Priority Research Papers:
Inflammatory breast cancer tumor emboli express high levels of anti-apoptotic proteins: use of a quantitative high content and high-throughput 3D IBC spheroid assay to identify targeting strategies
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Abstract
Jay Arora1,4,*, Scott J. Sauer2,*, Michael Tarpley5, Peter Vermeulen6, Charlotte Rypens6, Steven Van Laere6, Kevin P. Williams5, Gayathri R. Devi1,2,**, Mark W. Dewhirst1,3,**
1Duke Cancer Institute, Duke University, Durham, NC, USA
2Department of Surgery, Division of Surgical Sciences, Duke University, Durham, NC, USA
3Department of Radiation Oncology and Imaging Program, Duke University, Durham, NC, USA
4Trinity College of Arts and Sciences, Duke University, Durham, NC, USA
5Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC, USA
6Translational Cancer Research Unit, Oncology Center, General Hospital Sint Augustinus, Center for Oncological Research (CORE), University of Antwerp, Antwerp, Wilrijk, Belgium
*These authors have contributed equally to this work
**Both are co-senior and corresponding authors
Correspondence to:
Gayathri R. Devi, email: [email protected]
Mark W. Dewhirst, email: [email protected]
Keywords: NFκB, XIAP, disulfiram, oxidative stress, apoptosis
Received: June 15, 2016 Accepted: January 19, 2017 Published: February 24, 2017
ABSTRACT
Inflammatory breast cancer (IBC) is one of the most lethal breast cancer variants; with existing therapy, 5-yr survival rate is only 35%. Current barriers to successful treatment of IBC include frequent infiltration and the presence of tumor cell clusters, termed tumor emboli, within the breast parenchyma and lymphatics. Prior studies have identified the role of anti-apoptotic signaling, in particular hyperactivation of NFκB and its target genes, in IBC pathobiology and therapeutic resistance. The objectives of this study were to: (1) determine if IBC tumor emboli express anti-apoptotic proteins and (2) develop a high content, multiparametric assay to assess the morphology of the IBC 3D spheroids and to optimize a high throughput format to screen for compounds that can inhibit the formation of the IBC tumor clusters/embolic structures. Immunohistochemical analysis of IBC patient tumor samples with documented tumor emboli revealed high NFκB (p65) staining along with expression of XIAP, a potent anti-apoptotic protein known to interact with NFκB signaling in enhancing survival of malignant cells. Subsequently, the high content assay developed allowed for simultaneous imaging and morphometric analysis, including count and viability of spheroids derived from SUM149, rSUM149 and SUM190 cells and its application to evaluate XIAP and NFκB inhibitory agents. We demonstrate the efficacy of the off-patent drug disulfiram when chelated with copper, which we had previously reported to inhibit NFκB signaling, was highly effective in disrupting both IBC spheroids and emboli grown in vitro. Taken together, these results identify a high-throughput approach to target tumor spheroid formation for drug discovery. Finally, disulfiram is a safe and approved drug for management of alcohol abuse, warranting its evaluation for repurposing in IBC therapy.
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