Ink4a/Arf tumor suppressor does not modulate the degenerative conditions or tumor spectrum of the telomerase-deficient mouse.

The Rb/p16(Ink4a) and p53/p19Arf tumor suppressor pathways have been linked to diverse cancer-relevant processes, including those governing the cellular responses to telomere dysfunction. In this study, we sought to provide direct genetic evidence of a role for the Ink4a/Arf tumor suppressor gene, encoding both p16(Ink4a) and p19(Arf), in modulating the cellular and tissue phenotypes associated with telomere dysfunction by using the mTerc Ink4a/Arf mouse model. In contrast to the rescue associated with p53 deficiency, Ink4a/Arf deficiency did not attenuate the degenerative phenotypes elicited by telomere dysfunction in the late-generation mTerc-/- mice. Furthermore, in contrast to accelerated cancer onset and increased epithelial cancers of late-generation mTerc-/- p53 mutant mice, late-generation mTerc-/- Ink4a/Arf mutant mice experienced a delayed tumor onset and maintained the lymphoma and sarcoma spectrum. Consistent with the negligible role of Ink4a/Arf in the telomere checkpoint response in vivo, late-generation mTerc-/- Ink4a/Arf-/- tissues show activated p53, and derivative tumor cell lines sustain frequent loss of p53 function, whereas all early generation mTerc Ink4a/Arf-/- tumor cell lines remain intact for p53. In addition, the late-generation mTerc-/- Ink4a/Arf-/- tumors showed activation of the alternative lengthening of telomere mechanism, underscoring the need for adaptation to the presence of telomere dysfunction in the absence of p16(Ink4a) and p19(Arf). These observations highlight the importance of genetic context in dictating whether telomere dysfunction promotes or suppresses age-related degenerative conditions as well as the rate of initiation and type of spontaneous cancers.

Telomere-based crisis: functional differences between telomerase activation and ALT in tumor progression.

Telomerase activation is a common feature of most advanced human cancers and is postulated to restore genomic stability to a level permissive for cell viability and tumor progression. Here, we used genetically defined transformed mouse embryonic fibroblast (MEF) cultures derived from late generation mTerc(-/-) Ink4a/Arf(-/-) mice to explore more directly how telomere-based crisis relates to the evolution of cancer cell genomes and to tumor biology. An exhaustive serial analysis of cytogenetic profiles over extensive passage in culture revealed that the emergence of chromosomal fusions (including dicentrics) coincided with onset of deletions and complex nonreciprocal translocations (NRTs), whereas mTerc-transduced cultures maintained intact chromosomes and stable genomes. Despite a high degree of telomere dysfunction and genomic instability, transformed late passage mTerc(-/-) Ink4a/Arf(-/-) cultures retained the capacity to form subcutaneous tumors in immunocompromised mice. However, even moderate levels of telomere dysfunction completely abrogated the capacity of these cells to form lung metastases after tail-vein injection, whereas mTerc reconstitution alone conferred robust metastatic activity in these cells. Finally, serial subcutaneous tumor formation using late passage transformed mTerc(-/-) Ink4a/Arf(-/-) cultures revealed clear evidence of telomerase-independent alternative lengthening of telomeres (ALT). Significantly, despite a marked increase in telomere reserve, cells derived from the ALT+ subcutaneous tumors were unable to generate lung metastases, indicating in vivo functional differences in these principal mechanisms of telomere maintenance. Together, these results are consistent with the model that although telomere dysfunction provokes chromosomal aberrations that initiate carcinogenesis, telomerase-mediated telomere maintenance enables such initiated cells to efficiently achieve a fully malignant endpoint, including metastasis.