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Experimental Therapeutics |
Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Biologia e Patologia Cellulare e Molecolare "Luigi Califano" [F. Ca., D. V., T. G., G. V., A. F., M. S.] and Dipartimento di Endocrinologia e Oncologia Molecolare e Clinica [F. Ci., G. T.], University Federico II, 80131 Naples, Italy; Department of Oncology, Transplants and Advanced Technologies in Medicine, University of Pisa, 56100 Pisa, Italy [G. F.]; and Cancer Discovery, Astra Zeneca Mereside, Macclesfield, Cheshire, SK10 4TG, United Kingdom [A. J. R.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RET/PTC, RET/MEN2A, RET/MEN2B, and RET/FMTC alleles induce transformed foci, anchorage-independent growth and tumorigenicity in nude mice when introduced into NIH3T3 cells (2) . This transforming capacity is linked to their constitutive ligand-independent kinase activity. In RET/PTC, coiled-coil sequences of the fusion partners induce constitutive dimerization of the oncoprotein (10) . Disulfide-bond-mediated dimerization of RET occurs in MEN2A and FMTC cases bearing mutations of the extracellular cysteines (11) . The MEN2B-associated M918T mutation probably modifies the structure of the activation loop of the RET catalytic domain, thereby switching on the enzymatic function and altering its substrate specificity (11 , 12) .
Quinazolines are some of the most promising inhibitors of growth factor receptor tyrosine kinases (13)
. An anilinoquinazoline derivative, ZD1839 (Iressa is a potent and selective inhibitor of the EGFR and is currently in advanced clinical development (14)
. Another anilinoquinazoline, ZD6474, has recently been shown to be a selective inhibitor of the VEGF receptor-2 (flk-1/KDR) tyrosine kinase (15)
. ZD6474 has an IC50 of 
0.04 µM against the isolated KDR enzyme and blocks VEGF-stimulated endothelial cell migration and proliferation. In vivo, ZD6474 reverses VEGF-mediated hypotension and produces a dose-dependent increase in hypertrophy of the femoro-tibial epiphyseal growth plate zone, consistently with the inhibition of VEGF signaling and angiogenesis (15)
. ZD6474 inhibits, to a lesser extent, the activity of other tyrosine kinases such as the EGFR (IC50 = 0.5 µM) and the platelet-derived growth factor receptor (IC50 = 1.1 µM). Chronic once-daily oral administration of ZD6474 produced significant broad-spectrum antitumor activity on human tumor xenografts implanted in nude mice. ZD6474 is currently under Phase I evaluation in cancer patients (15)
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Here, we report a hitherto unknown property of ZD6474, i.e., it inhibits the enzymatic and transforming activity of RET oncoproteins and arrests the development of RET/PTC3-induced tumors in nude mice.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture.
Parental NIH3T3 and NIH3T3 cells transfected with EGFR (donated by P. P. Di Fiore; Ref. 16
), EGFR/RET (17)
, RET/PTC3 (4)
, MEN2A-associated RET C634R mutant, MEN2B-associated M918T RET mutant (11)
and v-Ha-Ras (18)
were cultured in DMEM supplemented with calf serum (10% for normal cells and 5% for transformed cells), 2 mM L-glutamine, and 100 units/ml penicillin-streptomycin (Life Technologies, Inc., Paisley, United Kingdom). After overnight starvation, NIH-EGFR and NIH-EGFR/RET were stimulated or not with 100 ng/ml EGF (Upstate Biotechnology Inc., Lake Placid, NY) for 10 min. Human thyroid-carcinoma cell lines [TPC1 (19)
; FB2 (a gift of F. Basolo; Ref. 20
), from papillary carcinomas harboring the RET/PTC1 rearrangement; and ARO (a gift of J. A. Fagin; Ref. 21
), derived from an anaplastic carcinoma negative for RET/PTC rearrangements], were cultured in RPMI with 10% FCS, 2 mM L-glutamine, and 100 units/ml penicillin-streptomycin (Life Technologies, Inc.). HEK293 cells were from American Type Culture Collection (Manassas, VA) and were grown in DMEM supplemented with 10% FCS. Transient transfections were carried out with the LipofectAMINE reagent used according to the manufacturer’s instructions (Life Technologies, Inc.). Briefly, cells were seeded at a density of 1.5 x 106/dish the day before transfection, then transfected with 5 µg of DNA, and harvested 48 h later.
