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RET Activation Inhibits Doxorubicin- induced in SK-N-MC Cells MICHAEL A. SKINNER 1, KAREN E. LACKEY 2 and ALEX J. FREEMERMAN 3

1Department of Pediatric Surgery, Children’s Medical Center of Dallas and University of Texas Southwestern Medical Center, Dallas, TX 75235; 2GlaxoSmithKline, Research Triangle Park, NC 27709; 3Department of Surgery, Division of Pediatric Surgery, Box 2627, Duke University, Durham, NC 27710, U.S.A.

Abstract. Background: Medullary thyroid cancer (MTC) is three syndromes, is medullary thyroid carcinoma (MTC) (6). generally resistant to chemotherapy and the frequent MTC more commonly arises sporadically and in the absence constitutive activation of RET (rearranged during transfection of an inherited RET mutation, but a high percentage of even ) in these tumors might inhibit drug-induced apoptosis. these non-syndromic tumors result s from sporadic activating Materials and Methods: Each RET isoform was separately RET mutations (7-9). Moreover, RET gene translocations expressed in SK-N-MC cells (neural crest-derived tumor) and that constitutively activate RET are important in the the impact of RET activation on doxorubicin-induced development of papillary thyroid carcinomas (6, 10, 11). apoptosis was examined. Results: The activation of RET9 and The RET proto-oncogene encodes a RET51 in the SK-N-MC cells significantly reduced the kinase (RTK) that exists in one of two alternatively spliced doxorubicin-induced apoptosis by 50%, compared to isoforms termed RET9 and RET51 (12, 13). These two splice untreated cells. RET activation also induced isoforms are identical until amino acid 1063 and then differ of ERK (extracellular regulated kinase), but no changes in by the addition of either 9 or 51 C-terminal residues. AKT (serine/threonine kinase) phosphorylation were noted. In Although RET is an RTK, the protein itself does not have a the presence of a MAP (mitogen-activated protein) kinase cognate , but instead pairs with one of four glycosyl- inhibitor or a RET kinase inhibitor, the RET-activated/drug- phosphotidylinositol-linked coreceptors, known as GFRα1-4 treated cells displayed nearly 75% and 100% of the (glial-derived neurotrophic factor (GDNF) family receptor). doxorubicin-induced apoptosis of the drug-treated cells Each coreceptor, in turn, has its own preferred ligand: GDNF, without RET activation, respectively. Conclusion: In SK-N-MC neurturin, persephin, and artemin, respectively (14, 15). cells, downstream activation of MAP kinase, by both RET9 Normally, an activated RET signaling complex is formed by and RET51, appears to mediate the majority of RET-dependent a homoduplex of RET (with each RET bound to a coreceptor resistance to chemotherapeutically induced apoptosis. MTC and its ligand), which pairs prefere ntially with a like splice might be rendered more responsive to chemotherapeutic agents variant (RET9/RET9 or RET51/RET51) (16). Activated RET by the co-administration of a RET kinase inhibitor. then autophosphorylates intracellular tyrosine residues to initiate downstream signaling cascades (17-19). Inherited gain-of-function point mutations in the RET The two RET splice variants exhibit both similarities and (rearranged during transfection) gene are responsible for the differences in downstream signaling that may affect the cancer syndromes multiple endocrine neoplasia (MEN)2A, apoptotic threshold of relevant cells. For example, depending MEN2B, and familial medullary thyroid cancer (FMTC) (1- on the line examined, RET9 showed preferential binding 5). Individuals with these cancer syndromes develop several to the intracellular docking protein Shc at Tyr1062, whereas types of tumor, but the most virulent, and common to all in RET51 the Tyr1062 residue bound more avidly to the Grb2 protein (20). Additionally, RET51 contains two tyrosine residues not present in RET9: Tyr1090 and Tyr1096. The latter of these two residues is a docking site for Grb2 (20). Correspondence to: Alex J. Freemerman, Duke University Medical Activation of these downstream adapter proteins mediates the Center, Box 2627, Durham, NC 27710, U.S.A. Tel: +1 919 6818358, Fax: +1 919 6818353, e-mail: [email protected] activation of multiple pathways from both splice variants, many of which are known to be important Key Words: RET, apoptosis, doxorubicin, medullary thyroid cancer, regulators of apoptosis. Most notably, the mitogen-activated MAPK pathway. protein kinase (MAPK), phosphoinositide 3-kinase (PI3K),

