Biomedical subjects

Francesca Carlomagno

Publications and source records attributed to Francesca Carlomagno.

At least 19 recordsLinked to original sources

PubMed2006

The receptor-type protein tyrosine phosphatase J antagonizes the biochemical and biological effects of RET-derived oncoproteins.

Thyroid cancer is frequently associated with the oncogenic conversion of the RET receptor tyrosine kinase. RET gene rearrangements, which lead to the generation of chimeric RET/papillary thyroid carcinoma (PTC) oncogenes, occur in PTC, whereas RET point mutations occur in familial multiple endocrine neoplasia type 2 (MEN2) and sporadic medullary thyroid carcinomas (MTC). We showed previously that the expression of the receptor-type protein tyrosine phosphatase J (PTPRJ) is suppressed in neoplastically transformed follicular thyroid cells. We now report that PTPRJ coimmunoprecipitates with wild-type RET and with the MEN2A-associated RET(C634R) oncoprotein but not with the RET/PTC1 and RET-MEN2B isoforms. Using mutated forms of PTPRJ and RET-MEN2A, we show that the integrity of the respective catalytic domains is required for the PTPRJ/RET-MEN2A interaction. PTPRJ expression induces dephosphorylation of the RET(C634R) and, probably via an indirect mechanism, RET/PTC1 oncoproteins on two key RET autophosphorylation sites (Tyr1062 and Tyr905). This results in a significant decrease of RET-induced Shc and extracellular signal-regulated kinase 1/2 phosphorylation levels. In line with this finding, adoptive PTPRJ expression reduced the oncogenic activity of RET(C634R) in an in vitro focus formation assay of NIH3T3 cells. As expected from the coimmunoprecipitation results, the RET(M918T) oncoprotein, which is associated to MEN2B and sporadic MTC, was resistant to the dephosphorylating activity of PTPRJ. Taken together, these findings identify RET as a novel substrate of PTPRJ and suggest that PTPRJ expression levels may affect tumor phenotype associated with RET/PTC1 and RET(C634R) mutants. On the other hand, resistance to PTPRJ may be part of the mechanism of RET oncogenic conversion secondary to the M918T mutation.

Cell Line, Tumor↗

PubMed2006

BAY 43-9006 inhibition of oncogenic RET mutants.

BACKGROUND: Medullary and papillary thyroid carcinomas are often associated with oncogenic activation of the RET tyrosine kinase. We evaluated whether the biaryl urea BAY 43-9006, which is known to inhibit several other tyrosine kinases, blocks RET kinase function and oncogenic activity. METHODS: We examined BAY 43-9006 activity against oncogenic RET in vitro and in cellular RET signaling in oncogenic RET-transfected NIH3T3 fibroblasts by using immunocomplex kinase assays and immunoblotting with phospho-specific antibodies. The effects of BAY 43-9006 on proliferation of human TPC1 and TT thyroid carcinoma cells, which harbor spontaneous oncogenic RET alleles, and on RAT1 fibroblasts transformed with oncogenic RET mutants, including mutants that are resistant to other chemotherapeutic agents, were determined using growth curves and flow cytometry. Growth of TT cell-derived xenograft tumors in athymic mice treated orally with BAY 43-9006 or with vehicle was measured. All statistical tests were two-sided. RESULTS: BAY 43-9006 inhibited oncogenic RET kinase activity at half-maximal inhibitory concentrations (IC50s) of 50 nM or less in NIH3T3 cells. It also arrested the growth of NIH3T3 and RAT1 fibroblasts transformed by oncogenic RET and of thyroid carcinoma cells that harbor spontaneous oncogenic RET alleles. Moreover, BAY 43-9006 inhibited the growth of cells carrying RET V804L (IC50 = 110 nM, 95% confidence interval [CI] = 88 to 133 nM) or RET V804M (IC50 = 147 nM, 95% CI = 123 nM to 170 nM), both mutants that are resistant to anilinoquinazolines and pyrazolopyrimidines. After 3 weeks of oral treatment with BAY 43-9006 (60 mg/kg/day), the volume of TT cell xenografts (n = 7) was reduced from 72.5 to 44 mm3 (difference = 28.5 mm3, 95% CI = 7 mm3 to 50 mm3), whereas in vehicle-treated mice (n = 7), mean tumor volume increased to 408 mm3 (difference = 320 mm3, 95% CI = 180 mm3 to 460 mm3; untreated versus treated, P =.02). This inhibition paralleled a decrease in RET phosphorylation. CONCLUSIONS: BAY 43-9006 is a powerful inhibitor of the RET kinase. Its potential as a therapeutic tool for RET-positive thyroid tumors, including those expressing V804 mutations merits study.

