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Background: Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer (TC). Several genomic and transcriptomic studies explored the molecular landscape of follicular cell-derived TCs, and BRAF V600E, RAS mutations, and gene fusions are well-established drivers. DICER1 mutations were described in specific sets of TC patients but represent a rare event in adult TC patients., Methods: Here, we report the molecular characterization of 30 retrospective follicular cell-derived thyroid tumors, comprising PTCs (90%) and poorly differentiated TCs (10%), collected at our Institute. We performed DNA whole-exome sequencing using patient-matched control for somatic mutation calling, and targeted RNA-seq for gene fusion detection. Transcriptional profiles established in the same cohort by microarray were investigated using three signaling-related gene signatures derived from The Cancer Genome Atlas (TCGA)., Results: The occurrence of BRAF V600E (44%), RAS mutations (13%), and gene fusions (13%) was confirmed in our cohort. In addition, in two patients lacking known drivers, mutations of the DICER1 gene (p.D1709N and p.D1810V) were identified. DICER1 mutations occur in two adult patients with follicular-pattern lesions, and in one of them a second concurrent DICER1 mutation (p.R459*) is also observed. Additional putative drivers include ROS1 gene (p.P2130A mutation), identified in a patient with a rare solid-trabecular subtype of PTC. Transcriptomics indicates that DICER1 tumors are RAS-like, whereas the ROS1 -mutated tumor displays a borderline RAS-/BRAF-like subtype. We also provide an overview of DICER1 and ROS1 mutations in thyroid lesions by investigating the COSMIC database., Conclusion: Even though small, our series recapitulates the genetic background of PTC. Furthermore, we identified DICER1 mutations, one of which is previously unreported in thyroid lesions. For these less common alterations and for patients with unknown drivers, we provide signaling information applying TCGA-derived classification., Competing Interests: GP received honoraria from Lilly, AstraZeneca, Novartis, Illumina, and Roche and is part of the advisory board of ADS Biotec. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Minna, Devecchi, Pistore, Paolini, Mauro, Penso, Pagliardini, Busico, Pruneri, De Cecco, Borrello, Sensi and Greco.)

Background: Papillary thyroid cancer (PTC) is the most frequent thyroid tumor. The tissue inhibitor of metalloproteinase-3 (TIMP3) gene encodes a matrix metalloproteinases inhibitor that exerts a tumor suppressor role in several tumor types. TIMP3 is frequently downregulated in PTC by promoter methylation. We have previously functionally demonstrated that TIMP3 exerts an oncosuppressor role in PTC: TIMP3 restoration in the PTC-derived NIM1 cell line affects in vitro migration, invasion and adhesive capability, while reduces tumor growth, angiogenesis and macrophage recruitment in vivo. To get a deeper insight on the mediators of TIMP3 oncosuppressor activity in thyroid tumors, here we focused on the TIMP3 related transcriptome., Methods: TCGA database was used for investigating the genes differentially expressed in PTC samples with low and high TIMP3 expression. Genome wide expression analysis of clones NIM1-T23 (expressing a high level of TIMP3 protein) and NIM1-EV (control empty vector) was performed. Gene sets and functional enrichment analysis with clusterProfiler were applied to identify the modulated biological processes and pathways. CIBERSORT was used to evaluate the distribution of different immunological cell types in TCGA-PTC tumor samples with different TIMP3 expression levels. Real time PCR was performed for the validation of selected genes., Results: Thyroid tumors with TIMP3-high expression showed a down-modulation of inflammation-related gene sets, along with a reduced protumoral hematopoietic cells fraction; an enrichment of cell adhesion functions was also identified. Similar results were obtained in the TIMP3-overexpessing NIM1 cells in vitro model, where a down-regulation of immune-related function gene sets, some of which also identified in tumor samples, was observed. Interestingly, through enrichment analysis, were also recognized terms related to cell adhesion, extracellular matrix organization, blood vessel maintenance and vascular process functions that have been found modulated in our previous in vitro and in vivo functional studies., Conclusions: Our results highlight the correlation of TIMP3 expression levels with the regulation of inflammatory functions and the immune infiltration composition associated with different PTC prognosis, thus providing a broader view on the oncosuppressor role of TIMP3 in PTC., (© 2022. The Author(s).)