0250-7005/2008 $2.00+.40 2019 ANTICANCER RESEARCH 28 : 2019-2026 (2008) extracellular regulated kinase (ERK) , c-Jun N-terminal kinase due to an extracellular mutation. Lastly, the downstream (JNK), and signal transducer and activator of transcription 3 phosphorylation events related to the activation of the RET (STAT3) pathways have all been shown to be activated by isoforms was also investigated. RET (21-25). In cells that normally express RET, mutated RET might exert Materials and Methods its oncogenic activity mainly by increasing the apoptotic RET constructs. To generate a RET construct that can be activated by threshold. This hypothesis is supported by several lines of EGF, the extracellular region and transmembrane domain of the evidence from clinical observations of patients with MTC. First, human EGF receptor (amino acids 1-668) was combined with the in the neck of patients with MEN 2B, numerous, prominent intracellular region of RET (either RET9 [amino acids 658-1072] or cutaneous nerves, that are usually not discernable, are evident RET51 [amino acids 658-1114], both amplified from normal human (personal observation, MAS). Similarly, normally identified cDNA). Each construct was ligated into the pLNX2 retroviral vector nerves are much larger than in patients with MEN 2B than in (Clontech, Mountain View, CA, USA). These constructs were then other patients. The presence of larger-than-normal nerves might transfected into AmphoPack ™ cells (Clontech) to generate a retroviral supernatant. The parental human cell line, SK-N-MC, was be induced by activated, mutant RET inhibiting normal nerve utilized to establish cell lines expressing both the RET constructs and regression (apoptosis) during development. Second, MTC is an empty vector (EV) control. The cell lines were established by generally an indolent, slow growing tumor. Indeed, the mean mixing 1 ml retroviral supernatant with 1 ml minimal essential survival of patients with MTC is about nine years, and patients medium (MEM) supplemented with 10% fetal bovine serum ( FBS), have been observed to survive for decades with metastatic 1x non-essential amino acids, and penicillin/streptomycin antibiotic disease (26) which suggests that oncogenic RET does not mix (Sigma Chemical Co., St. Louis, MO, USA). The cells were primarily drive uncontrolled cellular mitosis. Third, MTC is incubated for 24 hours and then selected in 800 μg/ml geneticin (Invitrogen, Carlsbad, CA, USA) for 3 weeks. The transfectant cells unresponsive to both chemotherapy and radiation treatment (9, were maintained in 400 μg/ml geneticin. 27). Since these agents induce tumor cell death primarily by triggering the apoptotic process (28), oncogenic RET might Chemicals. Doxorubicin (Sigma) was diluted in sterile water at somehow increase the apoptotic threshold rendering MTC 50 mg/ml. The EGF was resuspended in sterile water at 100 μg/ml. The insensitive to standard antineoplastic treatments. substituted 4-(3-hydroxyanilino)-quinoline compound GSK550, a potent The inhibition of apoptosis mediated by RET signal small molecule RET kinase inhibitor (36) (GlaxoSmithKline, Research transduction has not been thoroughly examined. Moreover, Triangle Park, NC, USA) (compound 40) was dissolved in dimethyl sulfoxide ( DMSO) at 5 mg/ml. PD98059, a small molecule MAP kinase because ligand activation of the two protein isoforms RET9 inhibitor (EMD Biosciences, Inc. [Calbiochem], San Diego, CA, USA) and RET51 differs in both duration and specificity of tyrosine was dissolved in DMSO at 10 mg/ml and diluted with media. The stock phosphorylation (16), it is possible that downstream signaling reagents were stored at –20˚C under light-free conditions and diluted to from the RET isoforms may differentially impact drug- the appropriate final