Validating the mode of action of ergosterol peroxide as a selective breast cancer inhibitor

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PROJECT SUMMARY Triple Negative Breast Cancer (TNBC) is an aggressive and lethal breast cancer subtype more commonly diagnosed on younger (<40y) women of color (Hispanics and African American), who are more likely to have disease recurrence and who have an overall shorter survival. Furthermore, additional limitations (i.e. drug toxicity, and therapy resistance) persist in the clinic for TNBC patients. Thus, a critical need exists to discover more desirable targeted therapeutic modalities that selectively target TNBC tumor cells while leaving normal cells unaffected, enhancing a complete response and reducing TNBC-associated mortality rates. Accordingly, our goal is to reduce BC mortality by identifying unique vulnerabilities of TNBC that can be exploited through the development of selective therapeutic agents. We recently identified the natural product ergosterol peroxide a steroidal compound found in fungi, as a potent antiproliferative agent against TNBC cell models with little to no effect on noncancerous cells. We demonstrated that EP is more potent towards TNBC than HER2-positive models. EP induces ROS and apoptosis in TNBC cells and reduces tumor volume in TNBC models in vivo. However, EP’s mode of action (MOA) remains to be defined. Extended periods of oxidative stress in the mitochondria lead to accumulation of damaged proteins, which are normally removed by ubiquitination via the VCP/ANKZF1 pathway. We have synthesized EP in gram scale and show that EP accumulates in the cytosol and affects VCP and ANKZF1 interactions, raising the possibility that the VCP/ANKZF1 complex is targeted during its assembly prior to arriving to the mitochondria. Thus, taking advantage of TNBC’s dependency on sterol uptake, removal of misfolded proteins and altered redox homeostasis, we propose that EP serves as a VCP/ANKZF1 complex protein-protein inhibitor (PPI). We hypothesize that EP targets the VCP/ANKZF1 complex, impairing the ability of cancer cells to clear damaged proteins and causing mitochondria dysfunction to induce TNBC cell death. To test the hypothesis we will identify the mechanism by which EP targets the VCP/ANKZF1 complex in TNBC models using biophysical assays and synthesized EP-probes (Aim 1). The results from this Aim will validate that targeting VCP/ANKZF1 complex induces increases misfolded and aggregated proteins, which lead to cancer cell death. We will determine the efficacy of EP using syngeneic models injected with doxycycline inducible ANKZF1 silenced cells, and in TNBC PDXs. EP safety and bio- availability will also be determined and studied by MTD and PK/PD (Aim 2). The result of this aim will provide data needed for the next step towards the translational development of EP. Student participation in this hypothesis-driven research will improve the pipeline for URM students in biomedical research and lead to improved BC treatment.