Immunoblotting Analysis.
Cells or tumor tissues were lysed in a buffer containing 50 mM HEPES (pH 7.5), 1% (vol/vol) Triton X-100, 50 mM NaCl, 5 mM EGTA, 50 mM NaF, 20 mM sodium PPI, 1 mM sodium vanadate, 2 mM phenylmethylsulphonyl fluoride (PMSF), and 1 µg/ml aprotinin. Lysates were clarified by centrifugation at 10,000 x g for 15 min. Lysates containing comparable amounts of proteins, estimated by a modified Bradford assay (Bio-Rad, Munich, Germany), were immunoprecipitated with the required antibody or subjected to direct Western blot. Immune complexes were detected with the enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom). Anti-phosphotyrosine (4G10) and anti-EGFR were from Upstate Biotechnology Inc. Anti-MAPK (no. 9101) and anti-phospho-MAPK (no. 9102) were from New England Biolabs (Beverley, MA). Anti-RET is a polyclonal antibody raised against the tyrosine kinase protein fragment of human RET (17)
. Secondary antibodies coupled to horseradish peroxidase were from Santa Cruz Biotechnology (Santa Cruz, CA).
In Vitro Kinase Assays.
Cells were solubilized in lysis buffer with phosphatase and protease inhibitors. NIH-EGFR and NIH-EGFR/RET were stimulated with 100 ng/ml EGF for 10 min before harvesting. Proteins (200 µg) were immunoprecipitated with the required antibodies; immunocomplexes were recovered with protein A Sepharose, washed five times with kinase buffer, and incubated (20 min at room temperature) in kinase buffer containing 200 µM poly-GT (Sigma Chemical Co.), 2.5 µCi [
-32P]ATP and unlabelled ATP to a final concentration of 20 µM in the presence of the inhibitory compound or vehicle. EGFR and EGFR/RET immunocomplexes were incubated with 100 ng/ml EGF. Samples were spotted on Whatman 3MM paper (Springfield Mill, United Kingdom) and 32P incorporation was measured with a ß-counter scintillator (Beckman Coulter, Unterschleissheim-Lohhof, Germany). The GST-RET/TK plasmid was generated by PCR amplification of the intracellular RET domain (residues 718-1072) and fusion to the GST coding sequence into the pEBG vector, kindly provided by S. Meakin (22)
. GST-RET/TK was purified from HEK293 cell lysates using glutathione Sepharose according to standard procedures.
Growth Curve and Cell Cycle Analysis.
For growth curves, NIH3T3 cells (10,000/dish) or human thyroid carcinoma cells (50,000/dish) were seeded on 60-mm dishes in complete medium. The next day (day 1) ZD6474 or vehicle was added to the medium and refreshed every 2 days. Cells were counted every day. For cytofluorimetric [fluorescence-activated cell sorting (FACS)] analysis, cells were grown to subconfluence, serum-starved for 24 h, and then subjected or not to 5.0 µM ZD6474 for an additional 24 h. After harvesting, cells were fixed in cold 70% ethanol in PBS. Propidium iodide (25 µg/ml) was added, and samples were analyzed with a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA) interfaced with a Hewlett Packard computer (Hewlett Packard, Palo Alto, CA).
Soft Agar Growth Assay.
Cells were seeded on 60-mm dishes (10,000 cells/dish) in 0.3% agar in complete medium on a base layer of 0.5% agar with or without various concentrations of the inhibitor; the compound solution was added to the top layer every 3 days. Colonies were counted 15 days later.
Tumorigenicity in Nude Mice.
NIH-RET/PTC3 and NIH-RAS cells (50,000/mouse) were inoculated s.c. into the right dorsal portion of 6-week-old male BALB/c nu/nu mice (The Jackson Laboratory, Bar Harbor, ME). ZD6474 (1 or 0.4 mg/mouse/day dissolved in PBS containing 0.5% v/v Tween 80) or vehicle alone was injected i.p. when established tumors (of 5–10 mm3) appeared. Tumor diameters were measured with calipers every 1–3 days. Tumor volumes (V) were calculated by the rotational ellipsoid formula: V = A x B2/2 (A = axial diameter; B = rotational diameter). No mouse showed signs of wasting or other signs of toxicity. Animals were maintained at the Dipartimento di Biologia e Patologia Cellulare e Molecolare Animal Facility. All of the manipulations were performed under isoflurane gas anesthesia and conducted in accordance with Italian regulations for experimentation on animals.