concentrations in media. Vehicle controls of water induced apoptosis. To address these issues, the ability of each or DMSO were included in all the experiments. of the RET9 and RET51 isoforms to inhibit drug-induced apoptosis was examined. To simplify our control of RET Cell culture. All the cultures were maintained under a fully humidified atmosphere of 95% air and 5% CO at 37˚C. The cultures were signaling, a chimeric protein containing the extracellular 2 routinely screened for mycoplasma contamination with a rapid region of the epidermal (EGF) receptor coupled hybridization assay for mycoplasma RNA (Gen-Probe, San Diego, to the intracellular portion of RET was used. This particular CA, USA) and consistently found to be mycoplasma-free. The cell RET construct has been used extensively in NIH3T3 and densities were determined by hematocytometer and trypan blue SK-N-MC cells to study RET signaling (29-32). Because of exclusion. For the experimental incubations, cells in log-phase growth 5 this previous work and because a cell line that did not express were plated at a density of 1×10 cells/ml and allowed to attach RET but would be likely to express the proper downstream overnight. The EV, EGFR/RET9 (ER9), and EGFR/RET51 (ER51) expressing cells were then treated with either vehicle control, EGF elements required for faithful RET signaling was required, (50 ng/ml), doxorubicin (250 nM) or both EGF and doxorubicin in SK-N-MC cells were used. SK-N-MC cells derive serum-free media for 24 hours in the presence or absence of the small embryologically from the neural crest (33), where RET is molecule RET inhibitor (50 nM) or the MAP kinase inhibitor normally expressed (34) and would therefore be more likely (10 μM). The experimental incubations were terminated by to possess an intracellular signaling milieu similar to that of collecting the cells, pelleting at 500×g for 5 minutes, and washing MTC. Doxorubicin was chosen to induce apoptosis on the with 1× phosphate-buffered saline (PBS) and either directly lysing basis of its use to treat MTC in patients (35). Even though cells in 1× Laemmli (1% SDS, 10% glycerol, 100 mM dithiothreitol, 50 mM Tris, pH 6.8) for Western blots or 1x lysis RET activation occurs within minutes following the addition buffer (25 mM HEPES [pH 7.4], 5 mM CHAPS, 5 mM DTT) from of EGF to the media, a 24-hour time point was chosen to the caspase-3 assay (Sigma). allow sufficient time for doxorubicin-induced apoptosis to occur, which may more faithfully model MTC, particularly in Western blots. The Laemmli lysates of the treated cells were the case of MEN 2A, where RET is constitutively activated sonicated and centrifuged at 12,800×g for 5 minutes. All the samples

2020 Skinner et al : Activated RET Decreases Drug-induced Apoptosis were boiled for 5 minutes, then equivalent volumes were separated by SDS-PAGE with a 4-20% gradient (Bio-Rad, Hercules, CA, USA), and electroblotted to 0.45 μM nitrocellulose membranes. The blots were stained with amido black (0.5% [w/v] in 5% acetic acid) and destained with water to confirm transfer and equal loading and then blocked in 1x Tris-buffered saline (0.8% NaCl, 2.7 mM KCl, 25 mM Tris, pH 7.4) with 0.05% Tween-20 (TBS-T) and 1% milk/1% bovine serum albumin (BSA) for 1 hour at 22˚C. The blots were incubated in fresh blocking solution and probed for 1 hour with a 1:1000 dilution of anti-phospho-RET (Tyr 1062-R) primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The blots were washed 3×5 minutes in TBS-T and then incubated with a 1:10,000 dilution of peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) in TBS-T for 1 hour at 22˚C. The blots were again washed 3×5 minutes in TBS-T and then developed by enhanced chemiluminesence (Pierce, Rockford, IL, USA). The individual blots were stripped for 30 minutes using Restore™ (Pierce) and reprobed sequentially as described above using either an anti-RET9 (C-19), anti-RET51 (C-20), anti-AKT1/2/3 (H-136), anti-phospho-AKT1/2/3 (SER-473-R), anti-ERK1/2 (C-16-G), anti-phospho-ERK1/2 (E4) (Santa Cruz Biotechnology), or anti- actin (MAB1501) (Chemicon International, Temecula, CA, USA) primary antibody.

