Oncotarget

Research Papers:

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

Patrizio Castagnola, Grazia Bellese, Filippo Birocchi, Maria Cristina Gagliani, Carlo Tacchetti and Katia Cortese _

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Oncotarget. 2016; 7:85411-85429. https://doi.org/10.18632/oncotarget.13392

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Abstract

Patrizio Castagnola2,*, Grazia Bellese1,*, Filippo Birocchi1, Maria Cristina Gagliani1, Carlo Tacchetti1,3, Katia Cortese1

1Dipartimento di Medicina Sperimentale, Anatomia Umana, Università di Genova, Italy

2Dipartimento di Terapie Oncologiche Integrate, IRCCS AOU San Martino – IST, Genova, Italy

3Centro Imaging Sperimentale, IRCCS Istituto Scientifico San Raffaele, Milano, Italy

*These authors have contributed equally to this work

Correspondence to:

Katia Cortese, email: cortesek@unige.it

Carlo Tacchetti, email: tacchetti.carlo@hrs.it

Keywords: ERBB2, geldanamycin (GA), polyubiquitin, proteasome, cleavage

Received: August 04, 2016     Accepted: October 28, 2016     Published: November 16, 2016

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.


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