Administration, Oral↗

PubMed2006

BRAF is a therapeutic target in aggressive thyroid carcinoma.

PURPOSE: Oncogenic conversion of BRAF occurs in approximately 44% of papillary thyroid carcinomas and 24% of anaplastic thyroid carcinomas. In papillary thyroid carcinomas, this mutation is associated with an unfavorable clinicopathologic outcome. Our aim was to exploit BRAF as a potential therapeutic target for thyroid carcinoma. EXPERIMENTAL DESIGN: We used RNA interference to evaluate the effect of BRAF knockdown in the human anaplastic thyroid carcinoma cell lines FRO and ARO carrying the BRAF V600E (V600EBRAF) mutation. We also exploited the effect of BAY 43-9006 [N-(3-trifluoromethyl-4-chlorophenyl)-N'-(4-(2-methylcarbamoyl pyridin-4-yl)oxyphenyl)urea], a multikinase inhibitor able to inhibit RAF family kinases in a panel of six (V600E)BRAF-positive thyroid carcinoma cell lines and in nude mice bearing ARO cell xenografts. Statistical tests were two sided. RESULTS: Knockdown of BRAF by small inhibitory duplex RNA, but not control small inhibitory duplex RNA, inhibited the mitogen-activated protein kinase signaling cascade and the growth of ARO and FRO cells (P < 0.0001). These effects were mimicked by thyroid carcinoma cell treatment with BAY 43-9006 (IC50 = 0.5-1 micromol/L; P < 0.0001), whereas the compound had negligible effects in normal thyrocytes. ARO cell tumor xenografts were significantly (P < 0.0001) smaller in nude mice treated with BAY 43-9006 than in control mice. This inhibition was associated with suppression of phospho-mitogen-activated protein kinase levels. CONCLUSIONS: BRAF provides signals crucial for proliferation of thyroid carcinoma cells spontaneously harboring the (V600E)BRAF mutation and, therefore, BRAF suppression might have therapeutic potential in (V600E)BRAF-positive thyroid cancer.

Animals↗

PubMed2006

Activation of the Erk8 mitogen-activated protein (MAP) kinase by RET/PTC3, a constitutively active form of the RET proto-oncogene.

Mitogen-activated protein (MAP) kinases have a central role in several biological functions, including cell adhesion and spreading, chemotaxis, cell cycle progression, differentiation, and apoptosis. Extracellular signal-regulated kinase 8 (Erk8) is a large MAP kinase whose activity is controlled by serum and the c-Src non-receptor tyrosine kinase. Here, we show that RET/PTC3, an activated form of the RET proto-oncogene, was able to activate Erk8, and we demonstrate that such MAP kinase participated in RET/PTC3-dependent stimulation of the c-jun promoter. By using RET/PTC3 molecules mutated in specific tyrosine autophosphorylation sites, we characterized Tyr(981), a known binding site for c-Src, as a major determinant of RET/PTC3-induced Erk8 activation, although, surprisingly, the underlying mechanism did not strictly depend on the activity of Src. In contrast, we present evidence that RET/PTC3 acts on Erk8 through Tyr(981)-mediated activation of c-Abl. Furthermore, we localized the region responsible for the modulation of Erk8 activity by the RET/PTC3 and Abl oncogenes in the Erk8 C-terminal domain. Altogether, these results support a role for Erk8 as a novel effector of RET/PTC3 and, therefore, RET biological functions.

Amino Acid Sequence↗

PubMed2006

Drug insight: Small-molecule inhibitors of protein kinases in the treatment of thyroid cancer.

Molecular targeting of protein kinases is a new paradigm in the treatment of cancer. The clinical efficacy of low-molecular weight inhibitors of ABL, stem-cell growth-factor receptor, and the epidermal growth factor receptor in different tumor types is witness to the power of this approach. The presence of activating mutations of a kinase, or an increased gene copy number, might anticipate tumor responsiveness to its targeting. Thyroid cancer is the most prevalent endocrine malignancy and is frequently associated with the oncogenic conversion of two specific protein kinases, RET and BRAF. Small-molecule inhibitors of both kinases have already reached the clinical testing stage. Protein kinases other than RET and BRAF are also being evaluated for their potential in thyroid-cancer treatment.