Detection of apoptosis. The caspase-3 colorimetric kit (Sigma) and protocol was used to assess apoptosis. Briefly, the treated cell lysates were centrifuged at 16,000×g for 10 minutes and the supernatant transferred to a new microfuge tube. The caspase-3 activity was measured in duplicate in a 96-well plate. Each well contained 5 μl lysate, 85 μl 1× assay buffer (20 mM HEPES [pH 7.4], 0.1% CHAPS, 5 mM DTT, 2 mM EDTA), and 10 μl substrate (2 mM acetyl-Asp-Glu-Val-Asp p-nitroanilide). The plates were incubated at 37˚C for 4 hours and the absorbance was measured at 405 nM on a Bio-Rad 680 plate reader and Microplate Manager™ software (Bio-Rad). Alternatively, the treated cells were stained with Hoechst dye 33342 (300 ng/ml) in medium for 10 minutes and visualized using a Nikon Eclipse TS100 inverted microscope (Nikon Instruments Inc., Melville, NY, USA) equipped with epifluorescence. The cells were then photographed with a Nikon Coolpix 990 at 200× (Nikon).

Statistics. The Student’s t-test was used to evaluate the statistical significance of the percentage of apoptotic cells in the treated versus untreated conditions. Software from GraphPad Prism 4 was used for graphing and analysis (GraphPad Software, San Diego, CA, USA).

Figure 1 . Induction of cell death in SK-N-MC cells was examined by Results cell morphology and caspase-3 activity and RET expression and downstream signaling was examined by Western blot. (A) Empty vector Drug-induced apoptosis and downstream signaling in SK-N-MC (EV), EGFR/RET9 expressing cells (ER9) or EGFR/RET51 expressing cells expressing RET isoforms. The control and EGF-treated cells (ER51) were either untreated or treated with 50 ng/ml EGF cells exhibited very few cells with bright nuclear staining, (+EGF) or 250 nM doxorubicin (+Dox), or 50 ng/ml EGF and 250 nM indicative of a paucity of apoptosis (Figure 1A). Treatment of doxorubicin for 24 hours. Cells were stained with Hoechst dye 33342 all SK-N-MC cell lines with 250 nM doxorubicin for 24 hours and photographed at ×200. (B) Quanti fic ation of caspase-3 activity (*p<0.005). (C) Western blots. Actin served as a loading control. The induced an equivalent amount of apoptosis as evidenced by the above assays and blots are representative of 3 separate experiments. punctate nuclear staining observed in the EV, ER9, and ER51 expressing cells. Importantly, when RET was activated by the addition of EGF to the media, the doxorubicin-treated ER9 and In agreement with the Hoechst staining results, the ER51 cells both exhibited a dramatic reduction in apoptosis untreated and EGF treated cells showed very little caspase-3 compared to the similarly treated EV cells. activity in all three cell lines (Figure 1B). Additionally, the

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EV, ER9 and ER51 expressing cells exhibited equivalently Discussion increased levels of caspase-3 activity when treated with doxorubicin alone. However, in the cells treated with both Activating RET in SK-N-MC cells significantly reduced EGF and doxorubicin, the RET9 and RET51 expressing cells apoptosis induced by doxorubicin. Furthermore, the degree exhibited just over a 50% reduction in caspase-3 activity of inhibition was the same with both RET splice forms. compared to similarly treated EV cells ( p< 0.005). Moreover, when RET activation was prevented by the Even though the experimental treatments were carried out administration of the small molecule RET inhibitor, in serum-free media, both ER9 and ER51 cells exhibited low apoptosis occurred at baseline levels suggesting that RET constitutive levels of RET phosphorylation by Western blot, signaling is critical in impeding apoptosis. Thus, the even in the absence of added EGF (Figure 1C). However, the activation of both RET isoforms increased the apoptotic cells exhibited robust activation (phosphorylation) of RET threshold and a constitutively activated RET might confer after exposure to 50 ng/ml EGF, alone and in combination a similar drug-resistance in actual MTC tumors. with doxorubicin. Interestingly, activation of both RET9 and The mechanisms by which activated RET might protect RET51 by EGF also seemed to up-regulate the total amount SK-N-MC cells (and MTC) from drug-induced apoptosis are of RET