Identification of an HSP90 modulated multi-step process for ERBB2 degradation in breast cancer cells

The receptor tyrosine kinase ERBB2 interacts with HSP90 and is overexpressedin aggressive breast cancers.Therapeutic HSP90 inhibitors, i.e. Geldanamycin (GA),target ERBB2 to degradation. We have previously shown that HSP90 is responsiblefor the missorting of recycling ERBB2 to degradation compartments. In this study,we used biochemical, immunofluorescence and electron microscopy techniquesto demonstrate that in SKBR3 human breast cancer cells, GA strongly inducespolyubiquitination and internalization of the full-length p185-ERBB2, and promotesits cleavage, with the formation of a p116-ERBB2 form in EEA1-positive endosomes(EE). p116-ERBB2 corresponds to a non-ubiquitinated, signaling-impaired, membraneboundfragment, which is readily sorted to lysosomes and degraded. To define thesequence of events leading to p116-ERBB2 degradation, we first blocked the EEmaturation/trafficking to late endosomes/lysosomes with wortmannin, and found anincrease in GA-dependent formation of p116-ERBB2; we then inhibited the proteasomeactivity with MG-132 or lactacystin, and observed an efficient block of p185-ERBB2cleavage, and its accumulation in EE, suggesting that p185-ERBB2 polyubiquitinationis necessary for proteasome-dependent p116-ERBB2 generation occurring in EE. Aspolyubiquitination has also been implicated in autophagy-mediated degradationof ERBB2 under different experimental conditions, we exploited this possibilityand demonstrate that GA strongly inhibits early autophagy, and reduces the levelsof the autophagy markers atg5-12 and LC3-II, irrespective of GA-induced ERBB2polyubiquitination, ruling out a GA-dependent autophagic degradation of ERBB2. Inconclusion, we propose that HSP90 inhibition fosters ERBB2 polyubiquitination andproteasome-dependent generation of a non-ubiquitinated and inactive p116-ERBB2form in EE, which is trafficked from altered EE to lysosomes.