Telomeres in the Initiation and Progression of Epithelial Cancer and in the Maintenance of Stem Cell Homeostasi

Kwok-Kin Wong*, Richard Maser*#, Andrew Aguirre*#, Nabeel Bardeesy*#, Eric Martin*#, Cameron Brennan*#, Lynda Chin*^ and Ronald A. DePinho*# 

* Department of Medical Oncology, Dana Farber Cancer Institute; ^ Department of Dermatology,
# Department of Medicine and Genetics, Harvard Medical School, Boston MA USA 

Telomeres are specialized nucleoprotein complexes at the ends of linear chromosomes consisting of long arrays of double stranded TTAGGG repeats, a G-rich 3’ single strand overhang, and associated telomeric repeat binding proteins 1, 2. The work of Muller and McClintock in the 1930’s led to the concept that telomeres function to ‘cap’ chromosomal termini and prevent end-to-end recombination, thereby maintaining chromosomal integrity.  Subsequent work on telomeres by many others has substantiated this model and advanced our understanding of the pathogenesis of complex human diseases including cancer. A large body of evidence from human tissues and model organisms has established that telomere maintenance and the cellular response to telomere dysfunction play crucial roles in the maintenance of chromosomal stability and longterm homeostasis of stem cells and organ function.  Notably, impaired telomere function has been linked to the evolution of aberrant cancer genomes and certain inherited and acquired chronic degenerative disorders.  With respect to cancer, the study of telomeres has provided mechanistic insights into how advancing age fuels the development of epithelial cancers as well as how chronic inflammation and degeneration may engender increased cancer risk in affected organs.  These advances in the basic understanding of telomere dynamics are now being translated into clinically relevant applications that may ultimately impact on the diagnosis and management of a broad spectrum of cancers. 

Contrasting Role of Telomeres in Cancer Genesis and Progression.  Robust telomerase activity is observed in greater than 80% of all human cancers 3, a profile consistent with its role in promoting malignant progression. There is however another side to the telomerase-cancer connection that has emerged from mouse models and increasing correlative data in staged human epithelial cancer.  These data have indicated that, during the early stages of neoplastic growth, the lack of telomerase and associated telomere attrition provides a mechanism that enables would-be cancer cells to achieve a threshold of cancer-promoting changes required to traverse the benign to malignant transition.  Indeed, telomeres of human cancer cells are often significantly shorter than their normal tissue counterparts, suggesting that telomere attrition has occurred at some time during the life history of these cancers presumably during early phases when telomerase activity is low. The subsequent reactivation of telomerase appears to restore telomere function, albeit at a shorter set length. Thus, although reactivation of telomerase is critical to the emergence of immortal human cells, a preceding and transient period of telomere shortening and dysfunction appears to contribute to carcinogenesis by leading to the formation of chromosomal rearrangements through breakage-fusion-bridge (BFB) cycles.  Serial BFB in turn begets rapid and wholesale genetic changes in the population with rare cells incurring a threshold number of relevant pro-carcinogenic changes needed to initiate the transformation process. While, at first glance, the cancer-promoting effects of telomere-based crisis seem contradictory to the hackneyed role of telomerase activation in cancer progression, this mechanism is less paradoxical if one considers that many early stage cancers deactivate pathways essential for telomere checkpoint responses, thus increasing the survival and proliferation of cells with increasing genomic instability 4, 5.   

Our hypothesis of ‘episodic instability’ derives support from genetic studies in the mouse showing that telomere-based crisis, coupled with loss of the p53-dependent DNA damage response can act cooperatively to effect malignant transformation. In humans, the accumulation of oncogenic lesions during normal aging or accelerated accumulation of DNA damage (e.g., environmental carcinogen exposure or oxidative damage) may deactivate the telomere checkpoint response, accelerate telomere attrition, and drive the affected premalignant cells into telomere-based crisis.  The rare transformed cells that emerges from crisis appears to do so by reactivating telomerase or the ALT mechanism.  Thus, telomeric shortening can been viewed both as a barrier to cancer development in the presence of intact checkpoint response and as a facilitator for numerous genetic changes necessary for the emergence of nascent cancer cells in the absence of the checkpoint response pathways.  The capacity of cells to utilize telomere-based crisis as a cancer-promoting mutational mechanism is highly dependent upon cell type (with epithelial cells able to tolerate a higher level of telomere dysfunction relative to hematopoeitic cells), the status of p53, and the degree of telomere dysfunction.  