expression which was reflected in both the total RET not entirely understood. RET activation seems to impact and the phospho-RET. numerous intracellular pathways, including the MAP kinase The activation of known downstream effectors of RET, and PI3 K cascades (21-24). In other tumor types, these AKT (PI3 K pathway) and ERK 1 and -2 p42/p44 (MAP pathways have been implicated in both tumor growth and kinase pathway) in the EV, ER9, and ER51 cells was also drug-resistance (28, 37). assayed. AKT showed a high baseline level of RET induced activation of one or more of these intracellular phosphorylation in all three cell lines and under all pathways might lead directly to drug-resistance by inhibiting conditions and there was no observable increase upon RET apoptosis. Indeed, in the SK-N-MC cells, RET activation stimulation. The ERK1/2 moities, on the other hand, were resulted in robust phosphorylation of ERK1/2, in the MAP dramatically phosphorylated by EGF activation of both ER9 kinase pathway, and the addition of the RET inhibitor and ER51 cells. The EV control cells showed no increase in completely inhibited ERK1/2 activation and restored the ability ERK1/2 phosphorylation in response to EGF (Figures 1C). of doxorubicin to induce apoptosis. More importantly, when the MAP kinase inhibitor was used, RET was fully activated, Impact of inhibiting RET and MAP kinase activity on drug- but ERK1/2 activation was abrogated, and a high proportion of induced apoptosis and downstream signaling. As demonstrated the drug sensitivity was restored. Thus, in SK-N-MC cells, in Figure 2A, the addition of the RET inhibitor appeared to MAP kinase pathway activation may be the main mechanism restore doxorubicin sensitivity to the cells expressing EGF- through which activated RET raises the apoptotic threshold. No activated RET9 and RET51. The cells treated with 50 nM RET changes in AKT phosphorylation levels, resulting from the inhibitor by itself or in combination with EGF all exhibited cell activation of either RET9 or RET51, were observed. death no greater than the untreated cells (data not shown). In Interestingly, SK-N-MC cells are reported by the agreement with the Hoechst staining experiments, the caspase-3 American Type Culture Collection (ATCC, www.atcc.org) activity results presented in Figure 2B confirmed that to have a 10 deletion and would, thus, lack doxorubicin-induced apoptosis, initially reduced by RET pTEN (phosphatase and tensin homolog), a major regulator activation, was restored by the addition of the RET inhibitor. of AKT phosphorylation. However, other studies of this cell Importantly, the inclusion of GSK550 in the culture media line have not noted an entire chromosome 10 deletion and, completely abrogated the EGF-induced phosphorylation of in fact, have demonstrated pTEN expression (38). Whether RET9 and RET51 and also abrogated the RET-dependent or not these cells express pTEN, even a small increase in downstream activation of ERK1/2 (Figure 2C). phosphorylation levels of AKT can have profound effects on As seen in Figure 3A, the inhibition of MAP kinase, by the apoptosis (39-42) and it is possible that such small changes addition of PD98059, mostly restored drug-sensitivity in the were simply not detected in the present study. Similarly, RET-activated ER9 and ER51 cells. The cells treated with 10 other investigators have noted that inhibitors of MAP kinase μM MAP kinase inhibitor by itself or in combination with and PI3 K have shown that both these pathways antagonize EGF all exhibited cell death no greater than the untreated cells apoptosis in neuronal cell lines (43-45). (data not shown). Again, in agreement with the Hoechst MTC is a particularly useful neoplasm in which to exploit staining, the inhibition of MAP kinase restored nearly 75% of the antiapoptotic signals that make the tumor difficult to treat. the caspase-3 activation in the RET-activated (ER9 and ER51) Unlike most carcinomas that arise from any of a range of cells (Figure 3B). PD98059 had no effect on EGF-activation genetic lesions, MTC is almost always associated with of RET or the apparent constitutive activation of AKT, but mutations in the RET protooncogene. This genetic completely inhibited ERK1/2 activation (Figure 3C). homogeneity might limit the number of signals that need to