Statistical Analysis.
The Student’s t test was used to evaluate the statistical significance of the results. Analyses were performed with the BMDP New System statistical package version 1.0 for Microsoft Windows (BMDP Statistical Software, Los Angeles, CA).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, ZD6474 effectively inhibited RET/PTC3; PP1 was slightly more efficient than ZD6474. Conversely, ZD1839 did not affect RET/PTC3. ZD6474 also inhibited RET/MEN2A and RET/MEN2B oncoproteins (Fig. 1B)
. The IC50 of ZD6474 for RET/PTC3, measured by the poly-GT phosphorylation assay, was 100 nM (Fig. 1C)
. We generated the isolated RET kinase domain fused to a GST tag. The GST-RET/TK protein expressed in HEK293 cells was purified by chromatography on glutathione-Sepharose beads. The ZD6474 IC50 for GST-RET/TK was identical to that for RET/PTC3 (Fig. 1C)
. Finally, we compared the ability of ZD6474 to inhibit EGFR or RET kinase activity. To this aim, we used an EGFR/RET chimeric protein in which the intracellular kinase domain of RET is fused to the extracellular domain of EGFR. Both EGFR and EGFR/RET kinases are stimulated by EGF (17)
. NIH-EGFR and NIH-EGFR/RET cells were stimulated with EGF (100 ng/ml, 10 min) and harvested; protein lysates were immunoprecipitated with specific antibodies, and the immunocomplexes were assayed for poly-GT phosphorylation in the presence of different concentrations of ZD6474 or ZD1839. ZD6474 efficiently blocked EGFR/RET (IC50 of 100 nM; Fig. 1D
). The IC50 for EGFR was 300 nM (Fig. 1D)
. Conversely, ZD1839 blocked EGFR kinase but did not affect EGFR/RET (Fig. 1E)
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, phosphotyrosine was detected in both EGFR/RET and EGFR only after EGF stimulation. ZD6474 abolished EGFR/RET autophosphorylation as early as 2 h after treatment, whereas ZD1839 had no effect. Both compounds affected EGFR, although the effect of ZD6474 was detected earlier (after 2 h). We also evaluated the effects exerted by ZD6474 on ligand-independent phosphorylation of RET/PTC3. NIH-RET/PTC3 cells were serum-starved for 24 h and then treated with 5.0 µM ZD6474 for different time points. As early as 2 h after treatment, ZD6474 reduced RET/PTC3 phosphorylation to undetectable levels (Fig. 2A
, panels on the right). After EGF stimulation, both EGFR/RET and EGFR activated the Ras/MAPK (also designated Ras/ERK) pathway. To determine whether ZD6474 and ZD1839 affected signaling, we monitored ERK activation by immunoblot with an antibody specific for p42- and p44-ERKs phosphorylated at threonine 202 and tyrosine 204. As early as 2 h after exposure to 5.0 µM ZD6474, there was a dramatic reduction of EGFR/RET-dependent ERK phosphorylation, whereas ZD1839 had no effect (Fig. 2A)
. EGFR-dependent ERK phosphorylation was significantly reduced in NIH-EGFR cells treated with ZD6474 or ZD1839 but only after 6 h of exposure. After ZD6474 treatment, also, RET/PTC3-dependent ERK activation was markedly inhibited (Fig. 2A)
. A dose-response experiment demonstrated a remarkable reduction of RET/PTC3 phosphorylation and signaling even on treatment with 1.0 and 0.5 µM ZD6474 (Fig. 2B
, panels on the left).
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(right panels), RET/MEN2B protein products migrate as a doublet (a mature, fully glycosylated Mr 170,000 and a partially glycosylated Mr 150,000 product). Notably, RET/MEN2B phosphorylation and RET/MEN2B-dependent MAPK activation, as well, were inhibited by ZD6474 in vivo at doses comparable with those effective on RET/PTC. Thus, ZD6474 is an effective inhibitor of different RET oncoproteins both in vitro and in intact cells.
Inhibition of the Transforming Effects of RET/PTC Oncogenes by ZD6474.
