortening of telomeres is The first way of intervening in telomerase action in human observed throughout life in many cell types in every part of the cancer cells exploited the fact that the telomeric DNA makes the body although the cells have a low level of telomerase. In cell molecular scaffold for the binding of telomere-protective culture, telomere shortening leads to cell senescence as the proteins, which include DNA sequence–specific binding telomeres no longer can sustain a capping structure at the ends proteins. As described above, the telomeric DNA sequence is of the chromosomes. In 1998, Bodnar et al. (4) showed in a specified by copying the telomerase RNA template. We mutated sufficiency experiment that forced ectopic overexpression of the telomerase RNA template so that now a mutated DNA telomerase is sufficient to overcome cellular senescence in sequence was added to the chromosomes, directed by the culture and maintain the telomeres at a steady length as cells mutated template. The mutated sequence could not bind the continue to divide. Although that experiment showed that with DNA sequence–specific protective proteins. Hence, the DNA at enough telomerase the telomeres can be maintained, the length the ends of the chromosomes was effectively naked. This had at which they are maintained in a cell is the result of a complex the consequence of leaving the telomeric tip uncapped (Fig. 1). interplay among many different factors. Today, many of those We found that the cellular response to this way of uncapping of factors are known, including proteins that aid in binding to the telomeres in cancer cells was a very robust apoptotic response telomere and protect it from increased by telomerase activity. (Fig. 2). For example, in the earliest experiments we did, we In the context of the human body, the fact that the action of transfected the mutant template telomerase RNA constructs into telomerase allows cells to keep multiplying can have different human cancer cell lines and selected for cells that stably kinds of consequences. Although in many normal adult human expressed the mutant-template telomerase RNA (5). These cell types telomerase is often expressed at very low, sometimes cancer cells already had high levels of telomerase, including undetectable, levels, it is clear that telomerase has some high levels of the normal wild-type telomerase RNA. We were protective role in such normal cells. In the context of cancer never able to obtain cells that stably expressed more than just a cells, particularly those that are well on the way to a malignant low fraction of the mutant-template kind of telomerase RNA. state, telomerase has cancer-promoting properties. It is in that Yet that small fraction of the total telomerase that contained latter context that we are interested in intervening in telomerase the mutant-template telomerase RNA was sufficient to elicit a action. robust apoptotic response.

FIGURE 1. Strategy for mutant telomere synthesis in human cancer cells. An example of mutated bases in the telomerase RNA and complementary mutant DNA bases incorpo- rated into telomeric DNA (red A’s and T’s, respectively) through copying of the mutant telomerase template. Lower left, schematic of the uncapping of telomeres. Ovals and dia- monds, sequence-specific DNA binding proteins that protect telomeric DNA and cap it; red, telomerase; blue Xs, mutated telomerase RNA template and correspondingly mutated telomeric DNA sequence. Reproduced from the Proceedings of the National Academy of Sciences, U.S.A., 2001;98:7982 – 7 by copyright permission of the National Academy of Sciences, U.S.A. (5).

FIGURE 2. Effects of low-level expression of mutant-template telomerase RNA in MCF-7 breast cancer cells. Apoptosis was quantified in clonal lines stably expressing low levels of a mutant-template telomerase RNA construct, a control wild-type RNA construct, or parental MCF-7 cells. Reproduced from the Proceedings of the National Academy of Sciences, U.S.A., 2001;98:7982 – 7 by copyright permission of the National Academy of Sciences, U.S.A. (5).