Medullary thyroid carcinoma (MTC) is a rare but aggressive tumor. Although RET and RAS genes are recognized drivers in MTC, associated downstream signaling pathways are largely unknown. In this study, we report 17 sporadic MTCs, collected at our institution, comprising patient-matched primary and lymph node metastatic tumors investigated for mutational and transcriptional profiles. As we identified two uncommon RET deletions (D898_E901del and E632_L633del), we also performed a literature review and meta-analysis to assess the occurrence of unconventional alterations in MTC, focusing on next-generation sequencing studies. We found that new gene alterations are emerging, along with the known RET/RAS drivers, involving not only RET by multiple concurrent mutations or deletions but also other previously underestimated cancer-related genes, especially in sporadic MTCs. In our MTC gene profiles, we found transcriptome similarity between patient-matched tissues and expression of immune genes only by a few samples. Furthermore, we defined a gene signature able to stratify samples into two distinct signaling types, termed MEN2B-like and MEN2A-like. We provide an updated overview of the MTC mutational spectrum and describe how transcriptional profiles can be used to define distinct MTC signaling subtypes that appear to be shared by various gene drivers, including the unconventional ones.

Inflammation plays a critical role in thyroid cancer onset and progression. We previously characterized the in vitro interplay between macrophages and senescent human thyrocytes and thyroid tumor-derived cell lines, modeling the early and the late thyroid tumor phases, respectively. We reported that both models are able to induce pro-tumoral M2-like macrophage polarization, through the activation of the COX2-PGE2 axis. Here, we investigated the presence of macrophage infiltrating cells in mouse xenografts derived from the above described cells models. We showed that subcutaneous injection in immunodeficient mice of both senescent human thyrocytes and thyroid tumor-derived cell lines elicits macrophage recruitment. Furthermore, considering the type of macrophage infiltrate, we observed a stronger infiltration of Arginase I positive cells (M2-like). Overall, these results demonstrate the in vivo capability of senescent and tumor thyroid cells to recruit and polarize macrophages, suggesting that the promotion of a pro-tumoral microenvironment through tumor associated macrophages may occurs in late as well as in early thyroid tumor stages, favoring tumor onset and progression.

BRAF V600E is the most frequent oncogenic mutation identified in papillary thyroid cancer (PTC). In PTC patients who do not respond to standard treatment, BRAF inhibitors are currently tested as alternative strategies. However, as observed for other targeted therapies, patients eventually develop drug resistance. The mechanisms of BRAF inhibitors response are still poorly understood in a thyroid cancer (TC) context. In this study, we investigated in BRAF V600E mutated TC cell lines the effects of Vemurafenib and Dabrafenib, two BRAF inhibitors currently used in a clinical setting. We assessed cell proliferation, and the expression and activity of the thyroid function related transporter NIS following the treatment with BRAF inhibitors. In addition, we investigated the global gene expression by microarray, the relevant modulated biological processes by gene set enrichment analysis (GSEA), and TC specific gene signatures related to MAPK pathway activation, thyroid differentiation, and transcriptional profile associated with BRAF V600E or RAS mutation. We found that both inhibitors induce antiproliferative and redifferentiative effects on TC cells, as well as a rewiring of the MAPK pathway related to RAS signaling. Our results suggest a possible mechanism of drug response to the BRAF inhibitors Vemurafenib or Dabrafenib, supporting very recent findings in TC patients treated with targeted therapies.

