ZD6474 (vandetanib, Zactima, Astra Zeneca) is an anilinoquinazoline used to target the receptor tyrosine kinase RET in familial and sporadic thyroid carcinoma (IC50: 100 nM). The aim of this study was to identify molecular determinants of RET sensitivity to ZD6474. Here, we show that mutation of RET tyrosine 806 to cysteine (Y806C) induced RET kinase resistance to ZD6474 (IC50: 933 nM). Y806 maps close to the gate-keeper position at the RET kinase nucleotide-binding pocket. Although tyrosine 806 is a RET auto-phosphorylation site, its substitution to phenylalanine (Y806F) did not markedly affect RET susceptibility to ZD6474 (IC50: 87 nM), suggesting that phosphorylation of Y806 is not required for compound binding. Accordingly, the introduction of a phosphomimetic residue (Y806E) also caused resistance to ZD6474, albeit of a lesser degree (IC50: 512 nM) than the cysteine mutation. Y806C/E RET mutants were also resistant to ZD6474 with respect to intracellular signalling and activation of an AP1-responsive promoter. We conclude that Y806 is a molecular determinant of RET sensitivity to ZD6474. Y806C is a natural RET mutation identified in a patient affected by multiple endocrine neoplasia type 2B. Based on its rare occurrence, it is unlikely that Y806C will be a frequent cause of refractoriness to ZD6474; however, it may be envisaged that mutations at this site can mediate secondary resistance formation in patients treated with the compound.
Francesca Carlomagno, Teresa Guida, Suresh Anaganti, Livia Provitera, Svend Kjaer, Neil Q McDonald, Anderson J Ryan and Massimo Santoro
Maria Rosaria Rusciano, Marcella Salzano, Sara Monaco, Maria Rosaria Sapio, Maddalena Illario, Valentina De Falco, Massimo Santoro, Pietro Campiglia, Lucio Pastore, Gianfranco Fenzi, Guido Rossi and Mario Vitale
RET/papillary thyroid carcinoma (PTC), TRK-T, or activating mutations of Ras and BRaf are frequent genetic alterations in PTC, all leading to the activation of the extracellular-regulated kinase (Erk) cascade. The aim of this study was to investigate the role of calmodulin-dependent kinase II (CaMKII) in the signal transduction leading to Erk activation in PTC cells. In normal thyroid cells, CaMKII and Erk were in the inactive form in the absence of stimulation. In primary PTC cultures and in PTC cell lines harboring the oncogenes RET/PTC-1 or BRafV600E, CaMKII was active also in the absence of any stimulation. Inhibition of calmodulin or phospholipase C (PLC) attenuated the level of CaMKII activation. Expression of recombinant RET/PTC-3, BRafV600E, or RasV12 induced CaMKII activation. Inhibition of CaMKII attenuated Erk activation and DNA synthesis in thyroid papillary carcinoma (TPC-1), a cell line harboring RET/PTC-1, suggesting that CaMKII is a component of the Erk signal cascade in this cell line. In conclusion, PTCs contain an active PLC/Ca2+/calmodulin-dependent signal inducing constitutive activation of CaMKII. This kinase is activated by BRafV600E, oncogenic Ras, and by RET/PTC. CaMKII participates to the activation of the Erk pathway by oncogenic Ras and RET/PTC and contributes to their signal output, thus modulating tumor cell proliferation.
Donata Vitagliano, Valentina De Falco, Anna Tamburrino, Sabrina Coluzzi, Giancarlo Troncone, Gennaro Chiappetta, Fortunato Ciardiello, Giampaolo Tortora, James A Fagin, Anderson J Ryan, Francesca Carlomagno and Massimo Santoro
Oncogenic conversion of the RET tyrosine kinase is a frequent feature of medullary thyroid carcinoma (MTC). ZD6474 (vandetanib) is an ATP-competitive inhibitor of RET, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor receptors kinases. In this study, we have studied ZD6474 mechanism of action in TT and MZ-CRC-1 human MTC cell lines, carrying cysteine 634 to tryptophan (C634W) and methionine 918 to threonine (M918T) RET mutation respectively. ZD6474 blunted MTC cell proliferation and RET, Shc and p44/p42 mitogen-activated protein kinase (MAPK) phosphorylation. Single receptor knockdown by RNA interference showed that MTC cells depended on RET for proliferation. Adoptive expression of the ZD6474-resistant V804M RET mutant rescued proliferation of TT cells under ZD6474 treatment, showing that RET is a key ZD6474 target in these MTC cells. Upon RET inhibition, adoptive stimulation of EGFR partially rescued TT cell proliferation, MAPK signaling, and expression of cell-cycle-related genes. This suggests that simultaneous inhibition of RET and EGFR by ZD6474 may overcome the risk of MTC cells to escape from RET blockade through compensatory over-activation of EGFR.