Aging and Cancer.  The study of telomeres has also provided some insights into the link between advancing age and increased cancer risk. In humans, there is a dramatic escalation in cancer risk between the ages 40 to 80, resulting primarily from a marked increase in epithelial malignancies such as carcinomas of the breast, lung, colon, and prostate. A conventional view is that the cancer-prone phenotype of older humans reflects the combined effects of cumulative mutational load, decreased DNA repair capabilities, increased epigenetic gene silencing, and altered hormonal and stromal milieus. While these factors are almost certain to contribute to increasing cancer incidence in aged humans, it is less evident why such processes would spur the preferential development of epithelial cancers. Moreover, these mechanisms do not readily explain one of the cardinal features of adult epithelial carcinomas -- namely, a radically altered genome typified by marked aneuploidy and complex non-reciprocal chromosomal translocations.  

The study of telomere dynamics in normal and neoplastic cells of the mouse has provided a potential explanation for the observed tumor spectrum and associated cytogenetic profiles in aged humans. In Terc p53 compound mutant mice, the presence of telomere dysfunction results in a dramatic shift in the tumor spectrum towards epithelial cancers including those of the lung, colon and skin.  Moreover, in contrast to the somewhat normal cytogenetic profiles of cancers arising in mice with intact telomeres, the cancers generated in the Terc p53 compound mutant mice had cytogenetic profiles with striking resemblance to human epithelial cancer genomes 6. Thus, telomere-based crisis seem to provide the means to generate many additional mutations required reach the early stages of malignant transformation. The subsequent reactivation of telomerase in transformed clones would serve to stabilize the genome to a level compatible with cell viability, allowing these initiated neoplasms to mature further 7.  It is unclear whether additional somatic mutations, beyond telomerase activation, would be needed to produce a fully malignant phenotype that includes invasive and metastatic potential.  Thus, a transient period of explosive chromosomal instability prior to telomerase reactivation appears to be required for the stochastic acquisition of the relatively high number of mutations thought to be required for adult epithelial carcinogenesis.

 

The "episodic instability" model of epithelial carcinogenesis fits well with current knowledge regarding the timing of telomerase activation and evolving genomic changes during various stages of human carcinoma development, particularly those of the breast, esophagus and colon. Comparative genome hybridization (CGH) has demonstrated that dysplastic human breast, esophageal and colon lesions sustain widespread gains and losses of regions of chromosomes early in their development, often well before these tissues exhibit carcinoma in situ or  invasive growth 8-10. The ploidy changes detected by CGH appear to correlate tightly with the presence of complex chromosomal rearrangements, and these markers of genomic instability are evident in the stages of advanced dysplasia of these tissues (e.g. DCIS, Barrett’s esophagus, etc).  As these cancers progress through invasive and metastatic stages, genomic instability continues, apparently at a moderate rate, but further mutations would be predicted to derive from non-telomere based mechanisms. Correspondingly, the measurement of telomerase activity in adenomatous polyps and colorectal cancers has established that telomerase activity is low or undetectable in small and intermediate sized polyps, reflecting less intact telomere function. In contrast, telomerase increases markedly in large adenomas and colorectal carcinomas, reflecting stabilization of telomere function 11. Therefore, it appears that there is widespread and severe chromosomal instability early on during human tumorigenesis at a time when telomerase activity is low. Additional support for this episodic instability model derives from the documentation of anaphase bridging (a non-specific but reasonable correlate of telomere-based crisis) in evolving human colorectal cancers 12 and in genomically unstable pancreatic cancers 13,14. This suggests that the DSB-induced conditions (including but not limited to telomere dysfunction) coupled with mutations that allow survival in the face of a DSB, could provide an amplification/deletion mechanism across the genome.  Biological forces would in turn lead to the selection of clones with the amplifications and deletions that target cancer-relevant loci.  Studies in the telomerase mutant mouse have begun to provide mechanistic insight into how BFB leads to cancer-relevant changes. In particular, telomerase p53 compound mutant mice with telomere dysfunction have increased end-to-end fusions, and the ensuing BFB process is associated with chromosomal regional gains and losses that appear linked to non-reciprocal translocations 15, 16.

