RET Is a Heat Shock Protein 90 (HSP90) Client Protein and Is ...

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RET Is a Heat Shock Protein 90 (HSP90) Client Protein and Is Knocked Down upon HSP90 Pharmacological Block Luigi Alfano,* Teresa Guida,* Livia Provitera, Giancarlo Vecchio, Marc Billaud, Massimo Santoro, and Francesca Carlomagno Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche (L.A., T.G., L.P., G.V., M.S., F.C.), Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universita` Federico II, 80131 Naples, Italy; and Laboratoire de Genetique (M.B.), Centre National de la Recherche Scientifique, 69008 Lyon, France

Context: 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. Design: 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 MEN2associated 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 Hsc70interacting protein. In turn, C-terminus of Hsc70-interacting protein polyubiquitinated RET, promoting its proteasomal degradation. 17-AAG blocked RET downstream effectors and RETdependent 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. Conclusion: RET and MEN2-associated RET mutants rely on HSP90 for protein stability, and HSP90 blockade by 17-AAG promotes RET degradation. (J Clin Endocrinol Metab 95: 3552–3557, 2010)

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earrangements of RET are found in approximately 20% of papillary thyroid carcinoma (PTC) (1). Germline missense mutations of RET cause multiple endocrine neoplasia type 2 (MEN2) syndromes. Somatic mutations of RET are found in approximately half of the sporadic medullary thyroid carcinoma (MTC) cases and correlate with an aggressive disease (2). Through different mechanisms, these mutations induce constitutive kinase activation and RET oncogenic conversion (1).

A previous report showed that RET/PTC1 (the H4RET fusion product) was degraded upon treatment with the geldanamycin (GA) derivative 17-allyl-amino-17-demethoxygeldanamycin (17-AAG) (3). Moreover, 17-AAG was able to block RET C634W phosphorylation in MTCderived cells without affecting total RET protein levels (4). GA and 17-AAG are benzoquinoid ansamycin antibiotics acting as specific inhibitors of heat shock protein (HSP) 90. HSP family members assist correct folding of newly

ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2010 by The Endocrine Society doi: 10.1210/jc.2009-2315 Received November 2, 2009. Accepted April 7, 2010. First Published Online May 5, 2010 * L.A. and T.G. contributed equally to the work.

Abbreviations: 17-AAG, 17-Allyl-amino-17-demethoxygeldanamycin; BrdU, bromodeoxyuridine; CHIP, C-terminus of Hsc70-interacting protein; GA, geldanamycin; HA, hemagglutinin; HSP, heat shock protein; MEN2, multiple endocrine neoplasia type 2; MTC, medullary thyroid carcinoma; PTC, papillary thyroid carcinoma; TPR, tetratricopeptide repeat; wt, wild-type.

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J Clin Endocrinol Metab, July 2010, 95(7):3552–3557


translated polypeptides, functioning as molecular chaperones. In particular, HSP90 mediates folding of several oncogenic client protein kinases, such as BRAF, HER2, and AKT (5). Drugs belonging to the GA family bind to the ATP pocket of HSP90 and block interaction with ATP, inducing the chaperone to adopt an inactive conformation. This results in the stabilization of the client protein interaction with HSP70, its polyubiquitination by C-terminus of Hsc70-interacting protein (CHIP), and degradation by the 26S proteasome (6). Currently, 17-AAG is being used alone or in combination with chemotherapeutic agents in clinical trials for different types of cancer such as multiple myeloma, breast and pancreatic carcinoma, melanoma, leukemia, lymphoma, and thyroid cancer (7).

Materials and Methods Compounds 17-AAG and radicicol were purchased by Calbiochem (Merck KGaA, Darmstad, Germany).

Cell culture and plasmids Plasmids and cell lines used in the study are described in the Supplemental Data (published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org).

Immunoblotting Protein lysates were prepared according to standard procedures. Methods are included in the Supplemental Data.

Cellular assays Luciferase activity, bromodeoxyuridine (BrdU) incorporation, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assays were performed according to standard procedures. Methods are included in the Supplemental Data. The ANOVA post hoc Tukey-Kramer multiple comparison test was used to assess statistical significance of luciferase assay. InStat3 (GraphPad Software, La Jolla, CA) was used.