Background: Papillary thyroid cancer (PTC) is the most frequent endocrine tumor. Radioiodine (RAI) treatment is highly effective in these tumors, but up to 60% of metastatic cases become RAI-refractory. Scanty data are available on either the molecular pattern of radioiodine refractory papillary thyroid cancers (PTC) or the mechanisms responsible for RAI resistance., Methods: We analyzed the molecular profile and gene/miRNA expression in primary PTCs, synchronous and RAI-refractory lymph node metastases (LNMs) in correlation to RAI avidity or refractoriness. We classified patients as RAI+/D+ (RAI uptake/disease persistence), RAI-/D+ (absent RAI uptake/disease persistence), and RAI+/D- (RAI uptake/disease remission), and analyzed the molecular and gene/miRNA profiles, and the expression of thyroid differentiation (TD) related genes., Results: A different molecular profile according to the RAI class was observed: BRAF V600E cases were more frequent in RAI-/D+ (P = 0.032), and fusion genes in RAI+/D+ cases. RAI+/D- patients were less frequently pTERT mutations positive, and more frequently wild type for the tested mutations/fusions. Expression profiles clearly distinguished PTC from normal thyroid. On the other hand, in refractory cases (RAI+/D+ and RAI-/D+) no distinctive PTC expression patterns were associated with either tissue type, or RAI uptake, but with the driving lesion and BRAF-/RAS-like subtype. Primary tumors and RAI-refractory LNMs with BRAF V600E mutation display transcriptome similarity suggesting that RAI minimally affects the expression profiles of RAI-refractory metastases. Molecular profiles associated with the expression of TPO, SLC26A4 and TD genes, that were found more downregulated in BRAF V600E than in gene fusions tumors., Conclusions: The present data indicate a different molecular profile in RAI-avid and RAI-refractory metastatic PTCs. Moreover, BRAF V600E tumors displayed reduced differentiation and intrinsic RAI refractoriness, while PTCs with fusion oncogenes are RAI-avid but persistent, suggesting different oncogene-driven mechanisms leading to RAI refractoriness.

Thyroid carcinoma (TC) comprises several histotypes with different aggressiveness, from well (papillary carcinoma, PTC) to less differentiated forms (poorly differentiated and anaplastic thyroid carcinoma, PDTC and ATC, respectively). Previous reports have suggested a functional role for cancer-associated fibroblasts (CAFs) or senescent TC cells in the progression of PTC. In this study, we investigated the presence of CAFs and senescent cells in proprietary human TCs including PTC, PDTC, and ATC. Screening for the driving lesions BRAFV600E and N/H/KRAS mutations, and gene fusions was also performed to correlate results with tumor genotype. In samples with unidentified drivers, transcriptomic profiles were used to establish a BRAF- or RAS-like molecular subtype based on a gene signature derived from The Cancer Genome Atlas. By using immunohistochemistry, we found co-occurrence of stromal CAFs and senescent TC cells at the tumor invasive front, where deposition of collagen (COL1A1) and expression of lysyl oxidase (LOX) enzyme were also detected, in association with features of local invasion. Concurrent high expression of CAFs and of the senescent TC cells markers, COL1A1 and LOX was confirmed in different TC histotypes in proprietary and public gene sets derived from Gene Expression Omnibus (GEO) repository, and especially in BRAF mutated or BRAF-like tumors. In this study, we show that CAFs and senescent TC cells co-occur in various histotypes of BRAF-driven thyroid tumors and localize at the tumor invasive front., Competing Interests: The authors declare no conflict of interest.

Background: Thyroid carcinoma includes several variants characterized by different biological and clinical features: from indolent microcarcinoma to undifferentiated and aggressive anaplastic carcinoma. Inflammation plays a critical role in thyroid tumors. Conditions predisposing to cancer, as well as oncogene activity, contribute to the construction of an inflammatory microenvironment that facilitates thyroid tumor progression. Moreover, oncogene-induced senescence, a mechanism tightly connected with inflammation, and able to restrain or promote cancer progression, is involved in thyroid cancer. The interactions between thyroid tumor cells and the microenvironment are not completely clarified., Methods: We characterize in vitro the interplay between macrophages and senescent thyrocytes and tumor-derived cell lines, modeling early and late thyroid tumor stages, respectively. Purified peripheral blood-derived human monocytes were exposed to thyroid cell-derived conditioned medium (CM) and assessed for phenotype by flow cytometry. The factors secreted by thyroid cells and macrophages were identified by gene expression analysis and ELISA. The protumoral effect of macrophages was assessed by wound healing assay on K1 thyroid tumor cells. The expression of PTGS2 and M2 markers in thyroid tumors was investigated in publicly available datasets., Results: Human monocytes exposed to CM from senescent thyrocytes and thyroid tumor cell lines undergo M2-like polarization, showing high CD206 and low MHC II markers, and upregulation of CCL17 secretion. The obtained M2-like macrophages displayed tumor-promoting activity. Among genes overexpressed in polarizing cells, we identified the prostaglandin-endoperoxide synthase enzyme (PTGS2/COX-2), which is involved in the production of prostaglandin E2 (PGE2). By using COX-2 inhibitors we demonstrated that the M2-like polarization ability of thyroid cells is related to the production of PGE2. Co-expression of PTGS2 and M2 markers is observed a significant fraction of human thyroid tumors., Conclusions: Our results demonstrate that both senescent thyrocytes and thyroid tumor cell lines trigger M2-like macrophage polarization that is related to PGE2 secretion. This suggests that the interaction with the microenvironment occurs at both early and late thyroid tumor stages, and favors tumor progression. The co-expression of PTGS2 gene and M2 markers in human thyroid carcinoma highlights the possibility to counteract tumor growth through COX-2 inhibition.