Hence, all one needed to do was spike the endogenous markedly decreased. In contrast, the controls, which received telomerase in the cancer cells with a little mutant-template either a wild-type-template telomerase RNA construct or just telomerase RNA that assembled into the telomerase to make the empty lentiviral vector, grew normally. We did these mutant repeats. The population of cells stably expressing the experiments in a manner that made the test of the effects as various different mutant-template RNAs at low levels could stringent as possible because we did not include a drug be cultured, although they grew very slowly. Interestingly, selection step after infecting the cells with the lentiviral vectors. despite being continually serially passaged for months, we Not all cells—only about 90% to 95% of the cells—were never observed any reversion or any fast-growing resistant infected. Various lines of evidence showed that the cells that subpopulations growing out from 80 to 100 different clonal grew out were those that either did not receive the lentivector lines that were followed. We interpret these findings to mean or failed to overexpress the mutant-template telomerase RNA. that as the cells grew, with some probability they would die For example, we could reinfect those outgrowing cells and by apoptosis. Such cell death is, in effect, a one-way street— recapitulate the same growth kinetics compared with the once they make the decision to enter apoptosis, the cells controls for at least three cycles of reinfections. Thus, again, cannot recover to generate any further daughter cells. Similar no evidence was found for any resistant subpopulation of cells findings were also made in the setting of a simple xenograft that evade the effect of mutant-template telomerase RNA model: When we xenografted human cancer cells expressing expression. the mutant-template telomerase RNA into a mouse model, we An intriguing aspect of these results was the rapidity of saw greatly decreased tumor growth and some regression of the cell growth response, which we could now examine and the tumors, accompanied by high rates of apoptosis in the analyze. The response to mutant-template telomerase RNA is tumors (5). highly specific for cells that have high levels of telomerase. To better understand the nature of this apoptotic response, This response does not require the telomeres to shorten. We we turned to a system in which we could perform more short- used a particular cell line, the LOX human melanoma cell line, term analyses. The problem was that previously, the only stable which happens to have particularly long telomeres, about 40 kb. lines that grew out were those that had expressed extremely low DNA Southern blot experiments, done to examine the length of levels of mutant-template telomerase RNA. Therefore, Shang the telomeric DNA, showed that there was no bulk shortening Li developed a short-term expression system for mutant- of the telomeres. We also showed that a kind of DNA damage template telomerase RNA (6). We expressed the telomerase response was elicited, as might be predicted because the RNA under a promoter that allowed telomerase to be expressed telomeres were expected to become uncapped. For example, and active, even in cell lines that normally do not express any up-regulation of p21 expression was seen, consistent with a telomerase. These experiments demonstrated that the telomer- DNA damage response. To determine whether the telomeres ase RNA was being expressed in a way that supported its had become uncapped, we looked for the appearance of DNA assembly into functional telomerase. We chose as examples for damage foci at telomeres (Fig. 3). For these immunostaining study two mutant-template telomerase RNAs, which were experiments, we used proteins that are known to accumulate in mutated in such a way that they dictated the synthesis of DNA DNA damage foci at sites of DNA damage, such as the protein that would be unable to bind the sequence-specific telomere- hRif1. Shang Li and Lifeng Xu showed that when we protective proteins. Within a few days, the cell growth rate was expressed the mutant-template telomerase RNA, even as early

as 3 or 4 days after infection, DNA damage foci appeared at the of cancer cells—which is typically a couple of orders of telomeres, as visualized by immunostaining of the telomere- magnitude higher in cancer cells than in the differentiated specific protein TRF2. The mutant-template telomerase RNA normal cells around it—usually does not lead to the cancer cells caused uncapping of the telomeres. The slowing of cellular having long telomeres. Their telomeres are mostly maintained proliferation did not require the protein p53, which is ordinarily at a rather short length; the 40 kb telomeres in LOX melanoma involved in the responses of cells to DNA damage. One line of cells are the exception rather than the rule. Thus, there exists a evidence for the lack of p53 protein requirement came from the puzzle that the abundant telomerase activity is apparently not use of a p53-null human colon cancer cell line kindly provided there to make long telomeres. We therefore investigated what by Dr. Burt Vogelstein. When compared with the parent cell happens when you knock down the high levels of telomerase in line, in which the p53 locus is left intact, the responses between human cancer cells. Through this work, we learned, somewhat the lines were similar. The p53 response pathway is commonly to our surprise, that we might also be able to incur a more rapid abrogated in human cancers. Hence, such a p53-independent effect on cancer cell growth than expected and even change apoptotic response is desirable if one is interested in their cancerous properties in ways that were hitherto unsus- intervening in telomerase action as an anticancer approach. pected (7). Finally, we observed the same kinds of rapid growth inhibitory Inhibition of telomerase activity leads to a very simple effects in the context of a more in vivo –like setting for tumor expectation: Failure to counteract telomere shortening, the cells. In collaboration with Jerry Cunha’s lab at University of result of incomplete DNA replication, would cause the California, San Francisco, human bladder tumor cells were telomeres to get shorter and shorter and eventually they would xenografted under the renal capsule in a mouse. In control become too short to sustain capping, and some signaling cells, the wild-type telomerase RNA was introduced, and big process would direct the cells to cease multiplying or even robust tumors formed. The tumors that expressed the mutant- undergo apoptosis. Indeed, that expected result is exactly what template telomerase RNA were smaller and much less is seen when the telomerase activity in cells is inhibited by, for vascularized, and the tumors weighed less (6). example, expressing an excess of a catalytically dead version of This work comprises one approach by which one might the protein TERT, which, although it can still assemble into the exploit the increased expression of telomerase, which is so telomerase ribonucleoprotein complex, forms a complex that is common in cancer cells by forcing them to make toxic not catalytically active. This inactive complex swamps out telomeres. Encouragingly, this is effective even with very low the endogenous telomerase. A delay ensues, during which levels of expression of the mutant-template RNAs. We are very telomeres shorten, before the cells finally cease to grow. interested in understanding the signaling pathways leading to Similarly, using inhibitors of telomerase activity, which prevent the apoptosis, which is under active investigation right now. telomeric DNA synthesis but do not affect the level of telomerase ribonucleoprotein, elicits the same expected time Unexpected Short-term Effects of Knocking course of events (Table 1). Down Telomerase Activity in Cancer Cells We did something somewhat different: We knocked down Thus far, everything discussed has related to telomerase the telomerase RNA levels so that now, not only was there less acting in its familiar mode: making telomeric DNA longer. telomerase enzymatic activity but also the cell was depleted of Curiously, however, the high level of telomerase in the majority telomerase ribonucleoprotein itself. The component of the