Adenocarcinoma, Follicular↗

PubMed2005

Clinical case seminar: in vivo and in vitro characterization of a novel germline RET mutation associated with low-penetrant nonaggressive familial medullary thyroid carcinoma.

CONTEXT: RET mutation analysis provides useful information on the clinical outcome of medullary thyroid carcinomas (MTCs) and the risk of disease in the family members. OBJECTIVE: The objective of this study was to document genotype-phenotype relationships in an Italian family with a novel RET mutation. DESIGN/SETTING: RET gene alterations were investigated in a patient with unifocal MTC and her relatives. The identified mutation was subjected to in vitro functional testing. PATIENTS: Patients included a female proband who developed MTC at age 60, her five children, and three grandchildren. MAIN OUTCOME MEASURES: DNA extracted from the blood and the proband's tumor were analyzed for RET alterations. The transforming potential and mitogenic properties of the identified mutation were investigated. RESULTS: A novel heterozygous germline RET mutation at codon 777 (AAC-->AGC, N-->S) (RET/N777S) was identified in the proband and three of her relatives. Two of the latter presented thyroid nodules, but none had MTC or C cell hyperplasia. The proband's MTC was characterized by late onset and limited aggressiveness, with no evidence of regional lymph node or distant metastases 10 yr after total thyroidectomy. This phenotype is consistent with the RET/N777S mutant's low-grade transforming potential and limited activation of RET tyrosine kinase. CONCLUSION: Our findings indicate that the newly identified RET/N777S mutation is a low-penetrant cause of MTC disease. This phenotype might be less aggressive than that associated with MEN2A of familial MTC, although close clinical follow-up of carriers is essential.

Carcinoma, Medullary↗

PubMed2005

Mitogenic effects of the up-regulation of minichromosome maintenance proteins in anaplastic thyroid carcinoma.

CONTEXT: Anaplastic thyroid carcinomas (ATC) are among the most aggressive human malignancies and are characterized by high mitotic activity. Minichromosome maintenance proteins (MCM) 2-7 are required to initiate eukaryotic DNA replication, and their overexpression has been associated with dysplasia and malignancy. OBJECTIVE: In an attempt to cast light on the mechanisms governing ATC, we evaluated MCM5 and MCM7 expression in human normal, papillary (PTC), and anaplastic thyroid samples, as well as in primary culture cells and transgenic mouse models. RESULTS: MCM5 and MCM7 expression was high in 65% of ATC and negligible in normal thyroid tissue and papillary thyroid carcinomas. In ATC, high MCM5 and MCM7 expression was paralleled by high levels of MCM2 and MCM6. An analysis of human ATC primary cell cultures and of a transgenic mouse model of ATC confirmed these findings. An increased transcription rate accounted for MCM7 up-regulation, because the activity of the MCM7 promoter was more than 10-fold higher in ATC cells compared with normal thyroid cells. Adoptive overexpression of wild-type p53, but not of its inactive (R248W and R273H) mutants, strongly down-regulated transcription from the MCM7 promoter, suggesting that p53 knock-out contributes to MCM7 up-regulation in ATC. Treatment with small inhibitory duplex RNAs, which decrease MCM7 protein levels, reduced the rate of DNA synthesis in ATC cells. CONCLUSION: MCM proteins are overexpressed in ATC and sustain the high proliferative capacity of ATC cells.

Animals↗

PubMed2005

Receptor tyrosine kinases as targets for anticancer therapeutics.

Oncogenic conversion of receptor protein tyrosine kinases (RTK) is a frequent feature of malignant cells. This knowledge has fostered efforts to develop target-specific low molecular weight therapeutics able to obstruct RTK signalling. The clinical efficacy of the ABL- and KIT-inhibitors are paradigmatic of the power of this approach. Here, we focus on small-molecule inhibitors for RTKs involved in human cancer. In particular, we examine the KIT, MET and RET receptors that are targeted by genetic alterations in both sporadic and familial human tumours.

Antineoplastic Agents↗

PubMed2004

Minireview: RET: normal and abnormal functions.

The RET gene encodes a single-pass transmembrane receptor tyrosine kinase. RET is the oncogene that causes papillary thyroid carcinoma and medullary thyroid carcinoma. The latter may arise as a component of multiple endocrine neoplasia type 2 syndromes; germline mutations in RET are responsible for multiple endocrine neoplasia type 2 inheritance. In this report we review data on the mechanisms leading to RET oncogenic conversion and on RET targeting as a strategy in thyroid cancer treatment.