Even if thyroid tumors are generally curable, a fraction will develop resistance to therapy and progress towards undifferentiated forms, whose treatment remains a demanding challenge. To identify potential novel targets for treatment of thyroid cancer, in a previous study using siRNA-mediated functional screening, we identified several genes that are essential for the growth of thyroid tumor, but not normal cells. Among the top-ranking hits, we found microtubule associated serine/threonine kinase-like (MASTL), which is known to play an essential role in mitosis regulation, and is also involved in the DNA damage response. Herein, we examine the effects of MASTL depletion on growth and viability of thyroid tumor cells. MASTL depletion impaired cell proliferation and increased the percentage of cells presenting nuclear anomalies, which are indicative of mitotic catastrophe. Furthermore, MASTL depletion was associated with enhanced DNA damage. All these effects eventually led to cell death, characterized by the presence of apoptotic markers. Moreover, MASTL depletion sensitized thyroid tumor cells to cisplatin. Our results demonstrate that MASTL represents vulnerability for thyroid tumor cells, which could be explored as a therapeutic target for thyroid cancer., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This study aimed to identify circulating miRNAs as novel non-invasive biomarkers for prognosis and vandetanib response in advanced medullary thyroid cancer (MTC) patients. We prospectively recruited two independent cohorts of locally advanced/metastatic MTC patients including a subgroup of vandetanib-treated subjects: a discovery cohort ( n  = 20), including matched plasma/tissue samples ( n  = 17/20), and a validation cohort, yielding only plasma samples ( n  = 17). Plasma samples from healthy subjects ( n  = 36) and MTC patients in remission ( n  = 9) were used as controls. MTC ( n  = 17 from 8 patients included in discovery cohort) and non-neoplastic thyroid specimens ( n  = 3) were assessed by microarray profiling to identify candidate circulating miRNAs. qRT-PCR and in situ hybridization were carried out to validate the expression and localization of a selected miRNA within tissues, and qRT-PCR was also performed to measure miRNA levels in plasma samples. By microarray analysis, we identified 51 miRNAs differentially expressed in MTC. The most overexpressed miR, miR-375, was highly expressed by C cells compared to other thyroid cells, and more expressed in MTC than in reactive C-cell hyperplasia. MTC patients had significantly higher miR-375 plasma levels than healthy controls ( P  < 0.0001) and subjects in remission ( P  = 0.0004) as demonstrated by qRT-PCR analysis. miR-375 plasma levels were not predictive of vandetanib response, but, notably, high levels were associated with significantly reduced overall survival (HR 10.61, P  < 0.0001) and were a strong prognostic factor of poor prognosis (HR 6.24, P  = 0.00025) in MTC patients. Overall, our results unveil plasma miR-375 as a promising prognostic marker for advanced MTC patients, to be validated in larger cohorts., (© 2018 Society for Endocrinology.)

[This corrects the article DOI: 10.18632/oncotarget.7262.].

Medullary thyroid cancer (MTC) relies on the aberrant activation of RET proto-oncogene. Though targeted approaches (i.e., tyrosine kinase inhibitors) are available, the absence of complete responses and the onset of resistance mechanisms indicate the need for novel therapeutic interventions. Due to their role in regulation of gene expression, G-quadruplexes (G4) represent attractive targets amenable to be recognized or stabilized by small molecules. Here, we report that exposure of MTC cells to a tri-substituted naphthalene diimide (NDI) resulted in a significant antiproliferative activity paralleled by inhibition of RET expression. Biophysical analysis and gene reporter assays showed that impairment of RET expression was consequent to the NDI-mediated stabilization of the G4 forming within the gene promoter. We also showed for the first time that systemic administration of the NDI in mice xenotransplanted with MTC cells resulted in a remarkable inhibition of tumor growth in vivo. Overall, our findings indicate that NDI-dependent RET G4 stabilization represents a suitable approach to control RET transcription and delineate the rationale for the development of G4 stabilizing-based treatments for MTC as well as for other tumors in which RET may have functional and therapeutic implications., Competing Interests: No conflict of interest to be disclosed.