Katrin-Janine Heiliger, Julia Hess, Donata Vitagliano, Paolo Salerno, Herbert Braselmann, Giuliana Salvatore, Clara Ugolini, Isolde Summerer, Tatjana Bogdanova, Kristian Unger, Gerry Thomas, Massimo Santoro and Horst Zitzelsberger
For an identification of novel candidate genes in thyroid tumourigenesis, we have investigated gene copy number changes in a Trk-T1 transgenic mouse model of thyroid neoplasia. For this aim, 30 thyroid tumours from Trk-T1 transgenics were investigated by comparative genomic hybridisation. Recurrent gene copy number alterations were identified and genes located in the altered chromosomal regions were analysed by Gene Ontology term enrichment analysis in order to reveal gene functions potentially associated with thyroid tumourigenesis. In thyroid neoplasms from Trk-T1 mice, a recurrent gain on chromosomal bands 1C4–E2.3 (10.0% of cases), and losses on 3H1–H3 (13.3%), 4D2.3–E2 (43.3%) and 14E4–E5 (6.7%) were identified. The genes Twist2, Ptma, Pde6d, Bmpr1b, Pdlim5, Unc5c, Srm, Trp73, Ythdf2, Taf12 and Slitrk5 are located in these chromosomal bands. Copy number changes of these genes were studied by fluorescence in situ hybridisation on 30 human papillary thyroid carcinoma (PTC) samples and altered gene expression was studied by qRT-PCR analyses in 67 human PTC. Copy number gains were detected in 83% of cases for TWIST2 and in 100% of cases for PTMA and PDE6D. DNA losses of SLITRK1 and SLITRK5 were observed in 21% of cases and of SLITRK6 in 16% of cases. Gene expression was significantly up-regulated for UNC5C and TP73 and significantly down-regulated for SLITRK5 in tumours compared with normal tissue. In conclusion, a global genomic copy number analysis of thyroid tumours from Trk-T1 transgenic mice revealed a number of novel gene alterations in thyroid tumourigenesis that are also prevalent in human PTCs.
Monica Fedele, Dario Palmieri, Gennaro Chiappetta, Rosa Pasquinelli, Ivana De Martino, Claudio Arra, Giuseppe Palma, Teresa Valentino, Giovanna M Pierantoni, Giuseppe Viglietto, Jay L Rothstein, Massimo Santoro and Alfredo Fusco
Impairment of the p27kip1 function, caused by a drastic reduction of its expression or cytoplasmic mislocalization, has been frequently observed in thyroid carcinomas. To understand the role of p27kip1 impairment in thyroid carcinogenesis, we investigated the consequences of the loss of p27kip1 expression in the context of a mouse modeling of papillary thyroid cancer, expressing the TRK-T1 oncogene under the transcriptional control of thyroglobulin promoter. We found that double mutant mice homozygous for a p27 kip1 null allele (TRK-T1/p27 −/−) display a higher incidence of papillary thyroid carcinomas, with a shorter latency period and increased proliferation index, compared with p27 kip1 wild-type compounds (TRK-T1/p27 +/+). Consistently, double mutant mice heterozygous for a p27 kip1 null allele (TRK-T1/p27 +/−) show an incidence of thyroid carcinomas that is intermediate between TRK-T1/p27 −/− and TRK-T1/p27 +/+ mice. Therefore, our findings suggest a dose-dependent role of p27 kip1 function in papillary thyroid cancer development.
