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Brief Report—Endocrine Research

The Effects of Four Different Tyrosine Kinase Inhibitors on Medullary and Papillary Thyroid Cancer Cells

Hans H. G. Verbeek,* Maria M. Alves,* Jan-Willem B. de Groot, Jan Osinga, John T. M. Plukker, Thera P. Links, and Robert M. W. Hofstra

Departments of Genetics (H.H.G.V., M.M.A., J.O., R.M.W.H.), Medical Oncology (J.-W.B.d.G.), Surgical Oncology (J.T.M.P.), and Internal Medicine—Endocrinology Section (H.H.G.V., T.P.L.), University Medical Center Groningen, University of Groningen, 9700 RB Groningen, The Netherlands

Context: Medullary and papillary thyroid carcinoma (MTC and PTC) are two types of thyroid cancer that can originate from activating mutations or rearrangements in the RET gene. Therapeutic options are limited in recurrent disease, but because RET is a tyrosine kinase (TK) receptor involved in cellular growth and proliferation, treatment with a TK inhibitor might be promising. Several TK inhibitors have been tested in clinical trials, but it is unknown which inhibitor is most effective and whether there is any specificity for particular RET mutations.

Objective: We aimed to compare the effect of four TK inhibitors (axitinib, sunitinib, vandetanib, and XL184) on cell proliferation, RET expression and autophosphorylation, and ERK activation in cell lines expressing a MEN2A (MTC-TT), a MEN2B (MZ-CRC-1) mutation, and a RET/PTC (TPC-1) rearrangement.

Design: The three cell lines were cultured and treated with the four TK inhibitors. Effects on cell proliferation and RET and ERK expression and activation were determined.

Results: XL184 and vandetanib most effectively inhibited cell proliferation, RET autophosphory- lation in combination with a reduction of RET expression, and ERK phosphorylation in MTC-TT and MZ-CRC-1, respectively. TPC-1 cells showed a decrease in RET autophosphorylation after treatment with XL184, but no effect was observed on ERK activation.

Conclusion: There is indeed specificity for different RET mutations, with XL184 being the most potent inhibitor in MEN2A and PTC and vandetanib the most effective in MEN2B in vitro.NoTK inhibitor was superior for all the cell lines tested, indicating that mutation-specific therapies could be beneficial in treating MTC and PTC. (J Clin Endocrinol Metab 96: E991–E995, 2011)

edullary thyroid carcinoma (MTC) and papillary and originates from the follicular epithelial cells of the M thyroid carcinoma (PTC) can be caused by ac- thyroid. In 2.5–40% of PTC, a RET rearrangement is tivating mutations or rearrangements of the rearranged found, although this percentage is higher in patients during transfection (RET) gene. MTC originates from exposed to radiation (1). the calcitonin-producing C cells of the thyroid and can Total thyroidectomy and extensive lymph node dissec- occur sporadically (75%) or as part of a familial cancer tion is the curative treatment for MTC and PTC, followed syndrome (25%). The latter occurs as multiple endo- by radioiodine ablation in PTC. However, recurrent dis- crine neoplasia (MEN) type 2 syndrome (MEN2A and ease is often seen in sporadic MTC, and until recently, MEN2B) or as familial MTC (1–3). PTC is the most reoperation was the only therapeutic option. In iodine- common thyroid cancer (80% of all thyroid cancers) refractory PTC, no effective adjuvant therapy is available

ISSN Print 0021-972X ISSN Online 1945-7197 * H.H.G.V. and M.M.A. contributed equally. Printed in U.S.A. Abbreviations: DMSO, Dimethylsulfoxide; MEN, multiple endocrine neoplasia; MTC, med- Copyright © 2011 by The Endocrine Society ullary thyroid carcinoma; PTC, papillary thyroid carcinoma; RET, rearranged during trans- doi: 10.1210/jc.2010-2381 Received October 8, 2010. Accepted March 18, 2011. fection; TK, tyrosine kinase. First Published Online April 6, 2011