RET/PTC3 induces morphological transformation, serum- and anchorage-independent proliferation and tumorigenicity in nude mice (4)
. We treated NIH-RET/PTC3 cells with 5.0 µM ZD6474 for 24 h and analyzed the morphological changes induced by the drug. As controls, we used parental and v-Ha-Ras-transformed NIH3T3 cells (NIH-RAS). We selected Ras as a control because it acts downstream from most receptors including RET (1)
. As shown in Fig. 3
, ZD6474 caused a complete morphological reversion of NIH-RET/PTC3 cells, whereas neither DMSO nor ZD1839 had any effect. Neither parental nor NIH-RAS cells were affected by ZD6474 (Fig. 3)
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, top panel). No effect was seen on NIH-RAS cells (Fig. 4B
, top panel). Flow cytometry showed that the inhibition of proliferation corresponded to increased G0-G1 cell fraction (Fig. 4, A and B
, bottom panels). The thyroid carcinoma TPC1 and FB2 cell lines bear the RET/PTC1 rearrangement. Treatment of those cells with 5.0 µM ZD6474 for 24 h induced growth arrest, resulting in an increased G0-G1 fraction, and markedly reduced the S-phase and G2-M fractions (Fig. 4C)
. There was also a significant increase of the sub-G1 (hypodiploid) fraction, which suggests that the drug had also an apoptotic effect in these cell lines. Another human thyroid carcinoma cell line, ARO, which does not contain a RET/PTC rearrangement, was not affected by ZD6474 treatment (Fig. 4C)
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, and the average results ± SD of three independent experiments are reported in Fig. 5B
. ZD6474 (5.0 µM) completely inhibited NIH-RET/PTC3 but not NIH-RAS colony formation in soft agar; 1.0 µM ZD6474 caused a 50% inhibition (Fig. 5B)
. Conversely, ZD1839 did not affect NIH-RET/PTC3 colony formation.
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5–10 mm3, animals (six for each group) were treated i.p. with ZD6474 at either 1 or 0.4 mg/mouse/day (equivalent to 50 or 20 mg/kg/day, respectively) or with vehicle. Fig. 6A
shows that vehicle-treated animals developed a tumor that grew rapidly to reach about 1–2 cm3 in 14 days. Treatment with ZD6474 at 0.4 mg daily strongly reduced tumor growth in NIH-RET/PTC3-injected animals. Daily treatment with 1 mg of ZD6474 completely arrested tumor development. NIH-RAS-induced tumors were unaffected by 0.4 mg ZD6474 daily, but with 1 mg/day/mouse there was a reduction of tumor growth, although over the time course of this experiment, it did not reach statistical significance (P > 0.05). The growth-inhibitory effects on NIH-RAS tumors are probably attributable to antiangiogenic effects of KDR inhibition. We counted blood vessels by light microscopy (x40) and immunoperoxidase staining with anti-factor VIII antibody, as described previously (24)
. Tumors (n = 3) established in NIH-RET/PTC3- and NIH-RAS-injected animals showed comparable vessel density (14 ± 4 and 13 ± 4 per microscopic field, respectively). ZD6474 treatment caused a significant (P > 0.03) reduction of vessel numbers in both tumor types (5 ± 1 and 4 ± 1 in NIH-RET/PTC3 and RAS tumors, respectively; not shown).
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, tumor-growth inhibition was associated with a remarkable reduction of RET/PTC3 phosphotyrosine content. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The RET gene is activated by somatic rearrangements in PTCs and by point mutations in sporadic and familial (MEN2) medullary thyroid carcinomas. Thus, RET kinase inhibitors might be beneficial in the treatment of these tumors, especially medullary thyroid carcinoma, which responds poorly to conventional chemotherapeutics (29) .
ZD6474 is a novel inhibitor of KDR with potent antiangiogenetic effects. Chronic oral administration of ZD6474 produces significant, broad-spectrum antitumor activity in different human tumor xenografts. Importantly, ZD6474 is active p.o. and has pharmacokinetics that are compatible with once-daily oral administration. An ongoing Phase I trial of ZD6474 in patients with solid tumors has revealed that the drug is well tolerated at doses of up to 300 mg per day (15) .