FIGURE 3. DNA damage foci accumulate at a subset of telomeres in cells expressing a mutant-template telomerase RNA. Immunos- taining was done for telomere-protective protein TRF2 (green) and for the DNA damage response protein hRif1 (red). Note that not all telomeres (TRF2 spots) overlap with DNA damage foci, although each of the large DNA damage focal spots overlaps with one or more telomeres. Reproduced from The Journal of Cell Biology, 2004;167:819 – 830 by copyright permission of The Rockefeller University Press (10).

telomerase ribonucleoprotein we targeted for knockdown by RNA interference (short interfering RNA or siRNA) was the telomerase RNA. The siRNA was directed against either the wild-type template sequence or the conserved pseudoknot structure of human telomerase RNA. We could achieve up to about 90% knockdown either by using short hairpin siRNAs expressed in the cells from a lentiviral vector expression cassette or by treating the cancer cells with the short synthetic siRNA oligonucleotides with or without modified backbones (Fig. 4). In all cases, we obtained a surprising result: rapid inhibition of the growth of human telomerase-positive cancer cells. The unexpectedly rapid response to telomerase knockdown prompted us to carry out many controls to rule out possibilities such as targets other than the telomerase RNA being responsible for the effects on cell growth. Satisfied that the effects indeed resulted specifically from knockdown of the FIGURE 4. Knockdown of endogenous WT-hTER by a short hairpin intended telomerase RNA, we analyzed the effects in multiple RNA (siRNA) directed specifically against the human WT telomerase RNA cell lines. In contrast to the effects of mutant-template template. Northern blot analysis of RNA extracted from a human cancer telomerase RNA expression on telomerase RNA knockdown, cell line, MCF-7. The human telomerase RNA, a non-mRNA, can be efficiently knocked down by expression of a short hairpin RNA from no telomere shortening or telomere uncapping, or DNA damage a lentiviral construct. GFP lane, Northern blot analysis of RNA from cells response, accompanied the cell growth rate decrease. Once expressing GFP alone from the control lentiviral vector [adapted from again, the effects did not require p53 (7). Li et al. (7)].