J Clin Endocrinol Metab, June 2011, 96(6):E991–E995 jcem.endojournals.org E991 E992 Verbeek et al. Effect of TK Inhibitors on MTC and PTC Cells J Clin Endocrinol Metab, June 2011, 96(6):E991–E995 as well (4, 5). New systemic therapies are therefore needed mental Materials and Methods (published on The Endocrine Soci- for both recurrent MTC and PTC. ety’s Journals Online web site at http://jcem.endojournals.org). With RET being the gene involved in a subset of MTC and PTC, it is logical to consider the encoded receptor as Tyrosine kinase inhibitors an important target for systemic therapy. RET is expressed XL184 (Exelixis, San Francisco, CA), vandetanib (AstraZen- in all tumor cells and continuous autophosphorylation on eca, Zoetermeer, The Netherlands), and sunitinib and axitinib (Pfizer, Capelle a/d IJssel, The Netherlands) were made up as a its tyrosine kinase (TK) residues caused by mutations stock solution of 10 mM in dimethylsulfoxide (DMSO). (MEN2) or rearrangements (PTC) on RET results in a constant activation of downstream signaling pathways Cell proliferation assays that ultimately lead to tumor growth (1). Therefore, in- MTC-TT and MZ-CRC-1 cells were plated in 200 ␮l medium hibition of RET phosphorylation and its downstream at concentrations of 4 ϫ 104 cells per well. TPC-1 and HEK293 pathways could be of great value. cells were plated at a density of 2 ϫ 103 cells per well. After Small-molecule inhibitors that selectively inhibit TK overnight incubation, increasing concentrations of TK inhibitor have been proven to be effective in the treatment of several solutions (0, 0.005, 0.05, 0.1, 0.5, and 5 ␮M) were added. A neoplastic diseases (6–8). A number of these clinically concentration of 0.1% DMSO was used in all experiments. Con- useful inhibitors target TK receptors that belong to the trol cells were grown without DMSO or TK inhibitor. Prolifer- same family group of proteins as RET (9). Several TK ation was measured at 1, 4, and 7 d using a cell proliferation kit inhibitors have already been tested in vitro and evaluated (MTT assay; Roche, Almere, The Netherlands) according to the manufacturer’s instructions. The concentration that led to in phase II clinical trials (Table 1). In a large number of 50% growth inhibition (IC50) was determined using linear patients (25–81%), a stable disease state can be estab- ϭ interpolation at r 0.5 (Supplemental Table 1). If IC50 con- lished, and some patients even show a partial response centrations were between 0.5 and 5 ␮M, additional cell pro- (2–33%) (10–19). liferation assays were performed (Supplemental Fig. 1, A–D). Because most studies have focused on one particular All experiments were performed in triplicate. TK inhibitor and have not looked for mutation specificity, it is hard to compare these compounds for the different Cell lysates and Western blot analysis patient groups. We therefore set out to compare the effi- MZ-CRC-1, MTC-TT, and TPC-1 cells were treated with 0, ciency of four recently developed TK inhibitors, XL184, IC50, and maximal concentrations of the different TK inhibitors for vandetanib, sunitinib, and axitinib, using three cell lines: 0, 2, and 5 d. Cell lysates were prepared as described in Supple- MTC-TT reported to be derived from a sporadic MTC mental Materials and Methods, and supernatants were stored at Ϫ expressing a C634W RET mutation, MZ-CRC-1 derived 80 C before they were further processed for SDS-PAGE followed by Western blot analysis. The antibodies used are described in Sup- from a patient with metastatic sporadic MTC expressing plemental Materials and Methods. All experiments were performed an M918T RET mutation, and TPC-1 derived from a pa- in duplicate. tient with PTC expressing a RET/PTC-1 rearrangement. RNA extraction and RT-PCR

Materials and Methods MTC-TT and MZ-CRC-1 cells, treated with 0, IC50, and maximal concentrations (Supplemental Table 1) of XL184 and Cell culture vandetanib, respectively, were collected after 0, 2, and 5 d. RNA MZ-CRC-1, MTC-TT, TPC-1, and HEK293 (human embry- extraction and RT-PCR procedures are described in the Supple- onic kidney cells) cell lines were cultured as described in Supple- mental Materials and Methods.

TABLE 1. Molecular targets and clinical trials performed with TK inhibitors in MTC patients

Molecular No. of Tumor response Stable disease targets patients ͓% (n)͔ ͓% (n)͔ Refs. Imatinib Bcr-Abl, PDGFR-␣ and -␤, c-Fms, c-Kit, RET 24 0 38 (9) 10, 11 Sorafenib RAF, VEGFR2 and -3, PDGFR, RET 21 10 (2) 86 (18) 12 Motesanib VEGFR1, -2, and -3, PDGFR, c-Kit, RET 91 2 (2) 81 (74) 13 Gefitiniba EGFR 4 0 0 14 Vandetanib VEGFR, EGFR, RET 30 20 (6) 53 (16) 15 19 16 (3) 53 (10) 16 Axitinib VEGFR1, -2, and -3 11 18 (2) 27 (3) 17 Sunitiniba RET, VEGFR, PDGFR 6 0 83 (5) 18 XL184b MET, VEGFR2, RET 37 (35 with 29 (10) 41 (15) 19 measurable disease) a Clinical trial performed in advanced thyroid cancer patients, not only MTC patients. b Phase 1 trial. J Clin Endocrinol Metab, June 2011, 96(6):E991–E995 jcem.endojournals.org E993