Results 17-AAG-induced degradation of RET and RET/MEN2 mutant proteins We performed a dose-response treatment of RAT1 cells stably transfected with wild-type (wt) RET and several RET/MEN2 mutants carrying mutations in the extracellular (RET C634R) or tyrosine kinase (V804M and M918T) domain. RET C634R is the most frequent extracellular substitution, whereas RET M918T is the MEN2B mutation and the most frequent mutation found in sporadic MTC (2). RET V804 is mutated in sporadic and familial MTC cases and corresponds to RET gatekeeper site-mediating resistance to some kinase inhibitors (8).


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RET protein abundance was measured by Western blotting. Anti-RET antibody recognized two molecular species of 150 and 170 kDa, which corresponded to the high mannose immature form of the receptor and the plasma membrane-associated mature form of RET, respectively (1). After 8 h of treatment, 20 nM 17-AAG was able to induce partial degradation of all RET proteins, whereas 50 nM reduced protein levels to 50% or less (Fig. 1A). Both 150 and 170 kDa isoforms decreased upon 17-AAG treatment, although the 170 kDa one, being less abundant, almost disappeared. RET and RET/MEN2 extracellular (RET C634R) or intracellular (RET E768D, L790F, Y791F, V804M, A883F, F891A, and M918T) mutant protein degradation was examined in a time-course experiment. As shown in Fig. 1B, upon treatment with 0.5 ␮M 17-AAG, both wt RET and RET/MEN2 mutants were degraded with a similar kinetics with a half-life of about 4 h, reaching 70 – 80% of degradation after 8-h treatment. Radicicol, a HSP90 inhibitor structurally unrelated to 17AAG and GA, provoked RET degradation as well (Supplemental Fig. 1). Fibroblasts transformed by RET C634R display constitutive RET-mediated phosphorylation of SHC that in turn activates the RAS/MAPK pathway (9). We performed Western blotting experiments using total and phosphospecific antibodies to analyze protein extracts from RAT1 cells expressing RET C634R treated with increasing concentrations of 17-AAG. As expected, decrease of RET protein was accompanied to a proportional decrease of RET phosphorylation in these cells (Fig. 1C). Phosphorylation of SHC and ERK1/2 was decreased as well, without any effect on total protein levels (Fig. 1C). We also tested whether 17-AAG could obstruct RET-mediated activation of transcription from specific gene promoters. Cells were transfected with an AP1-responsive and the MYC gene promoter, fused to the luciferase reporter, along with RET C634R or the empty vector as a control. As shown in Supplemental Fig. 2, the compound reduced RET C634R activity to less than 50% at 50 nM and completely abolished promoter activation at 100 nM, in agreement with reduction of RET protein levels and downstream signaling. No effect was observed in control cells transfected with the empty vector. Finally, we evaluated tyrosine phosphorylation and protein abundance of a few other RET intracellular mutants (e.g. RET E768D, L790F, and A883F) expressed in RAT1 cells and treated with increasing doses of 17AAG. Also for these mutants, inhibition of RET phosphorylation was proportional to protein degradation, similar to what was observed for RET C634R (Supplemental Fig. 3).

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RET Protein Stability Is Dependent on HSP90


FIG. 1. RET wt and RET/MEN2 mutant protein degradation by 17-AAG. A and B, RAT1 cells stably transfected with the indicated expressing vectors were treated with 17-AAG for the indicated time points with the indicated concentrations. Equivalent amounts of protein lysates were subjected to Western blotting with ␣RET or ␣Tubulin for normalization. The 150- and 170-kDa RET forms are indicated. Average densitometric analysis of three independent experiments ⫾ SD are reported. Values represent the percentage of signal compared with nontreated cells. C, RAT1 cells stably transfected with RET C634R were treated for 4 h with 17-AAG. Protein lysates were subjected to Western blotting with the indicated antibodies.


Molecular pathway mediating RET degradation upon 17-AAG treatment We investigated whether reduction of RET protein levels upon 17-AAG treatment was mediated by polyubiquitination and degradation of the receptor by 26S proteasome. To this aim, we transiently transfected a hemagglutinin (HA)-tagged ubiquitin vector in RAT1 cells expressing RETC634R and treated cells with 17-AAG and/or the proteasome inhibitor MG132. Protein lysates were immunoprecipitated with anti-RET antibody and then blotted with anti-RET or anti-HA to check for con-