This study shows that ZD6474 is a potent (IC50 of 100 nM) inhibitor of RET oncoproteins. In our experience, RET enzymatic activity, with the agents tried until now, is refractory to the effects of many kinase inhibitors. In a previous screening of compounds of different chemical classes, we found that only the pyrazolo-pyrimidine PP1 and two related compounds were efficient inhibitors of RET (23) . We also found that potent kinase inhibitors such as several tyrphostins (23) and ZD1839 (this report) do not affect RET. ZD6474-mediated block of RET enzymatic activity resulted in the inhibition of the signaling and transforming capacity of RET oncoproteins. Furthermore, ZD6474 exerted a powerful growth-inhibitory effect on thyroid carcinoma cell lines that spontaneously harbor RET/PTC rearrangements. Because, these thyroid carcinoma cells do not express detectable levels of KDR (not shown), we think that ZD6474 effects are mediated by RET inhibition and that the possibility that they are also mediated by KDR inhibition can be excluded. ZD6474 prevented the growth of NIH-RET/PTC3 xenografts. It also had inhibitory effects, albeit less strong, on NIH-RAS tumors. These last effects are likely attributable to the antiangiogenic properties of ZD6474.
Treating both cancer and endothelial cells in a tumor has the potential to be more effective than treating cancer cells alone. Tumor cells often are selected to bypass the effects of antineoplastic agents, and the occurrence of therapy-resistant clones is frequently the reason for treatment failure. A possible advantage of ZD6474 in RET-associated tumors is that it has the potential to act as both an antiangiogenetic and an antineoplastic drug. The simultaneous assault on both neoplastic and endothelial cells may offer a mechanism to circumvent the development of resistance. We have recently shown that PP1 is a powerful inhibitor of RET-derived oncoproteins with an IC50 (80 nM) similar to that for ZD6474. Nevertheless, ZD6474 has several possible advantages in the treatment of RET-related tumors, including antiangiogenetic effects, low toxicity, and the possibility of oral administration. On the other hand, PP1 and ZD6474 may be used alternately to treat RET-associated cancers. Such a strategy may further help to prevent the development of treatment resistance.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), by the Ministero per l’Istruzione, Università e Ricerca Scientifica (MIUR), and by the Ministero della Salute. 
2 To whom requests for reprints should be addressed, at Istituto di Endocrinologia ed Oncologia Sperimentale del CNR, Facoltà di Medicina e Chirurgia, via. S. Pansini 5, 80131 Naples, Italy. Phone: 0039-081-7463056; Fax: 0039-081-7463037; E-mail: masantor{at}unina.it
3 The abbreviations used are: PTC, papillary thyroid carcinoma; MEN2, multiple endocrine neoplasia type 2 (syndrome); EGF, epidermal growth factor; EGFR, EGF receptor; VEGF, vascular endothelial growth factor; MAPK, mitogen-activated protein kinase; GST, glutathione-S-transferase; poly-GT, poly(L-glutamic acid-L-tyrosine); ERK, extracellular signal-regulated kinase.
Received 5/23/02. Accepted 10/10/02.
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A. N. Cranston, C. Carniti, K. Oakhill, E. Radzio-Andzelm, E. A. Stone, A. S. McCallion, S. Hodgson, S. Clarke, P. Mondellini, J. Leyland, et al. RET Is Constitutively Activated by Novel Tandem Mutations that Alter the Active Site Resulting in Multiple Endocrine Neoplasia Type 2B. Cancer Res., October 15, 2006; 66(20): 10179 - 10187. [Abstract] [Full Text] [PDF]
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D. W. Kim, Y. S. Jo, H. S. Jung, H. K. Chung, J. H. Song, K. C. Park, S. H. Park, J. H. Hwang, S. Y. Rha, G. R. Kweon, et al. An Orally Administered Multitarget Tyrosine Kinase Inhibitor, SU11248, Is a Novel Potent Inhibitor of Thyroid Oncogenic RET/Papillary Thyroid Cancer Kinases J. Clin. Endocrinol. Metab., October 1, 2006; 91(10): 4070 - 4076. [Abstract] [Full Text] [PDF]