ϭ ␮ Statistical analysis (IC50 0.06 M) proliferation, whereas for MZ-CRC-1, The statistical analysis was performed using the software pro- vandetanib inhibited cell proliferation at the lowest concen- gram SPSS version 16.0. ϭ ␮ tration (IC50 0.26 M). DMSO did not seem to have a significant negative effect on cell growth of any of the cell lines tested (Supplemental Table 2). Results

Effect of different TK inhibitors on cell Effect of XL184 and vandetanib on RET proliferation autophosphorylation A dose-dependent decrease in cell proliferations was seen Inhibition of RET autophosphorylation on tyrosine for all four inhibitors (Fig. 1, A–C, and Supplemental Fig. 1, 1062 was observed for the three cell lines tested after 2 d A–D). In contrast, HEK293 cells, who do not endogenously of treatment with XL184 (MTC-TT and TPC-1) and van- express RET, showed only minor effects on proliferation at detanib (MZ-CRC-1). However, only vandetanib was concentrations lower than 0.5 ␮M (Supplemental Fig. 2A). able to induce this effect with IC50 levels (Fig. 1D). For

Based on the IC50 values (Supplemental Fig. 2B and Supple- MTC-TT and TPC-1, 5 d exposure to IC50 concentrations mental Table 1), we observed that XL184 was the most ef- of XL184 were necessary to reduce RET autophosphor- ϭ ␮ fective inhibitor of MTC-TT (IC50 0.04 M) and TPC-1 ylation levels (Fig. 1, E and F).

A MZ-CRC-1 D MZ-CRC-1 treated with vandetanib Sunitinib Vandetanib Axitinib XL184 2 days 5 days 125% C 0 IC50 max 0 IC50 max

100% RET

75% p-RET

50% ERK Proliferation 25% p-ERK 0% Actini 0 0.005 0.05 0.1 0.5 5 Concentration (µM) B MTC-TT E MTC-TT treated with XL184 Sunitinib Vandetanib Axitinib XL184 2 days 5 days 125% C 0 IC50 max 0 IC50 max 100% RET 75% p-RET

50% ERK Proliferation 25% p-ERK 0% 0 0.005 0.05 0.1 0.5 5 Acn Concentration (µM) C TPC-1 F TPC-1 treated with XL184 SitiibSunitinib VdtibVandetanib AitiibAxitinib XL184 2 days 5 days 125% C 0 IC50 max 0 IC50 max

100% RET

75% p-RET

50%

oliferation ERK Pro 25% p-ERK 0% Acn 0 0.005 0.05 0.1 0.5 5 Concentration (µM) FIG. 1. Effect of XL184, vandetanib, sunitinib, and axitinib on cell proliferation. A–C, Dose-response curves of MZ-CRC-1 (A), MTC-TT (B), and TPC-1 (C) cell lines incubated with different concentrations of XL184, vandetanib, sunitinib, and axitinib; D–F, effect of XL184 and vandetanib on

RET expression, RET autophosphorylation, ERK expression, and ERK phosphorylation: MZ-CRC-1 cells treated with IC50 concentration and 5 M vandetanib for 2 and 5 d (D), MTC-TT (E), and TPC-1 (F) cells treated with IC50 concentration and 5 M XL184 for 2 and 5 d. Error bars shown correspond to SD. E994 Verbeek et al. Effect of TK Inhibitors on MTC and PTC Cells J Clin Endocrinol Metab, June 2011, 96(6):E991–E995