Papillary Thyroid Carcinoma (PTC) is the most frequent thyroid cancer. Although several PTC-specific miRNA profiles have been reported, only few upregulated miRNAs are broadly recognized, while less consistent data are available about downregulated miRNAs. In this study we investigated miRNA deregulation in PTC by miRNA microarray, analysis of a public dataset from The Cancer Genome Atlas (TCGA), literature review and meta-analysis based on a univocal miRNA identifier derived from miRBase v21. A list of 18 miRNAs differentially expressed between PTC and normal thyroid was identified and validated in the TCGA dataset. Furthermore, we compared our signature with miRNA profiles derived from 15 studies selected from literature. Then, to select possibly functionally relevant miRNA, we integrated our miRNA signature with those from two in vitro cell models based on the PTC-driving oncogene RET/PTC1. Through this strategy, we identified commonly deregulated miRNAs, including miR-451a, which emerged also by our meta-analysis as the most frequently reported downregulated miRNA. We showed that lower expression of miR-451a correlates with aggressive clinical-pathological features of PTC as tall cell variant, advanced stage and extrathyroid extension. In addition, we demonstrated that ectopic expression of miR-451a impairs proliferation and migration of two PTC-derived cell lines, reduces the protein levels of its recognized targets MIF, c-MYC and AKT1 and attenuates AKT/mTOR pathway activation.Overall, our study provide both an updated overview of miRNA deregulation in PTC and the first functional evidence that miR-451a exerts tumor suppressor functions in this neoplasia.

Background: Hereditary medullary thyroid carcinoma (MTC) is caused by germ-line gain of function mutations in the RET proto-oncogene, and a phenotypic variability among carriers of the same mutation has been reported. We recently observed this phenomenon in a large familial MTC (FMTC) family carrying the RET-S891A mutation. Among genetic modifiers affecting RET-driven MTC, a role has been hypothesized for RET-G691S non-synonymous polymorphism, though the issue remains controversial. Aim of this study was to define the in vitro contribution of RET-G691S to the oncogenic potential of the RET-S891A, previously shown to harbour low transforming activity., Methods: The RET-S891A and RET-G691S/S891A mutants were generated by site-directed mutagenesis, transiently transfected in HEK293T cells and stably expressed in NIH3T3 cells. Their oncogenic potential was defined by assessing the migration ability by wound healing assay and the anchorage-independent growth by soft agar assay in NIH3T3 cells stably expressing either the single or the double mutants. Two RET-S891A families were characterised for the presence of RET-G691S., Results: The functional studies demonstrated that RET-G691S/S891A double mutant displays a higher oncogenic potential than RET-S891A single mutant, assessed by focus formation and migration ability. Moreover, among the 25 RET-S891A carriers, a trend towards an earlier age of diagnosis was found in the MTC patients harboring RET-S891A in association with RET-G691S., Conclusions: We demonstrate that the RET-G691S non-synonymous polymorphism enhances in vitro the oncogenic activity of RET-S891A. Moreover, an effect on the phenotype was observed in the RET-G691S/S891A patients, thus suggesting that the analysis of this polymorphism could contribute to the decision on the more appropriate clinical and follow-up management.

Thyroid cancer incidence is rapidly increasing. Papillary Thyroid Carcinoma (PTC), the most frequent hystotype, usually displays good prognosis, but no effective therapeutic options are available for the fraction of progressive PTC patients. BRAF and RET/PTC are the most frequent driving genetic lesions identified in PTC. We developed two complementary in vitro models based on RET/PTC1 oncogene, starting from the hypothesis that miRNAs modulated by a driving PTC-oncogene are likely to have a role in thyroid neoplastic processes. Through this strategy, we identified a panel of deregulated miRNAs. Among these we focused on miR-199a-3p and showed its under-expression in PTC specimens and cell lines. We demonstrated that miR-199a-3p restoration in PTC cells reduces MET and mTOR protein levels, impairs migration and proliferation and, more interesting, induces lethality through an unusual form of cell death similar to methuosis, caused by macropinocytosis dysregulation. Silencing MET or mTOR, both involved in survival pathways, does not recapitulate miR-199a-3p-induced cell lethality, thus suggesting that the cooperative regulation of multiple gene targets is necessary. Integrated analysis of miR-199a-3p targets unveils interesting networks including HGF and macropinocytosis pathways. Overall our results indicate miR-199a-3p as a tumor suppressor miRNA in PTC.