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Z. Zhu, R. Ciampi, M. N. Nikiforova, M. Gandhi, and Y. E. Nikiforov Prevalence of RET/PTC Rearrangements in Thyroid Papillary Carcinomas: Effects of the Detection Methods and Genetic Heterogeneity J. Clin. Endocrinol. Metab., September 1, 2006; 91(9): 3603 - 3610. [Abstract] [Full Text] [PDF]
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 A. Cranston, C. Carniti, S. Martin, P. Mondellini, Y. Hooks, J. Leyland, S. Hodgson, S. Clarke, M. Pierotti, B. A. J. Ponder, et al. A Novel Activating Mutation in the RET Tyrosine Kinase Domain Mediates Neoplastic Transformation Mol. Endocrinol., July 1, 2006; 20(7): 1633 - 1643. [Abstract] [Full Text] [PDF]
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T. Troiani, O. Lockerbie, M. Morrow, F. Ciardiello, and S. G. Eckhardt Sequence-dependent inhibition of human colon cancer cell growth and of prosurvival pathways by oxaliplatin in combination with ZD6474 (Zactima), an inhibitor of VEGFR and EGFR tyrosine kinases. Mol. Cancer Ther., July 1, 2006; 5(7): 1883 - 1894. [Abstract] [Full Text] [PDF]
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A. Morabito, E. De Maio, M. Di Maio, N. Normanno, and F. Perrone Tyrosine Kinase Inhibitors of Vascular Endothelial Growth Factor Receptors in Clinical Trials: Current Status and Future Directions Oncologist, July 1, 2006; 11(7): 753 - 764. [Abstract] [Full Text] [PDF]
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B. Ouyang, J. A. Knauf, E. P. Smith, L. Zhang, T. Ramsey, N. Yusuff, D. Batt, and J. A. Fagin Inhibitors of Raf Kinase Activity Block Growth of Thyroid Cancer Cells with RET/PTC or BRAF Mutations In vitro and In vivo. Clin. Cancer Res., March 15, 2006; 12(6): 1785 - 1793. [Abstract] [Full Text] [PDF]
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 F. Carlomagno, S. Anaganti, T. Guida, G. Salvatore, G. Troncone, S. M. Wilhelm, and M. Santoro BAY 43-9006 inhibition of oncogenic RET mutants. J Natl Cancer Inst, March 1, 2006; 98(5): 326 - 334. [Abstract] [Full Text] [PDF]
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P. Maione, C. Gridelli, T. Troiani, and F. Ciardiello Combining Targeted Therapies and Drugs with Multiple Targets in the Treatment of NSCLC. Oncologist, March 1, 2006; 11(3): 274 - 284. [Abstract] [Full Text] [PDF]
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C. J. Strock, J.-I. Park, D. M. Rosen, B. Ruggeri, S. R. Denmeade, D. W. Ball, and B. D. Nelkin Activity of Irinotecan and the Tyrosine Kinase Inhibitor CEP-751 in Medullary Thyroid Cancer J. Clin. Endocrinol. Metab., January 1, 2006; 91(1): 79 - 84. [Abstract] [Full Text] [PDF]
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S. Yano, H. Muguruma, Y. Matsumori, H. Goto, E. Nakataki, N. Edakuni, H. Tomimoto, S. Kakiuchi, A. Yamamoto, H. Uehara, et al. Antitumor Vascular Strategy for Controlling Experimental Metastatic Spread of Human Small-Cell Lung Cancer Cells with ZD6474 in Natural Killer Cell-Depleted Severe Combined Immunodeficient Mice Clin. Cancer Res., December 15, 2005; 11(24): 8789 - 8798. [Abstract] [Full Text] [PDF]
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 J. M. Vieira, S. C R. Santos, C. Espadinha, I. Correia, T. Vag, C. Casalou, B. M. Cavaco, A. L. Catarino, S. Dias, and V. Leite Expression of vascular endothelial growth factor (VEGF) and its receptors in thyroid carcinomas of follicular origin: a potential autocrine loop Eur. J. Endocrinol., November 1, 2005; 153(5): 701 - 709. [Abstract] [Full Text] [PDF]
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M. Vidal, S. Wells, A. Ryan, and R. Cagan ZD6474 Suppresses Oncogenic RET Isoforms in a Drosophila Model for Type 2 Multiple Endocrine Neoplasia Syndromes and Papillary Thyroid Carcinoma Cancer Res., May 1, 2005; 65(9): 3538 - 3541. [Abstract] [Full Text] [PDF]
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 X.-F. Zhu, B.-F. Xie, J.-M. Zhou, G.-K. Feng, Z.-C. Liu, X.-Y. Wei, F.-X. Zhang, M.-F. Liu, and Y.-X. Zeng Blockade of Vascular Endothelial Growth Factor Receptor Signal Pathway and Antitumor Activity of ON-III (2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone), a Component from Chinese Herbal Medicine Mol. Pharmacol., May 1, 2005; 67(5): 1444 - 1450. [Abstract] [Full Text] [PDF]