To investigate whether the decrease in RET autophos- transcription. It is possible that for XL184, lysosomal or phorylation was due to reduced levels of RET expression, proteasomal degradation is involved, as was described the membranes were stripped and probed for RET. for other inhibitors (20). For TPC-1 a marked decrease MTC-TT and MZ-CRC-1 cells showed a dose-dependent in RET phosphorylation levels was detected, but sur- response after 5 d incubation with XL184 and vandetanib, prisingly, RET/PTC expression levels increased after respectively. At IC50 values, a marked decreased in RET exposure to XL184. This dual effect might be the result expression was observed, and at maximal concentrations, of a negative feedback mechanism to compensate a re- RET was no longer detectable (Fig. 1, D and F). To de- duction in RET activation. Furthermore, it shows that termine whether this effect was due to inhibition of RET XL184 exerts its effect in PTC by direct inhibition of transcription, RET expression levels were determined. For RET autophosphorylation and that lysosomal or pro- MTC-TT, no effect on RET transcription levels was ob- teasomal degradation may not be effective due to the served. However, for MZ-CRC-1, 5 d exposure to max- presence of the fusion protein. imal concentrations of vandetanib led to a decrease in RET Finally, we explored the effect of these drugs in a down- expression (Supplemental Fig. 3, A–C). For TPC-1 cells, stream signaling pathway directly activated by RET, the the total amount of RET remained relatively unchanged MAPK pathway. For MTC-TT and MZ-CRC-1, exposure even after 5 d exposure to maximal concentrations of to XL184 and vandetanib, respectively, induced a marked XL184 (Fig. 1F). decrease in ERK phosphorylation. Interestingly, a reduc- tion on ERK expression was also observed for these cell Effect of XL184 and vandetanib on RET lines, suggesting a possible effect of XL184 and van- downstream signaling pathways detanib on ERK transcription. For the TPC-1 PTC model RET is involved in the activation of several signaling system, no change in ERK phosphorylation was detected pathways, including the MAPK pathway (20). Therefore, after exposure to XL184. ERK phosphorylation through we evaluated the effect of XL184 and vandetanib in ERK other TK receptors and TK effector molecules, e.g. BRAF, activation. For MTC-TT and MZ-CRC-1, ERK phos- are likely to exert a stronger effect in ERK phosphoryla- phorylation was markedly reduced with IC50 levels of tion than RET. It is possible that for PTC, a combination XL184 and vandetanib, respectively, and was totally in- of different inhibitors targeting RET and, e.g. BRAF (ser- hibited when maximal concentrations were used (Fig. 1, D ine/threonine-protein kinase B-Raf), could circumvent and E). However, XL184 was able to exert this effect after this problem and thus result in an even more effective only 2 d exposure. Interestingly, this reduction of ERK treatment for this type of cancer. activation was related to a decrease in ERK expression Because no TK inhibitor was superior for the cell lines (Fig. 1, D and E). For TPC-1, no effect on ERK expression tested, our in vitro results suggest that mutation-specific and activation was observed, even when maximal concen- therapies could be beneficial for the treatment of MTC and trations of XL184 were used (Fig. 1F). PTC. However, because only three different mutations were analyzed, additional mutational studies are neces- sary to confirm this specificity. Discussion To date, no distinction has been made between the dif- ferent RET-related mutational subtypes (MEN2A/MEN2B/ We compared the effects of four TK inhibitors, XL184, PTC) in clinical trials, and it is known that aspecific targeting vandetanib, sunitinib, and axitinib, on cell proliferation of TK inhibitors can also contribute to antitumor effects. and RET inhibition and looked for mutation specificity However, the risk of severe side effects for the patients in using cell lines harboring a MEN2A mutation (MTC-TT), combination with the development of resistance to the TK a MEN2B mutation (MZ-CRC-1), and a RET/PTC rear- inhibitors incorrectly used reinforces the need of mutation- rangement (TPC-1). Our results showed that all four TK specific therapies. Furthermore, the combined use of dif- inhibitors are capable of reducing cell proliferation to ferent TK inhibitors for multiple targeting will also benefit some extent. However, XL184 was found to be the most from this knowledge, because only then optimal combi- efficient inhibitor for MEN2A- and PTC-derived cell lines, nations of inhibitors can be chosen. whereas vandetanib proved to be the most potent inhibitor In conclusion, our results are in general agreement with for MEN2B. the outcome of the clinical trials, supporting the idea that We also showed that XL184 and vandetanib were able XL184 and vandetanib are two potent inhibitors for tu- to decrease RET autophosphorylation and expression lev- mor response in MTC. We also showed that there is spec- els in MTC-TT and MZ-CRC-1 cells, respectively. How- ificity of these inhibitors for the treatment of different RET ever, only vandetanib exerted this effect by inhibiting RET mutations, which suggest that mutation-specific therapies J Clin Endocrinol Metab, June 2011, 96(6):E991–E995 jcem.endojournals.org E995 will likely improve the outcome of ongoing studies. Thus, 8. 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