Abstract LB-077: Topoisomerase IIα mediates TCF-dependent epithelial-mesenchymal transition in colon cancer

The Wnt signaling pathway, which controls TCF/Lef/β-catenin (TCF) transcription, is well accepted as a dominate player in CRC tumor initiation and progression. More recently, this pathway has been implicated as a major driving force inducing epithelial-mesenchymal transition (EMT), acquiring a drug resistant tumor initiating cell (TIC) phenotype that may promote metastasis. As a result, this pathway has been seriously pursued for novel drug development that has yielded no real translational success due to the lack of “druggable” targets. Thus, the major challenge moving forward is to identify a druggable target within the Wnt/TCF pathway that may result in effective clinical translation. Using a chemical biology approach targeting EMT we have identified topoisomerase IIα (TopoIIα) as a master regulator EMT by participating as a required component of TCF-transcription in CRC. Specifically, we show that inhibiting TopoIIα using small molecule ATP-competitive inhibitors as well as shRNA results in the blockade of TCF-transcription using the TOP/FOP flash reporter system. Inhibition of TopoIIα-dependent TCF-transcription results in significant reversion of EMT characterized by Western blot analysis in a panel of CRC cell lines. Time course studies reveal that inhibiting TopoIIα results in downregulation of TCF-transcription followed by the induction of apoptosis using 3D multicellular tumor spheroid models. We show that TopoIIα participates in protein-protein complexation with β-catenin using co-immunoprecipitation studies, which is not prevented by small molecule inhibitors. However, chromatin immunoprecipitation (ChIP) studies reveal that ATP-competitive TopoIIα inhibitors can significantly block TCF-transcription by preventing TCF-complex-DNA binding. In addition, inhibiting TopoIIα-dependent TCF-transcription correlates with the inhibition of TIC function characterized by loss of colony formation using the clonogenic assay. Likewise, inhibiting TopoIIα-dependent TCF-transcription inhibits the invasive potential in a panel of CRC tumor cell lines. Interestingly, the clinically used TopoIIα drug, etoposide, had no effect on TCF-transcription, EMT, or invasion. In conclusion, we hypothesize that TopoIIα participates as a component of the TCF-complex via the C-terminus and is required for binding chromatin DNA and initiating gene transcription. TopoIIα participation in TCF-transcription may convey drug resistance due to altered poison binding sites. However, our data indicates that TopoIIα-dependent TCF-transcription requires ATP. Hence, central to our hypothesis is that N-terminal ATP sites are conserved providing an Achilles heel to drug resistance. Therefore, N-terminal ATP binding sites provide optimal druggable target sites that may be utilized for translational drug development preventing TCF-transcription, EMT and potentially metastasis in CRC patients. Citation Format: Daniel V. LaBarbera, Qiong Zhou, Adedoyin D. Abraham, Linfeng Li, Wells A. Messersmith. Topoisomerase IIα mediates TCF-dependent epithelial-mesenchymal transition in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-077. doi:10.1158/1538-7445.AM2015-LB-077