Abstract 1592: The role of T-oligo and its associated proteins in mediating anticancer responses in melanoma and lung cancer
Authors: Richard E. Mulnix, Ryan Pitman, Neelu Puri
Journal: Cancer Research (2010)

Abstract Telomeres are tandem repeats of the DNA sequence TTAGGG. The 3’ end of each telomere consists of a single stranded overhang that is normally concealed in a loop structure at the chromosome ends. We postulate that exposure of this sequence upon telomere disruption induces anticancer responses such as apoptosis and senescence. An 11-base oligonucleotide homologous to the telomere 3’ overhang sequence, (T-oligo) is thought to mimic exposure of the telomere overhang. T-oligo decreased NSCLC viability in six cell lines by 40-50% and induced a 3 fold increase in apoptosis and 10 fold increase in senescence. T-oligo did not effect cell proliferation or induce apoptosis or senescence in normal bronchial epithelial cells. To investigate proteins that bind to T-oligo which could induce apoptosis and senescence, cancer cells were treated with biotinylated T-oligo or complementary oligo, and T-oligo associated proteins were immunoprecipitated. Further analysis of unique protein bands after gel electrophoresis and mass spectrometry indicated that proteins such as hnRNP C, Purβ, and Msi-1 which are known to interact with the telomere and modulate p53 and pRb were bound to T-oligo. We hypothesize that T-oligo modulates hnRNP C, Purβ, and Msi-1 and induces various anticancer effects. We found in NSCLC cell lines H358 and SW1573 that hnRNP C was upregulated by T-oligo at 18 hrs and Purβ was found to be upregulated at 18 hrs in H358. Similar results were observed in two melanoma cells lines, MU and MM-AN. T-oligo upregulated hnRNP C at 18 hrs and 6 hrs in MM-AN and MU melanoma cells respectively. Additionally, Purβ and Msi-1 were upregulated at 24 and 48 hrs in MM-AN cells treated with T-oligo. To investigate hnRNP Cs involvement in senescence in H358 cells, hnRNP C was downregulated using shRNA in a lentiviral vector. It was found that 7 days after T-oligo treatment, 70% of the cells were positive for senescence associated β-galactosidase (β-gal) compared to 5% positive cells after control shRNA treatment. However, in cells treated with T-oligo after downregulation of hnRNP C only 8% of cells were positive for β-gal indicating that hnRNP C played an important role in mediating senescence. To test the ability of T-oligo as a therapeutic drug, subcutaneous tumors in nude mice were given daily intratumoral injections of 60 nmoles of T-oligo or a complementary oligonucleotide for 6 weeks. SW1573 and H358 tumors treated with T-oligo exhibited a 5.6 and 4.3 fold reduction in tumor size respectively. When tumor sections were further examined for senescence using β-gal, both H358 and SW1573 exhibited strong staining for senescence compared to minimal staining in controls. These results indicate that T-oligo not only reduced tumor size, but also induced senescence in tumor cells. These findings suggest that T-oligo may be a novel molecularly targeted therapy and its associated proteins may be novel cancer targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1592.