Abstract
606 Background: Topoisomerase I (topo-I) degradation by ubiquitin proteasomal pathway (UPP), in response to camptothecins (CPTs) is linked to the CPT response. But the mechanism is not understood. Methods: Improvised GST pull down experiments were performed to isolate topo-I interacting proteins which were analyzed by SDS-PAGE and identified by mass spectrometry. DNA-PKcs mediated phosphorylation of GST-topo-I was analyzed by SDS-PAGE and autoradiography. Immunoblot analysis of anti-BRCA1 precipitates from HCT15 cell lines was performed. GST-topo-I phosphorylated by DNA-PKcs, was incubated with the BRCA1/BARD1 heterodimer in the presence of E1, UbCH5c and ATP in ubiquitination buffer. Four triple negative breast cancer cell lines were selected to compare the degree of topo-I degradation. Colorectal cancer (CRC) tissues were immunostained with 1C1.H5.H7 to determine topo-I-pS10 level and identify a correlation between the topo-I-pS10 level and CPT response. Results: We isolated a topo-I interacting protein complex and determined that Ku70/Ku80/DNA-PKcs complex associates with topo-I, and DNA-PKcs phosphorylates topo-I at Serine10. We showed that cells with higher topo-I-pS10 level rapidly degrade topo-I and are CPT resistant. The cells with non-detectable level of topo-I-pS10 fail to degrade topo-I and are CPT sensitive. Retrospective study with 48 CRC tissues immuno-stained with anti-topo-I-pS10 supported our cell line data. Percent DAB positive nuclei demonstrated statistically significant differential staining between CPT responders and non-responders, and ROC analysis confirmed that nearly all non-responders exhibit >35% positive nuclei, indicating that topo-I-pS10 level determines topo-I degradation and a potential predictive biomarker for CPT response. Conclusions: Colorectal cancers are treated with topo-I inhibitors, but the response rate is low. We have shown that topo-I degradation rate is linked to CPT response. We have determined that high topo-I-pS10 level is indicative of rapid topoI degradation and can serve as a biomarker for CPT resistance.
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