Gene Expression Regulation, Neoplastic↗

PubMed2004

Disease associated mutations at valine 804 in the RET receptor tyrosine kinase confer resistance to selective kinase inhibitors.

We have recently demonstrated that the pyrazolopyrimidines PP1 and PP2 and the 4-anilinoquinazoline ZD6474 display a strong inhibitory activity (IC(50)< or =100 nM) towards constitutively active oncogenic RET kinases. Here, we show that most oncogenic MEN2-associated RET kinase mutants are highly susceptible to PP1, PP2 and ZD6474 inhibition. In contrast, MEN2-associated swap of bulky hydrophobic leucine or methionine residues for valine 804 in the RET kinase domain causes resistance to the three compounds. Substitution of valine 804 with the small amino- acid glycine renders the RET kinase even more susceptible to inhibition (ZD6474 IC(50): 20 nM) than the wild-type kinase. Our data identify valine 804 of RET as a structural determinant mediating resistance to pyrazolopyrimidines and 4-anilinoquinazolines.

Carcinoma, Medullary↗

PubMed2004

Functional expression of the CXCR4 chemokine receptor is induced by RET/PTC oncogenes and is a common event in human papillary thyroid carcinomas.

To identify genes involved in the transformation of thyroid follicular cells, we explored, using DNA oligonucleotide microarrays, the transcriptional response of PC Cl3 rat thyroid epithelial cells to the ectopic expression of the RET/PTC oncogenes. We found that RET/PTC was able to induce the expression of CXCR4, the receptor for the chemokine CXCL12/SDF-1alpha/beta. We observed that CXCR4 expression correlated with the transforming ability of the oncoprotein and depended on the integrity of the RET/PTC-RAS/ERK signaling pathway. We found that CXCR4 was expressed in RET/PTC-positive human thyroid cancer cell lines, but not in normal thyroid cells. Furthermore, we found CXCR4 expression in human thyroid carcinomas, but not in normal thyroid samples by immunohistochemistry. Since CXCR4 has been recently implicated in tumor proliferation, motility and invasiveness, we asked whether treatment with SDF-1alpha was able to induce a biological response in thyroid cells. We observed that SDF-1alpha induced S-phase entry and survival of thyroid cells. Invasion through a reconstituted extracellular matrix was also supported by SDF-1alpha and inhibited by a blocking antibody to CXCR4. Taken together, these results suggest that human thyroid cancers bearing RET/PTC rearrangements may use the CXCR4/SDF-1alpha receptor-ligand pathway to proliferate, survive and migrate.

Carcinoma, Papillary↗

PubMed2004

Regulation of p27Kip1 protein levels contributes to mitogenic effects of the RET/PTC kinase in thyroid carcinoma cells.

We show that treatment of a panel of thyroid carcinoma cell lines naturally harboring the RET/PTC1 oncogene, with the RET kinase inhibitors PP1 and ZD6474, results in reversible G(1) arrest. This is accompanied by interruption of Shc and mitogen-activated protein kinase (MAPK) phosphorylation, reduced levels of G(1) cyclins, and increased levels of the cyclin-dependent kinase inhibitor p27Kip1 because of a reduced protein turnover. MAP/extracellular signal-regulated kinase 1/2 inhibition by U0126 caused G(1) cyclins down-regulation and p27Kip1 up-regulation as well. Forced expression of RET/PTC in normal thyroid follicular cells caused a MAPK- and proteasome-dependent down-regulation of p27Kip1. Reduction of p27Kip1 protein levels by antisense oligonucleotides abrogated the G(1) arrest induced by RET/PTC blockade. Therefore, in thyroid cancer, RET/PTC-mediated MAPK activation contributes to p27Kip1 deregulation. This pathway is implicated in cell cycle progression and in response to small molecule kinase inhibitors.

Cell Cycle Proteins↗

PubMed2004

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.

