Epithelial-mesenchymal transition sensitizes breast cancer cells to cell death via the fungus-derived sesterterpenoid ophiobolin A

Abstract

The epithelial-mesenchymal transition (EMT) imparts properties of cancer stem-like cells, including resistance to frequently used chemotherapy, necessitating the identification of molecules that induce cell death specifically in stem-like cells with EMT properties. Herein, we demonstrate that breast cancer cells enriched for EMT features are more sensitive to cytotoxicity induced by ophiobolin A (OpA), a sesterterpenoid natural product. Using a model of experimentally induced EMT in human mammary epithelial (HMLE) cells, we show that EMT is both necessary and sufficient for OpA sensitivity. Moreover, prolonged, sub-cytotoxic exposure to OpA is sufficient to reduce migration, sphere formation, and resistance to doxorubicin. OpA is well-tolerated in mice and treatment with OpA alone reduces tumor burden. These data identify a driver of EMT-driven cytotoxicity with significant potential for use either in combination with standard chemotherapy or for tumors enriched for EMT features.in survival 14-17 . To improve TNBC patient outcomes, novel and specific approaches targeted at CSCs are needed.One proposed mechanism driving the emergence of CSC-like cells is the epithelialmesenchymal transition, EMT 18,19 . EMT is a trans-differentiation process characterized by spindle-like morphology, loss of apical-basal polarity, increased motility, and a tolerance to anoikis. These phenotypic shifts are driven by gene expression changes mediated by transcription factors Snail (SNAI1), Twist (TWIST1), and ZEB1, effects of which include upregulation of vimentin and N-cadherin, and downregulation of epithelial markers E-cadherin and miR-200c 20-26 . Cells that have undergone an EMT typically acquire CSC properties including decreased sensitivity to conventional chemotherapies used to treat TNBC. This chemoresistance is driven by drug efflux pumps, enhanced DNA repair capacity, mesenchymal-like properties, and epigenetic changes 16,27-32 . There are currently no approved therapies that specifically target CSCs. A leading pre-clinical compound is salinomycin, reported to decrease the subpopulation of CSCs, tumor initiating capability, and chemoresistance, with negligible side effects 33 . Other naturally occurring compounds such as curcumin, and quercetin have been reported to reduce the effects of EMT by inhibiting key proteins associated with migration (Snail, MMP-2/9), anoikis tolerance (Bcl-2), cell-to-cell adhesion (N-cadherin), and signaling cascades (JAK/STAT, ERK) 34-37 .Ophiobolin A (OpA) is a natural product produced from fungi in the genera Aspergillus, Bipolaris, Cephalosporium, Cochliobolus, is a secondary metabolite that has long been studied for its phytotoxic effects in a variety of plants and has begun to be evaluated as a cytotoxic compound 38 . Recently published cell Western blotting and antibodiesCells were lysed in the presence of 100 microliters radio-immunoprecipitation (RIPA) buffer containing protease inhibitors (Alfa Aesar, Stoughton, MA, USA) on ice. Protein was quantified using the Bradford Assay (BioRad,...

Pharmacophore-Directed Retrosynthesis Applied to Ophiobolin A: Simplified Bicyclic Derivatives Displaying Anticancer Activity

“…Several structure–activity relationship (SAR) studies of the anticancer effects of ophiobolins, derived mainly from derivatization of OpA or congeners, led us to apply our recently described pharmacophore-directed retrosynthesis (PDR) strategy , to elaborate on SAR information with respect to effects on stem-like cancer cells. In particular, the anticancer effects of various ophiobolins ,, and synthetic derivatives, in conjunction with our own work, point to the importance of the A/B-rings as an initial minimal pharmacophore consisting of a 1,4-keto unsaturated aldehyde moiety, the C5/C6-relative stereochemistry, the C3-hydroxyl, and syn-fused cyclooctene (Figure , highlighted in yellow). The potential reactivity of the 1,4-ketoaldehyde to primary amines including cellular protein lysines through a potential Paal–Knorr pyrrole reaction has been reported. , Isolation of a phosphatidyl­ethanolamine adduct from HEK293T cells treated with OpA supports this notion .…”

“…Moreover, the ability of certain ophiobolins to induce paraptosis ,, or autophagy makes these natural products valuable leads toward apoptosis-resistant cancer cells. Our interest in the ophiobolins stems from their complex structures, unique bioactivities and unknown cellular targets, and was further enhanced by our finding that OpA, but not 3-deoxy OpA, specifically induces cell death in stem-like cells with epithelial-mesenchymal transition (EMT) properties …”

Bridging the gap between natural product synthesis and drug discovery

“…Widespread interest in the ophiobolins has led to some preliminary SAR data, 49,53 which combined with our group's recent work on OpA and derivatives 54 suggested the importance of retaining the integrity of the A and B ring to maintain the desired biological effects since both 3-deoxy and 6-epi-OpA 49 both exhibited decreased potency. Furthermore, OpA contains a 1,4-ketoaldehyde moiety appended to the A ring which has the potential to react with primary amines (lysine) through a Paal-Knorr pyrrole reaction.…”

“…This led us to propose the following PDR route wherein the simplest form of the pharmacophore, monocyclic 1,4-keto aldehyde 75 is synthesized first in Stage I, followed by simplified bicyclic and tricyclic derivatives (74 and 73 respectively) which all contain the proposed pharmacophore and can provide valuable SAR information on both activity and selectivity of OpA toward CSCs. We recently described Stage I and II toward OpA 54 and Stage III will be the completion of OpA and closely related congeners synthesized in route to OpA followed by SAR gap filling as needed utilizing DTS in Stage IV.…”