Context: Tyrosine kinase inhibitors represent a new treatment option for patients with advanced medullary thyroid cancer (MTC). However, cures have not been achieved with current available agents used in monotherapy., Objective: Because RET has been shown to negatively regulate CD95 death receptor activation in preclinical models of RET-dependent MTC, we investigated the potential of the combination approach with the RET-targeting tyrosine kinase inhibitor sunitinib and cisplatin to enhance apoptosis activation through the extrinsic pathway., Design: The effects of sunitinib and cisplatin were examined in human MTC cell lines harboring oncogenic RET mutations. Experiments were designed to determine drug effects on RET signaling, cell growth, apoptosis, autophagy, and tumor growth in mice and to investigate the mechanisms of the drug interaction., Results: Sunitinib and cisplatin synergistically inhibited the growth of MZ-CRC-1 cells harboring the RET M918T activating mutation. The combination enhanced apoptosis activation through CD95-mediated, caspase-8-dependent pathway. Moreover, sunitinib induced a severe perturbation of the autophagic flux characterized by autophagosome accumulation and a remarkable lysosomal dysfunction, which was further enhanced, with lysosomal leakage induction, by cisplatin. Administration of the drug combination to mice xenografted with MZ-CRC-1 cells improved the antitumor efficacy, as compared with single-agent treatments, inducing complete responses in 30% of the treated mice, a significant increase in caspase-3 activation (P < .01 vs cisplatin, and P < .0005 vs sunitinib) and apoptosis in tumor cells., Conclusions: Addition of cisplatin to sunitinib potentiates apoptotic cell death and has promising preclinical activity in MTCs harboring the RET M918T oncogene.

Context: Papillary thyroid carcinoma (PTC) is the most frequent thyroid tumor and is responsible for the overall increase in thyroid cancer incidence. S100A11 (calgizzarin), a member of the S100 Ca(2+)-binding protein family, is involved in several different biological processes. S100A11 has been found up-regulated in PTC, both at the mRNA and protein levels., Objective: Through a combination of expression analysis and functional in vitro and in vivo studies, we have attempted to gain insight into the relevance of S100A11 overexpression in PTC biology., Design: The expression of the S100A11 gene in PTC was investigated in several gene expression data sets. The effect of S100A11 silencing on the hallmarks of the malignant phenotype of several PTC-derived cell lines was investigated. In NIH3T3 cells, the cooperation of S100A11 with the different PTC-specific oncogenes was assessed., Results: We found that the S100A11 gene expression is frequently up-regulated in PTC, anaplastic thyroid carcinoma, but not in follicular thyroid carcinoma. S100A11 overexpression was also detected in PTC-derived cell lines, which were then used for functional studies. S100A11 silencing in PTC-derived cell lines did not affect cell proliferation, whereas it reduced the loss of contact inhibition, anchorage-independent growth, and resistance to anoikis. Cotransfection experiments in NIH3T3 cells showed that overexpression of the S100A11 gene was able to enhance the transforming capabilities of the different PTC-associated oncogenes by affecting the loss of contact inhibition, anchorage-independent growth, and in vivo tumor formation., Conclusion: Our data indicate that S100A11 overexpression exerts a protumoral functional role in PTC pathogenesis.

Introduction: The RET gene encodes a receptor tyrosine kinase essential for ontogenesis of the enteric nervous system and kidney. Following identification of RET, it was found that somatic rearrangements of this gene, conventionally designated as RET/PTC, are frequently present in papillary thyroid carcinoma. Subsequently, activating germ line point mutations of RET were identified as being responsible for the hereditary medullary thyroid carcinoma syndromes MEN2A, MEN2B and FMTC. RET rearrangements have recently been identified in a small fraction of lung adenocarcinomas., Area Covered: The authors review the current field concerning the RET gene and protein, its involvement in cancer and the preclinical and clinical studies which highlight its role as a potentially important therapeutic target for several cancers., Expert Opinion: Many multitargeted inhibitors which crossreact with RET have been developed and investigated in clinical trials targeting many cancer indications. In particular, VEGFR/PDGFR inhibitors, widely explored as antiangiogenics, have been intensively studied in thyroid carcinoma patients. Notwithstanding the efficacy observed with such agents, their common clinical activity in thyroid carcinoma is of short duration and includes frequent and severe side effects, limiting their therapeutic action. These findings are discussed and the need for improved, more specific RET-targeting drugs is highlighted.