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jugation of the HA-ubiquitin molecules to RET. MG132 prevented 17-AAG-mediated degradation of RET, indicating that such degradation was mediated by the proteasome. Indeed, cells treated with MG132 and 17-AAG displayed an accrual of high molecular weight species of RET C634R (⬎200 kDa), corresponding to polyubiquitinated forms saved from proteasome degradation. These data suggested that RET represent a HSP90 client protein (Fig. 2A). To test this hypothesis, we performed coimmunoprecipitation of RET with HSP90 and HSP70 proteins in

FIG. 2. RET/C634R interaction with the HSP90/HSP70 chaperone complex and CHIP E3-ligase. A, RAT1 cells expressing RET C634R were transiently transfected with the PCDNA-ubiquitin-HA expressing vector and after 2 d treated for 4 h with vehicle or 40 ␮M MG132 and/or 0.5 ␮M 17-AAG. Equivalent amounts of protein lysates were subjected to immunoprecipitation with ␣RET antibody, and immunocomplexes were subjected to Western blotting with the indicated antibodies. Total lysates were immunoblotted with the ␣Tubulin antibody for normalization. The position of protein species that migrate above the 200-kDa marker is indicated (⬎200 kDa). B, RAT1 cells stably transfected with RET C634R were treated for 4 h with vehicle or 0.5 ␮M 17-AAG. Equivalent amounts of RET proteins were subjected to immunoprecipitation with preimmune (IgG) or ␣RET antibody followed by Western blotting with the indicated antibodies. C and D, HEK293 cells were transiently transfected with RET C634R in association with CHIP-myc, CHIP-⌬U-myc, or CHIP-TPR-myc or the empty vector. Equivalent amounts of protein lysates were subjected to (C) direct Western blotting with the indicated antibodies or (D) immunoprecipitation with preimmune (IgG) or ␣RET antibody followed by Western blotting with the indicated antibodies. Densitometric analyses of RET protein (expressed as percentage of signal intensity compared with the empty vector transfected sample) (C) or of CHIP protein present in the anti-RET immunoprecipitates (expressed as percentage of total CHIP protein) (D) are included. E, HEK293 cells were transiently transfected with PCDNA-ubiquitin-HA and RET C634R, together with CHIP-myc, CHIP-⌬U-myc, or CHIP-TPR-myc. Equivalent amounts of protein lysates were subjected to immunoprecipitation with ␣RET antibody and then subjected to Western blotting with ␣HA and ␣RET antibodies, as indicated. The position of protein species that migrate above the 200-kDa marker is indicated (⬎200 kDa).

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RAT1 cells expressing RET C634R. As shown in Fig. 2B, RET C634R was bound to endogenous HSP90 at the steady state, such an interaction being reduced upon addition of 17-AAG. Conversely, the stoichiometry of RET interaction with HSP70 was increased by the compound (Fig. 2B). The HSP70-interacting E3-ligase protein CHIP binds to HSP70 via an amino-terminal tetratricopeptide repeat (TPR) domain, whereas its enzymatic activity depends on the COOH-terminal U box motif responsible for interaction with ubiquitin (10). Deletion of TPR (CHIP-TPR) or the U box (CHIP-⌬U) domains impairs the CHIP ability of binding the client protein-HSP90/70 complex or the ubiquitin-bound E2 protein, respectively (10). We expressed RET C634R alone or in combination with myc-tagged CHIP, CHIP-TPR, or CHIP-⌬U. As shown in Fig. 2C, overexpression of CHIP, but not its mutants, reduced RET C634R levels. CHIP and CHIP-⌬U interacted with RET/C634R, whereas CHIP-TPR lost its ability to bind to the receptor, as expected (Fig. 2D). Finally, we coexpressed HA-tagged ubiquitin, RET, and either wt CHIP or its mutants and analyzed RET conjugation to ubiquitin by immunoprecipitation with anti-RET followed by Western blotting with anti-HA. As shown in Fig. 2E, polyubiquitinated RET species accumulated upon overexpression of CHIP but not of its inactive mutants; these last mutants (particularly CHIP-TPR), conversely, displayed a dominant negative effect on endogenous RET polyubiquitination. Effect of 17-AAG treatment on human MTC cell lines We used two different MTC-derived cell lines, TT and MZCRC1, which naturally express oncogenic RET C634W and RET M918T mutants, respectively (11). As shown in Supplemental Fig. 4, RET protein levels decreased upon 17-AAG treatment of MTC cells, although with a lower efficiency compared with the exogenous receptor expressed in rat fibroblasts. As expected also, RET phosphorylation levels and RET-dependent activation of SHC and ERK1/2 proteins were reduced. In addition, after 24- and 48-h treatment with 100 and 500 nM doses, in both cell lines 17-AAG caused a marked hindrance of DNA synthesis, measured as incorporation of BrdU (Supplemental Fig. 5). After 72-h treatment with 17-AAG (100 and 500 nM), there was only a weak induction of programmed cell death as demonstrated by the TUNEL assay and by Western blotting using an anti-poly(ADP-ribose) polymerase (anti-PARP) antibody, which detects fulllength PARP and the large fragment (89 kDa) produced by caspase cleavage (Supplemental Fig. 5).