 

Telomere dynamics, inflammatory diseases, and cancer.  The telomere dysfunction-induced genomic instability model also suggests some unanticipated opportunities for the therapies of other human diseases.  For example, this model provides a potential explanation for the high cancer incidence associated with diseases characterized by chronic cell destruction and renewal.  One of the most notable examples of this tight link is the high incidence of hepatocellular carcinoma in late stage cirrhotic livers.  Cirrhosis is the phenotypic endpoint of prolonged cycles of hepatocyte destruction and regeneration and cirrhotic livers show a documented reduction in telomere length over time.  Mouse models involving the telomerase null mouse have shown that critical reductions in telomere length and function can accelerate the development of cirrhosis and hepatocellular carcinoma in chronic liver injury experiments 12, 17.  Another example of a telomere-based pathogenetic relationship between chronic tissue turnover, telomere-based crisis and increased cancer risk is ulcerative colitis, a condition characterized by rapid cell turnover and oxidative injury to the intestines, and a high incidence of intestinal dysplasia or cancer 13. In addition to the progressive telomere attrition resulting from the cell turnover, accelerated telomere attrition might occur via increased oxidative stress and from the altered inflammatory microenvironment milieu. Together, such observations offer the intriguing possibility that early somatic reconstitution of telomerase could attenuate telomere attrition and paradoxically reduce the occurrence of cancers in these high-turnover disease states, a theory that will require additional preclinical studies. In addition, serial analyses of telomere length from these tissues may provide prognostic information regarding the rising risk of cancer development.  Thus, progress in our understanding of telomere biology has mechanistically connected diverse fields in medicine involving chronic inflammatory diseases, degenerative diseases, geriatrics and oncology. 

Telomere dynamics and stem cell homeostasis. There is also accumulating evidence that telomere dynamics might play a crucial role in stem cell homeostasis of various organs. Coincident with the onset of chromosomal instability, the mTerc-/- mice with telomere dysfunction exhibit impaired organ renewal signs of premature aging, shortened lifespan and reduced capacity to tolerate acute and chronic stress 18-20. In addition, it was recently shown that the telomerase and Atm deficient compound mutant mice with critically shortened telomeres, cause dramatically increased genomic instability, Atm-independent p53 activation, and accelerated aging with depleted stem/progenitor cell reserves in various organ compartments examined 21. Surprisingly, although these compound mutant mice have accelerated aging phenotypes, they have dramatically decreased cancer incidence – relating presumably to the inability to activate telomerase and intense level of genomic instability that is incompatible with cell viability. The Atm-independent p53 activation and apoptosis in stem/progenitor cell compartments, coupled with accelerated aging and resistance to lymphoma seen in the mTerc-/-Atm-/- mice with telomere dysfunction, are reminiscent of the premature aging and tumor resistance of mice harboring an activated p53 germ line allele 22. Thus, one mechanistic explanation for the premature aging and stem cell depletion in these mTerc-/-Atm-/- mice might be that chronic telomere dysfunction activates p53 constitutively in the various organs.  These results are the first experimental evidence that telomeres are essential to maintain stem cell reserves and provided a rational explanation for the progressive decline of organ homeostasis as a function of age.  The impact of telomere dysfunction on stem cell dynamics across many organ systems raises the possibility that the advent of stem cell transplantation could open new therapeutic options in the management of stem cell depletion related organ failures

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