Caspase cleavage of HER-2/neu Releases a BH3-like Cell Death Effector

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Breast cancer cells acquire many genetic alterations in apoptotic signaling pathways rendering them resistant to apoptosis. Human Epidermal growth factor Receptor-2 (HER-2/ErbB2/neu) is amplified or overexpressed in approximately 30% of breast and ovarian tumors and correlates with poor prognosis. Although HER-2 is an orphan receptor, it forms potent signaling heterodimers with other family members (HER-1/EGFR, HER-3 or HER-4) and plays a central role in both the establishment and progression of breast tumors. Using a novel expression cloning strategy, we identified HER-2 as a substrate of multiple caspases. HER-2 is cleaved by caspases at four aspartic acid residues (Asp1016, Asp1019, Asp1087, and Asp1125) in its cytoplasmic tail in vitro and in breast cancer cells during the induction of apoptosis. HER-2 is initially cleaved by caspases at Asp1016/Asp1019, releasing a 47-kDa product from the cell membrane. This product is subsequently processed after Asp1125 into a predicted 25 kDa product and an unstable 22-kDa fragment that is rapidly degraded by the proteasome. We demonstrate that expression of either a caspase cleavage-resistant or a truncated HER-2 conferred greater protection against apoptosis than wild-type HER-2 in breast cancer cells stably expressing these proteins, suggesting a pro-apoptotic function for one or more of the HER-2 cleavage products. Indeed, transfection of cDNAs encoding the 25 or 47-kDa HER-2 caspase cleavage products into breast cancer cells was sufficient to induce apoptosis in a caspase-dependent manner. Biochemical fractionation and confocal analyses documented the presence of the HER-2 cleavage products at the mitochondria after ectopic expression and in dying cells which endogenously express HER-2. Once at the mitochondria, these fragments induce the release cytochrome c in a Bcl-XL-inhibitable manner. Further, we identify a novel BH3-like domain in a sequence shared by the 47 and 25 kDa HER-2 products which is required for cell death. Recombinant peptides containing the HER-2 BH3 domain, but not a mutant (2XE) domain, were sufficient to release cytochrome c from isolated mitochondria. Collectively, our results indicate that caspases activate a previously unrecognized pro-apoptotic function of HER-2 by releasing a BH3 domain containing product, which translocates to the mitochondria , triggers cytochrome c release, and induces apoptosis.

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  • 07/30/2018
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