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Figure 2. Drug-induced cell death and downstream signaling in SK-N- MC cells in the presence of a small molecule RET inhibitor. EV, ER9, and ER51 cells were treated as in Figure 1 in the presence of 50 nM Figure 3. Drug-induced cell death and downstream signaling in SK-N-MC RET inhibitor (+GSK550). (A) Cells stained with Hoechst dye 33342 cells in the presence of a small molecule MAP kinase inhibitor. EV, ER9, and photographed at ×200. (B) Quanti fic ation of caspase-3 activity. (C) and ER51 cells were treated as in Figure 1 in the presence of 10 μM MAP Western blots. Actin served as a loading control. The above assays and kinase inhibitor (+PD98059). (A) Cells stained with Hoechst dye 33342 blots are representative of 3 separate experiments. and photographed at ×200. (B) Quanti fic ation of caspase-3 activity (*p<0.01). (C) Western blots. Actin served as a loading control. The above assays and blots are representative of 3 separate experiments. be controlled to allow apoptosis. Abrogating RET activation References might therefore hold promise for the increased success in the management of MTC in humans. In conclusion, RET 1 Donis-Keller H, Dou S, Chi D, Carlson KM, Toshima K, signaling in SK-N-MC cells raises the apoptotic threshold and Lairmore TC, Howe JR, Moley JF, Goodfellow P and Wells SA Jr: Mutations in the RET proto-oncogene are associated with selective inhibition of RET activity increases the sensitivity of MEN 2A and FMTC. Hum Mol Gen 2: 851-856, 1993. the neoplastic cells to doxorubicin-induced apoptosis. 2 Mulligan LM, Kwok JBJ, Healey CS, Elsdon MJ, Eng C, Gardner E, Love DR, Mole SE, Moore JK, Papi L, Ponder MA, Acknowledgements Telenius H, Tunnacliffe A and Ponder PAJ: Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type Supported in part by donations to The Children’s Miracle Network 2A. Nature 363 : 458-460, 1993. and a grant from GlaxoSmithKline. Karen Lackey is an employee 3 Carlson KM, Dou S, Chi D, Scavarda N, Toshima T, Jackson of GlaxoSmithKline. CE, Wells SA, Jr, Goodfellow PJ and Donis-Keller H: Single

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missense mutation in the domain of the RET 18 Takahashi M: The GDNF/RET signaling pathway and human protooncogene is associated with multiple endocrine neoplasia diseases. Growth Factor Rev 12 : 361-373, 2001. type 2B. Proc Nat Acad Sci 91 : 1579-1583, 1994. 19 Saarma M: GDNF recruits the signaling crew into lipid rafts. 4 Hofstra RMW, Landsvater RM, Ceccherini I, Stulp RP, Stelwagon Trends Neurosci 24 : 427-429, 2001. T, Luo Y, Pasini B, Hoppener JWM, van Amstel HKP, Romeo 20 Alberti L, Borrello MG, Ghizzoni S, Torriti F, Rizzetti MG and G,Lips CJM and Buys CHCM: A mutation in the RET proto- Pierotti MA: Grb2 binding to the different isoforms of Ret oncogene associated with multiple endocrine neoplasia type 2B and tyrosine kinase. Oncogene 17 : 1079-1087, 1998. sporadic medullary thyroid carcinoma. Nature 367 : 375-376, 1994. 21 Chiariello M, Visconti R, Carlomagno F, Melillo RM, Bucci C, 5 Eng C, Smith DP, Mulligan LM, Nagal MA, Healey CS, Ponder de Franciscis V, Fox GM, Jing S, Coso OA, Gutkind JS, Fusco A MA, Gardner E, Scheumann GFW, Jackson CE, Tunacliffe A and Santoro M: Signalling of the Ret and Ponder BAJ: Point mutation within the tyrosine kinase through the c-Jun NH 2-terminal protein kinases (JNKS): domain of the RET proto-oncogene in multiple endocrine evidence for a divergence of the ERKs and JNKs pathways neoplasia type 2B and related sporadic tumours. Hum Mol Gen induced by Ret. Oncogene 16 : 2435-2445, 1998. 3: 237-241, 1994. 22 Schuringa JJ, Wojtachnio K, Hagens W, Vellenga E, Buys CH, 6 Le HN and Norton JA: Perspective on RET proto-oncogene and Hofstra R and Kruijer W: MEN2A-RET-induced cellular thyroid cancer. Cancer J 6: 50-57, 2000. transformation by activation of STAT3. Oncogene 20 : 5350- 7 Jhiang SM: The RET proto-oncogene in human cancers. 5358, 2001. Oncogene 19 : 5590-5597, 2000. 23 Melillo RM, Barone MV, Lupoli G, Cirafici AM, Carlomagno F, 8 Santoro M, Melillo RM, Carlomagno F, Fusco A and Vecchio G: Visconti R, Matoskova B, Di Fiore PP, Vecchio G, Fusco A and Molecular mechanisms of RET activation in human cancer. Ann Santoro M: Ret-mediated mitogenesis requires Src kinase N Y Acad Sci 963 : 116-121, 2002. activity. Cancer Res 59 : 1120-1126, 1999. 