Activating germ-line point mutations in the RET receptor are responsible for multiple endocrine neoplasia type 2-associated medullary thyroid carcinoma (MTC), whereas somatic RET rearrangements are prevalent in papillary thyroid carcinomas (PTCs). Some rare kindreds, carrying point mutations in RET, are affected by both cancer types, suggesting that, under specific circumstances, point mutations in RET can drive the generation of PTC. Here we describe a family whose siblings, affected by both PTC and MTC, carried a germ-line point mutation in the RET extracellular domain, converting cysteine 634 into serine. We tested on thyroid follicular cells the transforming activity of RET(C634S), RET(K603Q), another mutant identified in a kindred with both PTC and MTC, RET(C634R) a commonly isolated allele in MEN2A, RET(M918T) responsible for MEN2B and also identified in kindreds with both PTC and MTC, and RET/PTC1 the rearranged oncogene that characterizes bona fide PTC in patients without MTC. We show that the various RET point mutants, but not wild-type RET, scored constitutive kinase activity and exerted mitogenic effects for thyroid PC Cl 3 cells, albeit at significantly lower levels compared to RET/PTC1. The low mitogenic activity of RET point mutants paralleled their reduced kinase activity compared to RET/PTC. Furthermore, RET point mutants maintained a protein domain, the intracellular juxtamembrane domain, that exerted negative effects on the mitogenic activity. In conclusion, RET point mutants can behave as dominant oncogenes for thyroid follicular cells. Their transforming activity, however, is rather modest, providing a possible explanation for the rare association of MTC with PTC.

Adenocarcinoma, Follicular↗

PubMed2003

Protein kinase Calpha activation by RET: evidence for a negative feedback mechanism controlling RET tyrosine kinase.

We have studied the role of protein kinase C (PKC) in signaling of the RET tyrosine kinase receptor. By using a chimeric receptor (E/R) in which RET kinase can be tightly controlled by the addition of epidermal growth factor (EGF), we have found that RET triggering induces a strong increase of PKCalpha, PKCdelta and PKCzeta activity and that PKCalpha, not PKCdelta and PKCzeta, forms a ligand-dependent protein complex with E/R. We have identified tyrosine 1062 in the RET carboxyl-terminal tail as the docking site for PKCalpha. Block of PKC activity by bisindolylmaleimide or chronic phorbol esters treatment decreased EGF-induced serine/threonine phosphorylation of E/R, while it caused a similarly sized increase of EGF-induced E/R tyrosine kinase activity and mitogenic signaling. Conversely, acute phorbol esters treatment, which promotes PKC activity, increased the levels of E/R serine/threonine phosphorylation and significantly decreased its phosphotyrosine content. A threefold reduction of tyrosine phosphorylation levels of the constitutively active RET/MEN2A oncoprotein was observed upon coexpression with PKCalpha. We conclude that RET binds to and activates PKCalpha. PKCalpha, in turn, causes RET phosphorylation and downregulates RET tyrosine kinase and downstream signaling, thus functioning as a negative feedback loop to modulate RET activity.

3T3 Cells↗

PubMed2003

Cloning and molecular characterization of a novel gene strongly induced by the adenovirus E1A gene in rat thyroid cells.

Expression of the adenovirus E1A gene in the rat thyroid differentiated cell line PC Cl 3 induces thyrotropin-independent cell growth and impairs differentiation. However, the malignant phenotype is achieved only when the PC E1A cells are infected with other murine retroviruses carrying the v-abl, v-raf or polyoma middle-T genes. To determine through which genes E1A affects thyroid cells, we differentially screened PC Cl 3 and PC E1A cells. Here we report a new gene, named CL2, that is upregulated in PC E1A cells. The CL2 transcript is 4.4 kb long and encodes a 949 amino-acid protein. Conceptual translation of the open reading frame showed one product with a signal peptide, multiple nuclear localization signals and three newly described domains. Furthermore, in vivo, this protein was located juxtanuclear, which is suggestive of Golgian localization, and also in cytoplasm and nucleus/nucleolus. Finally, CL2 gene expression was drastically downregulated in human thyroid neoplastic cell lines and tissues.

Adenovirus E1A Proteins↗

PubMed2003

Efficient inhibition of RET/papillary thyroid carcinoma oncogenic kinases by 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2).

Inappropriate activation of the RET receptor tyrosine kinase causes development of papillary and medullary thyroid cancer. We have previously shown that pyrazolopyrimidine is a potent inhibitor of the RET kinase. Here, we show that 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) (PP2), another pyrazolopyrimidine, blocks the enzymatic activity of the isolated RET kinase and RET/PTC1 oncoprotein at IC(50) in the nanomolar range. PP2 blocked in vivo phosphorylation and signaling of the RET/PTC1 oncoprotein. PP2 prevented serum-independent growth of RET/PTC1-transformed NIH3T3 fibroblasts and of TPC1 and FB2, two human papillary thyroid carcinoma cell lines that carry spontaneous RET/PTC1 rearrangements. Finally, PP2 blocked invasion of type I collagen matrix by TPC1 cells. Thus, pyrazolopirimidines hold promise for the treatment of human cancers sustaining oncogenic activation of RET.