Thyroid carcinomas derived from follicular cells comprise papillary thyroid carcinoma (PTC), follicular thyroid carcinoma, poorly differentiated thyroid carcinoma (PDTC) and undifferentiated anaplastic thyroid carcinoma (ATC). PTC, the most frequent thyroid carcinoma histotype, is associated with gene rearrangements that generate RET/PTC and TRK oncogenes and with BRAF-V600E and RAS gene mutations. These last two genetic lesions are also present in a fraction of PDTCs. The ERK1/2 pathway, downstream of the known oncogenes activated in PTC, has a central role in thyroid carcinogenesis. In this study, we demonstrate that the BRAF-V600E, RET/PTC, and TRK oncogenes upregulate the ERK1/2 pathway's attenuator cytoplasmic dual-phase phosphatase DUSP6/MKP3 in thyroid cells. We also show DUSP6 overexpression at the mRNA and protein levels in all the analysed PTC cell lines. Furthermore, DUSP6 mRNA was significantly higher in PTC and PDTC in comparison with normal thyroid tissues both in expression profile datasets and in patients' surgical samples analysed by real-time RT-PCR. Immunohistochemical and western blot analyses showed that DUSP6 was also overexpressed at the protein level in most PTC and PDTC surgical samples tested, but not in ATC, and revealed a positive correlation trend with ERK1/2 pathway activation. Finally, DUSP6 silencing reduced the neoplastic properties of four PTC cell lines, thus suggesting that DUSP6 may have a pro-tumorigenic role in thyroid carcinogenesis.

Background: Approximately 60% of sporadic medullary thyroid carcinomas (sMTC) remain orphan of a recognized genetic cause. Recently, a high percentage of RAS point mutations have been described in RET-negative sMTC. The aim of this study was to assess the prevalence of RAS point mutations in a large series of MTC collected in four Italian centers., Methods: For this purpose, we studied codons 12, 13, and 61 of H-, K-, and N-RAS genes in 188 MTC samples, either hereditary or sporadic, by direct sequencing. Correlations between the RAS mutational status and the clinical-pathological features of MTC patients as well as a meta-analysis of all published data were performed., Results: The prevalence of RAS mutations in the present series of MTC was 10.1%, and 17.6% when considering only RET-negative cases. RAS mutations were found in MTC tumoral tissue, but not in peripheral blood indicating their somatic origin. A novel mutation in codon 72 (M72I) was found, but with a low or null transforming potential. No association was found between the presence of RAS mutations and the clinical-pathological features of the patients. Although not statistically significant, a positive association between the presence of RAS mutations and a better outcome was observed. The meta-analysis of all published studies confirmed a prevalence of 8.8% for RAS mutations in MTC., Conclusions: The prevalence of RAS mutations in our MTC series was relatively low and consistent with the meta-analysis data. Only somatic RAS mutations were found and only in RET-negative sMTC. Likewise, MTCs that harbor a RAS mutation identify a subgroup of tumors with less aggressive behavior. To our knowledge, this is the largest series of MTCs studied for the presence of mutations in RAS genes and the first meta-analysis on this specific topic.