9 Moley JF and Fialkowski EA: Evidence-based approach to the 24 van Weering DH and Bos JL: Signal transduction by the receptor management of sporadic medullary thyroid carcinoma. World J tyrosine kinase Ret. Recent Results Cancer Res 154 : 271-281, Surg 31 : 946-956, 2007. 1998. 10 Santoro M, Dathan NA, Berlingieri MT, Bongarzone I, Paulin 25 Hwang JH, Kim DW, Suh JM, Kim H, Song JH, Hwang ES, C, Grieco M, Pierotti MA, Vecchio G and Fusco A: Molecular Park KC, Chung HK, Kim JM, Lee TH, Yu DY and Shong M: characterization of RET/PTC3: a novel rearranged version of the Activation of signal transducer and activator of transcription 3 RET proto-oncogene in a human thyroid papillary thyroid by oncogenic RET/PTC (rearranged in transformation/papillary carcinoma. Oncogene 9: 509-516, 1994. thyroid carcinoma) tyrosine kinase: roles in specific gene 11 Bongarzone I, Butti MG, Coronelli S, Borrello MG, Santoro M, regulation and cellular transformation. Mol Endocrinol 17 : 1155- Mondellini P, Pilotti S, Fusco A, Della Porta G and Pierotti MA: 1166, 2003. Frequent activation of RET protooncogene by fusion with a new 26 Bhattacharyya N: A population-based analysis of survival factors activating gene in papillary thyroid carcinomas. Cancer Res 54 : in differentiated and medullary thyroid carcinoma. Otolaryngol 2979-2985, 1994. Head Neck Surg 128 : 115-123, 2003. 12 Kwok JB, Gardner E, Warner JP, Ponder BA and Mulligan LM: 27 Chen H, Roberts JR, Ball DW, Eisele DW, Baylin SB, Udelsman Structural analysis of the human RET proto-oncogene using R and Bulkley GB: Effective long-term palliation of symptomatic, exon trapping. Oncogene 8: 2575-2582, 1993. incurable metastatic medullary thyroid cancer by operative 13 Tahira T, Ishizaka Y, Itoh F, Sugimura T and Nagao M: resection. Ann Surg 227 : 887-895, 1998. Characterization of RET proto-oncogene mRNAs encoding two 28 Fulda S and Debatin KM: Extrinsic versus intrinsic apoptosis isoforms of the protein product in a human neuroblastoma cell pathways in anticancer chemotherapy. Oncogene 25 : 4798-4811, line. Oncogene 5: 97-102, 1990. 2006. 14 Milbrandt J, de Sauvage FJ, Fahrner TJ, Baloh RH, Leitner ML, 29 Pandit SD, O’Hare T, Donis-Keller H and Pike LJ: Functional Tansey MG, Lampe PA, Heuckeroth RO, Kotzbauer PT, characterization of an epidermal growth factor receptor/RET Simburger KS, Golden JP, Davies JA, Vejsada R, Kato AC, Hynes chimera. J Biol Chem 272 : 2199-2206, 1997. M, Sherman D, Nishimura M, Wang LC, Vandlen R, Moffat B, 30 van Weering DH, Medema JP, van Puijenbroek A, Burgering Klein RD, Poulsen K, Gray C, Garces A, Henderson CE, Phillips BM, Baas PD and Bos JL: Ret receptor tyrosine kinase activates H and Johnson EM Jr: Persephin, a novel neurotrophic factor extracellular signal-regulated kinase 2 in SK-N-MC cells. related to GDNF and neuturin. Neuron 20 : 245-253, 1998. Oncogene 11 : 2207-2214, 1995. 15 Kouvaraki MA, Shapiro SE, Perrier ND, Cote GJ, Gagel RF, Hoff 31 van Weering DH and Bos JL: Glial cell line-derived neurotrophic AO, Sherman SI, Lee JE and Evans DB: RET proto-oncogene: a factor induces Ret-mediated lamellipodia formation. J Biol review and update of genotype-phenotype correlations in Chem 272 : 249-254, 1997. hereditary medullary thyroid cancer and associated endocrine 32 van Puijenbroek AA, van Weering DH, van den Brink CE, tumors. Thyroid 15 : 531-544, 2005. Bos JL, van der Saag PT, de Laat SW and den Hertog J: Cell 16 Tsui-Pierchala BA, Ahrens RC, Crowder RJ, Milbrandt J and scattering of SK-N-MC neuroepithelioma cells in response to Johnson EM Jr: The long and short isoforms of Ret function as Ret and FGF receptor tyrosine kinase activation is correlated independent signaling complexes. J Biol Chem 277 : 34618- with sustained ERK2 activation. Oncogene 14 : 1147-1157, 34625, 2002. 1997. 17 Mason I: The RET receptor tyrosine kinase: activation, signalling 33 Dunn T, Praissman L, Hagag N and Viola MV: ERG gene is and significance in neural development and disease. Pharm Acta translocated in an Ewing’s sarcoma cell line. Cancer Genet Helv 74 : 261-264, 2000. Cytogenet 76 : 19-22, 1994.