Roberto Bellelli, Maria Domenica Castellone, Ginesa Garcia-Rostan, Clara Ugolini, Carmelo Nucera, Peter M Sadow, Tito Claudio Nappi, Paolo Salerno, Maria Carmela Cantisani, Fulvio Basolo, Tomas Alvarez Gago, Giuliana Salvatore and Massimo Santoro
Anaplastic thyroid carcinoma (ATC) is a very aggressive thyroid cancer. forkhead box protein M1 (FOXM1) is a member of the forkhead box family of transcription factors involved in control of cell proliferation, chromosomal stability, angiogenesis, and invasion. Here, we show that FOXM1 is significantly increased in ATCs compared with normal thyroid, well-differentiated thyroid carcinomas (papillary and/or follicular), and poorly differentiated thyroid carcinomas (P=0.000002). Upregulation of FOXM1 levels in ATC cells was mechanistically linked to loss-of-function of p53 and to the hyperactivation of the phosphatidylinositol-3-kinase/AKT/FOXO3a pathway. Knockdown of FOXM1 by RNA interference inhibited cell proliferation by arresting cells in G2/M and reduced cell invasion and motility. This phenotype was associated with decreased expression of FOXM1 target genes, like cyclin B1 (CCNB1), polo-like kinase 1 (PLK1), Aurora B (AURKB), S-phase kinase-associated protein 2 (SKP2), and plasminogen activator, urokinase: uPA (PLAU). Pharmacological inhibition of FOXM1 in an orthotopic mouse model of ATC reduced tumor burden and metastasization. All together, these findings suggest that FOXM1 represents an important player in thyroid cancer progression to the anaplastic phenotype and a potential therapeutic target for this fatal cancer.
Lilja E Laatikainen, Maria D Castellone, Aline Hebrant, Candice Hoste, Maria C Cantisani, Juha P Laurila, Giuliana Salvatore, Paolo Salerno, Fulvio Basolo, Johnny Näsman, Jacques E Dumont, Massimo Santoro and Mikko O Laukkanen
Reactive oxygen species, specifically hydrogen peroxide (H2O2), have a significant role in hormone production in thyroid tissue. Although recent studies have demonstrated that dual oxidases are responsible for the H2O2 synthesis needed in thyroid hormone production, our data suggest a pivotal role for superoxide dismutase 3 (SOD3) as a major H2O2-producing enzyme. According to our results, Sod3 is highly expressed in normal thyroid, and becomes even more abundant in rat goiter models. We showed TSH-stimulated expression of Sod3 via phospholipase C–Ca2+ and cAMP–protein kinase A, a pathway that might be disrupted in thyroid cancer. In line with this finding, we demonstrated an oncogene-dependent decrease in Sod3 mRNA expression synthesis in thyroid cancer cell models that corresponded to a similar decrease in clinical patient samples, suggesting that SOD3 could be used as a differentiation marker in thyroid cancer. Finally, the functional analysis in thyroid models indicated a moderate role for SOD3 in regulating normal thyroid cell proliferation being in line with our previous observations.
Ornella Affinito, Paolo Salerno, Alfonso D’Alessio, Mariella Cuomo, Ermanno Florio, Francesca Carlomagno, Agnese Proietti, Riccardo Giannini, Fulvio Basolo, Lorenzo Chiariotti, Sergio Cocozza and Massimo Santoro
Molecular differentiation between benign (follicular thyroid adenoma (FTA)) and malignant (follicular thyroid carcinoma (FTC)) thyroid neoplasms is challenging. Here, we explored the genome-wide DNA methylation profile of FTA (n.10) and FTC (n.11) compared to normal thyroid (NT) (n.7) tissues. FTC featured 3564 differentially methylated CpGs (DMCpG), most (84%) of them hypermethylated, with respect to normal controls. At the principal component analysis (PCA), the methylation profile of FTA occupied an intermediate position between FTC and normal tissue. A large fraction (n. 2385) of FTC-associated DMCpG was related (intragenic or within 1500 bp from the transcription start site) to annotated genes (n. 1786). FTC-hypermethylated genes were enriched for targets of the Polycomb transcriptional repressor complex and the specific histone H3 marks (H3K4me2/me3-H3K27me3) found in chromatin domains known as ‘bivalent’. Transcriptome profiling by RNAseq showed that 7.9% of the DMCpGs-associated genes were differentially expressed in FTC compared to NT, suggesting that altered DNA methylation may contribute to their altered expression. Overall, this study suggests that perturbed DNA methylation, in particular hypermethylation, is a component of the molecular mechanisms leading to the formation of FTC and that DNA methylation profiling may help differentiating FTCs from their benign counterpart.