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M. Santoro, R. M. Melillo, F. Carlomagno, G. Vecchio, and A. Fusco Minireview: RET: Normal and Abnormal Functions Endocrinology, December 1, 2004; 145(12): 5448 - 5451. [Abstract] [Full Text] [PDF]
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 J A Fagin How thyroid tumors start and why it matters: kinase mutants as targets for solid cancer pharmacotherapy J. Endocrinol., November 1, 2004; 183(2): 249 - 256. [Abstract] [Full Text] [PDF]
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J. A. Fagin Challenging Dogma in Thyroid Cancer Molecular Genetics--Role of RET/PTC and BRAF in Tumor Initiation J. Clin. Endocrinol. Metab., September 1, 2004; 89(9): 4264 - 4266. [Full Text] [PDF]
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 R. M. Melillo, A. M. Cirafici, V. De Falco, M. Bellantoni, G. Chiappetta, A. Fusco, F. Carlomagno, A. Picascia, D. Tramontano, G. Tallini, et al. The Oncogenic Activity of RET Point Mutants for Follicular Thyroid Cells May Account for the Occurrence of Papillary Thyroid Carcinoma in Patients Affected by Familial Medullary Thyroid Carcinoma Am. J. Pathol., August 1, 2004; 165(2): 511 - 521. [Abstract] [Full Text] [PDF]
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S. A. Wells and J. R. Nevins Evolving Strategies for Targeted Cancer Therapy--Past, Present, and Future J Natl Cancer Inst, July 7, 2004; 96(13): 980 - 981. [Full Text] [PDF]
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G. Cuccuru, C. Lanzi, G. Cassinelli, G. Pratesi, M. Tortoreto, G. Petrangolini, E. Seregni, A. Martinetti, D. Laccabue, C. Zanchi, et al. Cellular Effects and Antitumor Activity of RET Inhibitor RPI-1 on MEN2A-Associated Medullary Thyroid Carcinoma J Natl Cancer Inst, July 7, 2004; 96(13): 1006 - 1014. [Abstract] [Full Text] [PDF]
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G. A. Kaltsas, G. M. Besser, and A. B. Grossman The Diagnosis and Medical Management of Advanced Neuroendocrine Tumors Endocr. Rev., June 1, 2004; 25(3): 458 - 511. [Abstract] [Full Text] [PDF]
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M. Braga-Basaria, E. Hardy, R. Gottfried, K. D. Burman, M. Saji, and M. D. Ringel 17-Allylamino-17-Demethoxygeldanamycin Activity against Thyroid Cancer Cell Lines Correlates with Heat Shock Protein 90 Levels J. Clin. Endocrinol. Metab., June 1, 2004; 89(6): 2982 - 2988. [Abstract] [Full Text] [PDF]
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D. Vitagliano, F. Carlomagno, M. L. Motti, G. Viglietto, Y. E. Nikiforov, M. N. Nikiforova, J. M. Hershman, A. J. Ryan, A. Fusco, R. M. Melillo, et al. Regulation of p27Kip1 Protein Levels Contributes to Mitogenic Effects of the RET/PTC Kinase in Thyroid Carcinoma Cells Cancer Res., June 1, 2004; 64(11): 3823 - 3829. [Abstract] [Full Text] [PDF]
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H. Namba, M. Nakashima, T. Hayashi, N. Hayashida, S. Maeda, T. I. Rogounovitch, A. Ohtsuru, V. A. Saenko, T. Kanematsu, and S. Yamashita Clinical Implication of Hot Spot BRAF Mutation, V599E, in Papillary Thyroid Cancers J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4393 - 4397. [Abstract] [Full Text] [PDF]
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C. J. Strock, J.-I. Park, M. Rosen, C. Dionne, B. Ruggeri, S. Jones-Bolin, S. R. Denmeade, D. W. Ball, and B. D. Nelkin CEP-701 and CEP-751 Inhibit Constitutively Activated RET Tyrosine Kinase Activity and Block Medullary Thyroid Carcinoma Cell Growth Cancer Res., September 1, 2003; 63(17): 5559 - 5563. [Abstract] [Full Text] [PDF]
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M. Braga-Basaria and M. D. Ringel Beyond Radioiodine: A Review of Potential New Therapeutic Approaches for Thyroid Cancer J. Clin. Endocrinol. Metab., May 1, 2003; 88(5): 1947 - 1960. [Abstract] [Full Text] [PDF]
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