3T3 Cells↗

PubMed2003

Antitumor effects of ZD6474, a small molecule vascular endothelial growth factor receptor tyrosine kinase inhibitor, with additional activity against epidermal growth factor receptor tyrosine kinase.

PURPOSE: Vascular endothelial growth factor (VEGF) is a major mitogen for endothelial cells and enhances vascular permeability. Enhanced VEGF secretion is found in human cancers and correlates with increased tumor neovascularization. ZD6474 is a p.o. bioavailable, VEGF flk-1/KDR receptor (VEGFR-2) tyrosine kinase inhibitor with antitumor activity in many human cancer xenografts and is currently in Phase I clinical development. EXPERIMENTAL DESIGN: We tested the effects of ZD6474 on EGFR phosphorylation in cell expressing functional epidermal growth factor receptor (EGFR) and the antiproliferative and the proapoptotic activity of ZD6474 alone or in combination taxanes in human cancer cell lines with functional EGFR but lacking VEGFR-2. The antitumor activity of this drug was also tested in nude mice bearing established GEO colon cancer xenografts. RESULTS: ZD6474 causes a dose-dependent inhibition of EGFR phosphorylation in mouse NIH-EGFR fibroblasts and human MCF-10A ras breast cancer cells, two cell lines that overexpress the human EGFR. ZD6474 treatment resulted in a dose-dependent inhibition of soft agar growth in seven human cell lines (breast, colon, gastric, and ovarian) with functional EGFR but lacking VEGFR-2. A dose-dependent supra-additive effect in growth inhibition and in apoptosis in vitro was observed by the combined treatment with ZD6474 and paclitaxel or docetaxel. ZD6474 treatment of nude mice bearing palpable GEO colon cancer xenografts (which are sensitive to inhibition of EGFR signaling) induced dose-dependent tumor growth inhibition. Immunohistochemical analysis revealed a significant dose-dependent reduction of neoangiogenesis. The antitumor activity of ZD6474 in GEO tumor xenografts was also found to be enhanced when combined with paclitaxel. Tumor regression was observed in all mice after treatment with ZD6474 plus paclitaxel, and it was accompanied by a significant potentiation in inhibition of angiogenesis. Six of 20 mice had no histological evidence of tumors after treatment with ZD6474 plus paclitaxel. CONCLUSIONS: This study suggests that in addition to inhibiting endothelial cell proliferation by blocking VEGF-induced signaling, ZD6474 may also be able to inhibit cancer cell growth by blocking EGFR autocrine signaling. These results provide also a rationale for the clinical evaluation of ZD6474 combined with taxanes in cancer patients.

Agar↗

PubMed2002

ZD6474, an orally available inhibitor of KDR tyrosine kinase activity, efficiently blocks oncogenic RET kinases.

RET/papillary thyroid carcinoma (PTC) oncogenes, generated by recombination of the tyrosine kinase-encoding domain of RET with different heterologous genes, are prevalent in papillary carcinomas of the thyroid. Point mutations of RET cause multiple endocrine neoplasia type 2 (MEN2) familial cancer syndrome and are found in sporadic medullary thyroid carcinomas. Here, we show that ZD6474, a low molecular weight tyrosine kinase inhibitor, blocks the enzymatic activity of RET-derived oncoproteins at a one-half maximal inhibitory concentration of 100 nM. ZD6474 blocked in vivo phosphorylation and signaling of the RET/PTC3 and RET/MEN2B oncoproteins and of an epidermal growth factor (EGF)-activated EGF-receptor/RET chimeric receptor. RET/PTC3-transformed cells-treated ZD6474 lost proliferative autonomy and showed morphological reversion. ZD6474 prevented the growth of two human PTC cell lines that carry spontaneous RET/PTC1 rearrangements. Finally, it blocked anchorage-independent growth of RET/PTC3-transformed NIH3T3 fibroblasts and the formation of tumors after injection of NIH-RET/PTC3 cells into nude mice. Thus, targeting RET oncogenes with ZD6474 might offer a potential treatment strategy for carcinomas sustaining oncogenic activation of RET.

3T3 Cells↗