Multiple strategies for HB-EGF inhibition: interference with receptor binding, ectodomain shedding and matricrine...

Nontoxic analogues of Corynebacterium diphtheriae exotoxin can inhibit the binding of heparin-binding EGF-like growth factor (HB-EGF) to tumor cell receptors, while targeting its heparin-binding site or preventing ectodomain shedding can further disrupt its signaling through paracrine and matricrine mechanisms. In this study, we used fluorescent HB-EGF derivatives to evaluate the effects of recombinant diphtheria toxin analogues, CRM197 and the B subunit (SbB), on the binding of HB-EGF to EGFRs via flow cytometry. Using an MTT assay, we evaluated the impact of HB-EGF-HSPG interactions and different protease inhibitor treatments on the ability of HB-EGF to promote cell proliferation. The IC50 values of single compounds and their mixtures were used to calculate a combination index (CI) for assessing the different means interactions. CRM197 and SbB effectively prevent exogenous sHB-EGF from interacting with cells. Moreover, the addition of protease inhibitors to cell cultures resulted in cytostatic and cytotoxic effects. Interestingly, heparin did not enhance the proliferative activity of treated cells, likely because it binds to the heparin-binding domain of HB-EGF, sequestering it from interaction with membrane-associated heparan sulfate proteoglycans and preventing matricrine activation. These findings suggest that combining HB-EGF-targeting strategies could significantly reduce mitogenic activation and induce cancer cell death in tumors with high EGFR and proHB-EGF expression.