Abstract 1592: The role of T-oligo and its associated proteins in mediating anticancer responses in melanoma and lung cancer

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.