RET is a heat shock protein 90 (HSP90) client protein and is knocked down upon HSP90 pharmacological block.
BACKGROUND
Mutations of the RET receptor tyrosine kinase are associated to multiple endocrine neoplasia type 2 (MEN2) and sporadic medullary thyroid carcinoma (MTC). The heat shock protein (HSP) 90 chaperone is required for folding and stability of several kinases. HSP90 is specifically inhibited by 17-allyl-amino-17-demethoxygeldanamycin (17-AAG).


OBJECTIVE
Our aim was to investigate whether RET protein half-life depends on HSP90 and to dissect the molecular pathway responsible for the degradation of RET upon HSP90 inhibition by 17-AAG.


METHODS
17-AAG effects were studied in RAT1 fibroblasts exogenously expressing MEN2-associated RET mutants and human MTC-derived cell lines.


RESULTS
17-AAG induced a 26S proteasome-dependent degradation of wild-type RET and MEN2-associated RET mutants. The compound hampered HSP90/RET interaction and stabilized RET binding to HSP70, leading to the recruitment of the HSP70-associated E3 ligase C-terminus of Hsc70-interacting protein. In turn, C-terminus of Hsc70-interacting protein polyubiquitinated RET, promoting its proteasomal degradation. 17-AAG blocked RET downstream effectors and RET-dependent transcriptional activation of gene promoters. In human MTC cells carrying oncogenic RET mutants, HSP90 inhibition induced receptor degradation and signaling hindrance leading to cell cycle arrest.


CONCLUSIONS
RET and MEN2-associated RET mutants rely on HSP90 for protein stability, and HSP90 blockade by 17-AAG promotes RET degradation.
Alfano
Luigi
L
Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universitá Federico II, 80131 Naples, Italy.
Guida
Teresa
T
Provitera
Livia
L
Vecchio
Giancarlo
G
Billaud
Marc
M
Santoro
Massimo
M
Carlomagno
Francesca
F
D016227:Benzoquinones / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D018942:Macrolides / Q000494:pharmacology; D010766:Phosphorylation / Q000235:genetics; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection
D016227:Benzoquinones; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D018942:Macrolides; C112765:tanespimycin; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; C035359:monorden