title: Identification of an HSP90 modulated multi-step process for ERBB2 degradation in breast cancer cells.
abstract: The receptor tyrosine kinase ERBB2 interacts with HSP90 and is overexpressed in aggressive breast cancers. Therapeutic HSP90 inhibitors, i.e. Geldanamycin (GA), target ERBB2 to degradation. We have previously shown that HSP90 is responsible for the missorting of recycling ERBB2 to degradation compartments. In this study, we used biochemical, immunofluorescence and electron microscopy techniques to demonstrate that in SKBR3 human breast cancer cells, GA strongly induces polyubiquitination and internalization of the full-length p185-ERBB2, and promotes its cleavage, with the formation of a p116-ERBB2 form in EEA1-positive endosomes (EE). p116-ERBB2 corresponds to a non-ubiquitinated, signaling-impaired, membrane-bound fragment, which is readily sorted to lysosomes and degraded. To define the sequence of events leading to p116-ERBB2 degradation, we first blocked the EE maturation/trafficking to late endosomes/lysosomes with wortmannin, and found an increase in GA-dependent formation of p116-ERBB2; we then inhibited the proteasome activity with MG-132 or lactacystin, and observed an efficient block of p185-ERBB2 cleavage, and its accumulation in EE, suggesting that p185-ERBB2 polyubiquitination is necessary for proteasome-dependent p116-ERBB2 generation occurring in EE. As polyubiquitination has also been implicated in autophagy-mediated degradation of ERBB2 under different experimental conditions, we exploited this possibility and demonstrate that GA strongly inhibits early autophagy, and reduces the levels of the autophagy markers atg5-12 and LC3-II, irrespective of GA-induced ERBB2 polyubiquitination, ruling out a GA-dependent autophagic degradation of ERBB2. In conclusion, we propose that HSP90 inhibition fosters ERBB2 polyubiquitination and proteasome-dependent generation of a non-ubiquitinated and inactive p116-ERBB2 form in EE, which is trafficked from altered EE to lysosomes.
authors.affiliation: Dipartimento di Terapie Oncologiche Integrate, IRCCS AOU San Martino - IST, Genova, Italy.
authors.forename: Patrizio
authors.initials: P
authors.lastname: Castagnola
authors.affiliation: Dipartimento di Medicina Sperimentale, Anatomia Umana, Università di Genova, Italy.
authors.forename: Grazia
authors.initials: G
authors.lastname: Bellese
authors.affiliation: Dipartimento di Medicina Sperimentale, Anatomia Umana, Università di Genova, Italy.
authors.forename: Filippo
authors.initials: F
authors.lastname: Birocchi
authors.affiliation: Dipartimento di Medicina Sperimentale, Anatomia Umana, Università di Genova, Italy.
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Gagliani
authors.affiliation: Dipartimento di Medicina Sperimentale, Anatomia Umana, Università di Genova, Italy.|Centro Imaging Sperimentale, IRCCS Istituto Scientifico San Raffaele, Milano, Italy.
authors.forename: Carlo
authors.initials: C
authors.lastname: Tacchetti
authors.affiliation: Dipartimento di Medicina Sperimentale, Anatomia Umana, Università di Genova, Italy.
authors.forename: Katia
authors.initials: K
authors.lastname: Cortese
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001343:Autophagy / Q000187:drug effects; D000071183:Autophagy-Related Proteins / Q000378:metabolism; D016227:Benzoquinones / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D045744:Cell Line, Tumor; D011992:Endosomes / Q000187:drug effects / Q000201:enzymology; D005260:Female; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology*; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D059748:Proteolysis; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D000903:Antibiotics, Antineoplastic; D000071183:Autophagy-Related Proteins; D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D008869:Microtubule-Associated Proteins; D046988:Proteasome Endopeptidase Complex; D061988:Proteasome Inhibitors; D018719:Erb-b2 Receptor Tyrosine Kinases; C477012:MAP1LC3A protein, human; C508053:ERBB2 protein, human; C001277:geldanamycin
keywords: ERBB2; cleavage; geldanamycin (GA); polyubiquitin; proteasome