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34 Biedler JL, Helson L and Spengler BA: Morphology and growth, 42 Sale EM and Sale GJ: Protein kinase B: signalling roles and tumorigenicity, and cytogenetics of human neuroblastoma cells therapeutic targeting. Cell Mol Life Sci 65 : 113-127, 2008. in continuous culture. Cancer Res 33 : 2643-2652, 1973. 43 Mograbi B, Bocciardi R, Bourget I, Busca R, Rochet N, Farahi- 35 Nocera M, Baudin E, Pellegriti G, Cailleux AF, Mechelany- Far D, Juhel T and Rossi B: Glial cell line-derived neurotrophic Corone C and Schlumberger M: Treatment of advanced medullary factor-stimulated phosphatidylinositol 3-kinase and Akt activities thyroid cancer with an alternating combination of doxorubicin- exert opposing effects on the ERK pathway: importance for the streptozocin and 5 FU-dacarbazine. Groupe d’Etude des Tumeurs rescue of neuroectodermic cells. J Biol Chem 276 : 45307-45319, a Calcitonine (GETC). Br J Cancer 83 : 715-718, 2000. 2001. 36 Robinett G, Freemerman AJ, Skinner MA, Shewchuk L and 44 De Vita G, Melillo RM, Carlomagno F, Visconti R, Castellone Lackey K: The discovery of substituted 4-(3-hydroxyanilino)- MD, Bellacosa A, Billaud M, Fusco A, Tsichlis PN and Santoro quinolines as potent RET kinase inhibitors. Bioorg Med Chem M: Tyrosine 1062 of RET-MEN2A mediates activation of Akt Lett 17 : 5886-5893, 2007. (protein kinase B) and mitogen-activated protein kinase 37 McCubrey JA, Steelman LS, Abrams SL, Bertrand FE, Ludwig pathways leading to PC12 cell survival. Cancer Res 60 : 3727- DE, Bäsecke J, Libra M, Stivala F, Milella M, Tafuri A, Lunghi 3731, 2000. P, Bonati A and Martelli AM: Targeting survival cascades 45 Baki L, Neve RL, Shao Z, Shioi J, Georgakopoulos A and induced by activation of Ras/Raf/MEK/ERK, PI3K/PTEN/Akt/ Robakis NK: Wild-type but not FAD mutant presenilin-1 prevents mTOR and Jak/STAT pathways for effective leukemia therapy. neuronal degeneration by promoting phosphatidylinositol Leukemia 22 : 708-722, 2008. 3-kinase neuroprotective signaling. J Neurosci 28 : 483-490, 2008. 38 Munoz J, Lazcoz P, Inda MM, Nistal M, Pestana A, Encio IJ and Castresana JS: Homozygous deletion and expression of PTEN and DMBT1 in human primary neuroblastoma and cell lines. Int J Cancer 109 : 673-679, 2004. 39 Hill MM and Hemmings BA: Inhibition of protein kinase B/Akt. implications for cancer therapy. Pharmacol Ther 93 : 243-251, 2002. 40 Lawlor MA and Alessi DR: PKB/Akt: a key mediator of cell proliferation, survival and insulin responses? J Cell Sci 114 : 2903-2910, 2001. 41 Parcellier A, Tintignac LA, Zhuravleva E and Hemmings BA: Received March 3, 2008 PKB and the mitochondria: AKTing on apoptosis. Cell Signal Revised May 22, 2008 20 : 21-30, 2008. Accepted May 26, 2008

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