Discussion Here we show that RET oncoproteins require HSP90 chaperone for stability and activity. Thus, in RAT1 cells, 17-AAG reduced protein half-life of both wt RET and MEN2-associated RET mutants, including those mutants (V804) that are resistant to some tyrosine kinase inhibitors (8). 17-AAG impaired RET C634R-dependent activation of signaling, resulting in hindrance of AP1- and MYCmediated gene transcription. 17-AAG induced 26S proteasome-dependent degradation of RET C634R by blocking HSP90/RET interaction and stabilizing the HSP70/ RET complex. We also demonstrate that RET C634R is polyubiquitinated by the E3 ligase CHIP. Finally, in MTCderived cells, 17-AAG induced RET degradation with a reduction of BrdU incorporation and a weak apoptosis induction. Such a block of proliferation is likely due to a pleiotropic effect of 17-AAG on several HSP90 client proteins, rather than RET alone. HSP90 inhibitors as well as proteasome inhibitors have been shown to cause “proteotoxic stress” to cancer cells, causing an accumulation of unfolded proteins and cell damage (12). Mutational activation of some receptor tyrosine kinases, like epidermal growth factor replication, was shown to be associated to increased dependence on HSP90 function compared with wt protein, probably due to a conformational effect on receptor structure (13). This does not apply to RET. Indeed, RET oncogenic mutants, carrying mutations in either the intracellular or extracellular domains, displayed a degradation kinetics virtually identical to the RET wt protein. This is also true for chimeric RET/PTC oncoproteins; in fact, in murine fibroblasts, RET/ PTC3 protein stability was affected by 17-AAG treatment to the same extent as wt RET (data not shown). HSP90 protein recognizes a common surface in the amino-terminal lobe of client kinases. In particular, the ␣C-␤4 loop surface electrostatics determines the interaction with the HSP90 chaperone complex, and therefore sensitivity to its inhibition (14, 15). A neutral/positive surface charge is characteristic of HSP90 client kinases, whereas HSP90-independent kinases display a negative surface charge (14). Mutations in this region might alter interaction with the chaperone and mediate evasion from the HSP90/70 control, resulting in hyperactivation (16). Residues L790 and Y791 in RET are localized in the ␣C-␤4 loop, which overall displays a neutral/positive surface charge. Nevertheless, MEN2-associated L790F and Y791F mutations did not alter sensitivity to 17-AAG, thereby suggesting that these mutations do not activate RET by increasing its stability; in fact, an accumulation of these mutant proteins was not noted with respect to wt RET.


Efficacy of RET targeting as a therapeutic tool for MTC has been proven in several preclinical settings (17). In addition, phase II/III clinical trials with multikinase inhibitors that include RET among their targets are in progress in patients affected by MTC (18 –20). Our results demonstrate that MEN2 RET mutants are HSP90 client proteins and require HSP90 for protein stability and function. Although the absence of in vivo data renders this study still preliminary as far as the use in patients is concerned, results suggest that 17-AAG may have the important property of causing degradation of RET in MTC cells.

Acknowledgments We gratefully acknowledge L. Neckers for the CHIP plasmids, S. Giordano for the HA-ubiquitin, S. J. Gutkind for MYC-Luc vector, and R. Gagel for MZCRC1 cells. We thank Ciotola Presentation for the art work. Address all correspondence and requests for reprints to: Massimo Santoro, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universita’ degli Studi Federico II, via Pansini 5, 80131 Napoli, Italy. E-mail: [email protected]. This study was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), the Naples Oncogenomic Center, Fondazione San Paolo, and by grants from Italian Ministero della Salute and Ministero dell’Universita` e della Ricerca. T.G. was supported by an AIRC fellowship. Disclosure Summary: The authors have nothing to disclose.

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