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 Mol Cancer Res 2005;3(9). September 2005 477 Downloaded from mcr.aacrjournals.org on September 29, 2021. © 2005 American Association for Cancer Research. 478 Blackburn 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.

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