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.

The HSP90 Family: Structure, Regulation, Function, and Implications in Health and Disease

“…Although the anti-cancer efficacy of GA has been tested in many in vivo and in vitro studies, it has not yet been approved for clinical uses due to its structural instability and hepatotoxicity. However, GA is still commonly used for in vitro studies concerning HSP90 inhibition [ 192 , 193 ]. Similarly, RD usage was limited due to its instability as well [ 194 ].…”

Targeting Heat Shock Proteins in Cancer: A Promising Therapeutic Approach

“…Although it was able to induce potent in vitro and in vivo cytotoxic effects, due to its structural instability and hepatotoxicity, its phase I clinical trial was suspended and it failed to progress further [ 253 ]. Despite the lack of success in the clinic, GM still plays fundamental roles as a potent HSP90 inhibitor for in vitro studies, especially in ERBB2+ breast cancer cells [ 254 , 255 ]. Another important natural inhibitor of HSP90 is radicicol (RD) ( Figure 9 (AII)), which was derived from Monosporium bonorden , which showed strong in vitro antitumor properties via attacking the core ATP-binding pocket of HSP90, but was proven ineffective in vivo due to its structural instability [ 256 ].…”

Inhibition of ezrin causes PKCα-mediated internalization of erbb2/HER2 tyrosine kinase in breast cancer cells

“…The different members of this signaling complex, which include ezrin, NHERF1, PMCA2, HSP90, EGFR or HER3, and HER2, appear to be involved in a series of interdependent interactions that stabilize and amplify HER2 expression and signaling within specific plasma membrane domains. Experience with HSP90 inhibitors has already suggested that disrupting the membrane stabilization of HER2 has clinical utility (12,57). Therefore, targeting other members of this complex singly or in combination might also be efficacious for the treatment of HER2-positive cancers.…”