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Disruption of telomere maintenance pathways leads to accelerated entry into cellular senescence, a stable proliferative arrest that promotes aging-associated disorders in some mammals. The budding yeast CST complex, comprising Cdc13, Stn1, and Ctc1, is critical for telomere replication, length regulation, and end protection. Although mammalian homologues of CST have been identified recently, their role and function for telomere maintenance in normal somatic human cells are still incompletely understood. Here, we characterize the function of human Stn1 in cultured human fibroblasts and demonstrate its critical role in telomere replication, length regulation, and function. In the absence of high telomerase activity, shRNA-mediated knockdown of hStn1 resulted in aberrant and fragile telomeric structures, stochastic telomere attrition, increased telomere erosion rates, telomere dysfunction, and consequently accelerated entry into cellular senescence. Oxidative stress augmented the defects caused by Stn1 knockdown leading to almost immediate cessation of cell proliferation. In contrast, overexpression of hTERT suppressed some of the defects caused by hStn1 knockdown suggesting that telomerase can partially compensate for hStn1 loss. Our findings reveal a critical role for human Stn1 in telomere length maintenance and function, supporting the model that efficient replication of telomeric repeats is critical for long-term viability of normal somatic mammalian cells.

In normal human somatic cells, telomere dysfunction causes cellular senescence, a stable proliferative arrest with tumour suppressing properties. Whether telomere dysfunction-induced senescence (TDIS) suppresses cancer growth in humans, however, is unknown. Here, we demonstrate that multiple and distinct human cancer precursor lesions, but not corresponding malignant cancers, are comprised of cells that display hallmarks of TDIS. Furthermore, we demonstrate that oncogenic signalling, frequently associated with initiating cancer growth in humans, dramatically affected telomere structure and function by causing telomeric replication stress, rapid and stochastic telomere attrition, and consequently telomere dysfunction in cells that lack hTERT activity. DNA replication stress induced by drugs also resulted in telomere dysfunction and cellular senescence in normal human cells, demonstrating that telomeric repeats indeed are hypersensitive to DNA replication stress. Our data reveal that TDIS, accelerated by oncogene-induced DNA replication stress, is a biological response of cells in human cancer precursor lesions and provide strong evidence that TDIS is a critical tumour suppressing mechanism in humans.

Telomerase is a reverse transcriptase associated with cellular immortality through telomere maintenance. This enzyme is activated in 90% of human cancers, and inhibitors of telomerase are currently in clinical trials to counteract tumor growth. Many aspects of telomerase biology have been investigated for therapy, particularly inhibition of the enzyme, but little was done regarding its subcellular shuttling. We have recently shown that mutations in the nuclear export signal of hTERT, the catalytic component of telomerase, led to a mutant ((NES-)hTERT) that failed to immortalize cells despite nuclear localization and catalytic activity. Expression of (NES-)hTERT in primary fibroblast resulted in telomere-based premature senescence and mitochondrial dysfunction. Here we show that expression of (NES-)hTERT in LNCaP, SQ20B and HeLa cells rapidly and significantly decreases their proliferation rate and ability to form colonies in soft agar while not interfering with endogenous telomerase activity. The cancer cells showed increased DNA damage at telomeric and extra-telomeric sites, and became sensitive to ionizing radiation and hydrogen peroxide exposures. Our data show that expression of (NES-)hTERT efficiently counteracts cancer cell growth in vitro in at least two different ways, and suggest manipulation with the NES of hTERT or its subcellular shuttling as a new strategy for cancer treatment.