Off-Label Telomerase metastasis in two in vivo models. The telomerase RNA of The rapid response of telomerase-positive cancer cells to an mouse melanoma cells (B16 cells) was targeted using abrupt depletion of their telomerase despite the lack of telomere ribozymes, and the human telomerase RNA in human uncapping prompted the hypothesis that these effects were melanoma cells was targeted using the siRNA approach unrelated to telomere maintenance or telomere function. described previously (8). Each ribozyme was delivered Supporting this model, Shang Li, in collaboration with Chris systemically after B16 cells had been introduced into mice by Haqq and his group at University of California, San Francisco, tail-vein injection. After 30 days, the tumor burden in the lungs, showed that global gene expression profile changed within 4 representing metastatic tumors in the lungs, was quantified. The days after the telomerase RNA was knocked down. The effects ribozyme treatment decreased the metastatic burden. were different from the gene expression effects elicited by The rapid inhibition of cell growth seen on knockdown of mutant-template telomerase RNA expression (7). This new telomerase indicated that cancer cells are particularly suscep- gene expression profile defined an interesting signature for tible to its depletion. This implies that they have become telomerase knockdown, marked by diminished expression of adapted to their high levels of telomerase. As Bernard 73 genes. These included some intriguing genes, such as cyclin Weinstein (9) has proposed for other signaling pathways in G2 or CDC27—genes that have been implicated in cell cycle tumors, cancer cells might be ‘‘addicted’’ to high levels of progression. Integrin aV, which has been implicated in telomerase, such that their physiology becomes habituated to it promoting tumor metastasis, was also suppressed. that when telomerase is withdrawn, a ‘‘cold turkey’’ response is Clinically, metastasis is arguably the most important factor deleterious to the cell. Clearly, our challenge is to understand in cancer. That telomerase knockdown produced down- how such a mechanism might work. regulation of a specific subset of genes that included those An important conclusion of this work is that telomerase implicated in tumor progression properties, such as metastasis, participates in cell responses in ways that do not seem to was intriguing. In collaboration with Mohammed Kashani- involve the telomere itself. Hence, our attempts to intervene in Sabet and his group at University of California, San Francisco, telomerase action have led us unexpectedly down a new avenue we examined the effect of telomerase RNA knockdown on in which we find telomerase doing things that we never

Table 1. Growth Inhibitory Effects of Knocking Down Telomerase in Cancer Cells

Old New

Need complete knockdown? f90% bulk knockdown is enough Delayed effect: bulk telomeres decline to critical short length? Effect is immediate*—as fast as can be measured Delay dependent on initial telomere length in the cancer cells? Independent of starting telomere length Telomere uncapping has to occur? No uncapping seen

NOTE: Comparison of human cancer cell growth inhibitory effects predicted and observed when telomerase enzymatic activity was only inhibited (Old) versus those observed in cells in which total telomerase RNA levels were knocked down (New). *Rapid down-regulation of genes promoting cell cycle progression and metastasis.

expected to see. We are hopeful that these avenues will lead us 5. Kim MM, Rivera MA, Botchkina IL, Shalaby R, Thor AD, Blackburn EH. A low threshold level of expression of mutant-template telomerase RNA to a greater understanding of the biology that is being played inhibits human tumor cell proliferation. Proc Natl Acad Sci U S A 2001;98: out in the progression to a more malignant state of cancer cells, 7982 – 7. particularly metastasis. 6. Li S, Rosenberg JE, Donjacour AA, et al. Rapid inhibition of cancer cell growth induced by lentiviral delivery and expression of mutant-template telomerase RNA and anti-telomerase short-interfering RNA. Cancer Res 2004; References 64:4833 – 40. 1. Greider CW, Blackburn EH. Identification of a specific telomere terminal 7. Li S, Crothers J, Haqq CM, Blackburn EH. Cellular and gene expression transferase activity in Tetrahymena extracts. Cell 1985;43:405 – 13. responses involved in the rapid growth inhibition of human cancer cells by RNA 2. Lin J, Ly H, Hussain A, et al. A universal telomerase RNA core structure interference-mediated depletion of telomerase RNA. J Biol Chem 2005;280: includes structured motifs required for binding the telomerase reverse 23709 – 17. transcriptase protein. Proc Natl Acad Sci U S A 2004;101:14713 – 8. 8. Nosrati M, Li S, Bagheri S, et al. Antitumor activity of systemically 3. Yu GL, Bradley JD, Attardi LD, Blackburn EH. In vivo alteration of telomere delivered ribozymes targeting murine telomerase RNA. Clin Cancer Res 2004; sequences and senescence caused by mutated Tetrahymena telomerase RNAs. 10:4983 – 90. Nature 1990;344:126 – 32. 9. Weinstein IB. Disorders in cell circuitry during multistage carcinogenesis: the 4. Bodnar AG, Ouellette M, Frolkis M, et al. Extension of life span by role of homeostasis. Carcinogenesis 2000;21:857 – 64. introduction of telomerase into normal human cells. Science 1998;279: 10. Xu L, Blackburn EH. Human Rifl protein binds aberrant telomeres and aligns 349 – 52. along anaphase midzone microtubules. J Cell Biol 2004;167:819 – 30.

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Elizabeth H. Blackburn

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