Genome stability necessitates a mechanism to protect the termini of linear chromosomes from inappropriate degradation or recombination. In many species this protection depends on 'capping' proteins that bind telomeric DNA. The budding yeast Cdc13p binds single-stranded telomeric sequences, prevents lethal degradation of chromosome ends and regulates telomere extension by telomerase. Two Cdc13-interacting proteins, Stn1p and Ten1p, are also required for viability and telomere length regulation. It has been proposed that Cdc13p DNA binding directs a Cdc13p-Stn1p-Ten1p complex to telomeres to mediate end protection. However, the functional significance of these protein interactions, and their respective roles in maintaining telomere integrity, remain undefined. Here, we show that co-overexpressing TEN1 with a truncated form of STN1 efficiently bypasses the essential role of CDC13. We further show that this truncated Stn1p binds directly to Pol12p, a polymerase alpha-primase regulatory subunit, and that Pol12 activity is required for CDC13 bypass. Thus, Stn1p and Ten1p control a Cdc13p-independent telomere capping mechanism that is coupled to the conventional DNA replication machinery.

The function of telomeres is twofold: to facilitate complete chromosome replication and to protect chromosome ends against fusions and illegitimate recombination. In the budding yeast Saccharomyces cerevisiae, interactions among Cdc13p, Stn1p, and Ten1p are thought to be critical for promoting these processes. We have identified distinct Stn1p domains that mediate interaction with either Ten1p or Cdc13p, allowing analysis of whether the interaction between Cdc13p and Stn1p is indeed essential for telomere capping or length regulation. Consistent with the model that the Stn1p essential function is to promote telomere end protection through Cdc13p, stn1 alleles that truncate the C-terminal 123 residues fail to interact with Cdc13p and do not support viability when expressed at endogenous levels. Remarkably, more extensive deletions that remove an additional 185 C-terminal residues from Stn1p now allow cell growth at endogenous expression levels. The viability of these stn1-t alleles improves with increasing expression level, indicating that increased stn1-t dosage can compensate for the loss of Cdc13p-Stn1p interaction. However, telomere length is misregulated at all expression levels. Thus, an amino-terminal region of Stn1p is sufficient for its essential function, while a central region of Stn1p either negatively regulates the STN1 essential function or destabilizes the mutant Stn1 protein.

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Epidermal growth factor receptor (EGFR) is a proven therapeutic target to treat a subset of NSCLC harboring activating mutations within the EGFR gene. However, many NSCLC patients are not sensitive to EGFR kinase inhibitors, suggesting that other factors may play a role in determining survival of NSCLC cells. Signal transducers and activators of transcription 3 (Stat3) functions as a transcription factor to mediate cell survival and differentiation and the dysregulation of Stat3 has been described in numbers of cancers. Our previous report showed that suppression of Stat3 activity by a pharmacological approach, WP1066, sensitized NSCLC cells. Along with Stat3 inhibition, we also found that WP1066 treatment up-regulated the expression of Fas, a death receptor, regulating numbers of physiological and pathological process of cell death. Although Fas-mediated apoptosis pathway has been studied mostly in the immune system, the identification of Fas mutations in non-lymphoid malignancies, such as NSCLC, suggests its implication in the pathogenesis of non-lymphoid malignancies as well. Results from this study showed that levels of Fas were increased in cells with down-regulated Stat3 by small interfering RNA (siRNA), suggesting that Stat3 may play an inhibitory role to regulate the expression of Fas. To examine whether the Fas pathway is responsible for the WP1066-mediated cytotoxicity, Fas apoptosis signaling was induced by anti-Fas antibody and Fas ligand. Our results revealed that NSCLC cells showed different degrees of sensitivity to anti-Fas antibody but resistance to Fas ligand. However, augmentation of Fas expression by WP1066 remarkably increased the sensitivity of NSCLC cells to both anti-Fas antibody and Fas ligand. Taken together, our findings suggest that regulation of the Fas system may be a potential strategy to treat NSCLC and the combined use of a Fas inducer, such as WP1066, may further strengthen its efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B211.