Somatotrophs produce growth hormone (GH) and are the most abundant secretory cells of the pituitary. Somatotrophs express the transcription factor Pit-1 and the dependence receptor RET, its co-receptor GFRa1 and ligand GDNF. Pit-1 is a transcription factor essential for somatotroph proliferation and differentiation and for GH expression. GDNF represses excess Pit-1 expression preventing excess GH. In the absence of GDNF, RET behaves as a dependence receptor, becomes intracellularly processed and induces strong Pit-1 expression leading to p53 accumulation and apoptosis. How accumulation of Pit-1 leads to p53 expression is unknown. We have unveiled the relationship of Pit-1 with the p19Arf gene. There is a parallel correlation of RET processing, Pit-1 increase and ARF protein and mRNA expression. Interfering the pathway with RET, Pit-1 or p19Arf siRNA blocked apoptosis. We have found a Pit-1 DNA-binding element within the ARF promoter. Pit-1 directly regulates the CDKN2A locus and binds to the p19Arft promoter inducing p19Arf gene expression. The Pit-1-binding element is conserved in rodents and humans. RET/Pit-1 induces p19Arf/p53 and apoptosis not only in a somatotroph cell line but also in primary cultures of pituitary somatotrophs, where ARF siRNA interference also blocks p53 and apoptosis. Analyses of the somatotrophs in whole pituitaries supported the above findings. Thus Pit-1, a differentiation factor, activates the oncogene-induced apoptosis (OIA) pathway as oncogenes exerting a tight control in somatotrophs to prevent the disease due to excess of GH (insulin-resistance, metabolic disease, acromegaly).

Oncogene-induced senescence (OIS) is a growth arrest triggered by the enforced expression of cancer-promoting genes and acts as a barrier against malignant transformation in vivo. In this study, by a combination of in vitro and in vivo approaches, we investigate the role of OIS in tumours originating from the thyroid epithelium. We found that expression of different thyroid tumour-associated oncogenes in primary human thyrocytes triggers senescence, as demonstrated by the presence of OIS hallmarks: changes in cell morphology, accumulation of SA-β-Gal and senescence-associated heterochromatic foci, and upregulation of transcription of the cyclin-dependent kinase inhibitors p16(INK4a) and p21(CIP1). Furthermore, immunohistochemical analysis of a panel of thyroid tumours characterised by different aggressiveness showed that the expression of OIS markers such as p16(INK4a), p21(CIP1) and IGFBP7 is upregulated at early stages, and lost during thyroid tumour progression. Taken together, our results suggest a role of OIS in thyroid carcinogenesis.

Activating mutations of RET, a gene encoding two isoforms of a tyrosine kinase receptor physiologically expressed in several neural crest-derived cell lineages, are associated with the inherited forms of medullary thyroid carcinoma (MTC). The identification and characterization of novel RET mutations involved in MTC is valuable, as RET gene testing plays a crucial role in the management of these patients. In an MTC patient, we have identified a germline c.1996A>G transition in heterozygosis leading to K666E substitution. In addition, the conservative S904S (c.2712C>G) and the non-conservative functional G691S (c.2071G>A) polymorphisms have been identified. Through functional studies, we demonstrate for the first time that K666E is a gain-of-function mutation with oncogenic potential, based on its ability to transform NIH3T3 cells. It was not possible to define whether K666E is a de novo or inherited RET variant in the patient, as the family history was negative for MTC, and the carrier status of family members could not be tested. Our results, together with a recent report of co-segregation of the mutation in three MTC families, suggest that K666E is a causative MTC mutation. As we have shown that the same patient allele carries both K666E and G691S variants, the latter known to increase downstream RET signaling, a possible role for the G691S polymorphism has also been investigated. We have demonstrated that, although RET-G691S is not oncogenic per se, it enhances the transforming activity of the RET-K666E mutant, thus suggesting a modifier role for this functional polymorphism.

Papillary thyroid carcinoma (PTC) arises from the thyroid follicular epithelium and represents the most frequent thyroid malignancy. PTC is associated with gene rearrangements generating RET/PTC and TRK oncogenes, and to the BRAFV600E activating point mutation. A role of tumor-suppressor genes in the pathogenesis of PTC has not been assessed yet. The tissue inhibitor of metalloproteinase-3 (TIMP3) gene, encoding a metalloproteinases inhibitor and capable of inhibiting growth, angiogenesis, invasion and metastasis of several cancers, was found to be silenced by promoter methylation in a consistent fraction of PTCs, in association with tumor aggressiveness and BRAFV600E mutation, thus suggesting an oncosuppressor role. To explore this possibility, in this study we performed gene expression and functional studies. Analysis of gene expression data produced in our laboratory as well as meta-analysis of publicly available data sets confirmed the downregulation of TIMP3 gene expression in PTC with respect to normal thyroid. The functional consequences of TIMP3 downregulation were investigated in the PTC-derived NIM1 cell line, in which the expression of TIMP3 is silenced. Restoration of TIMP3 expression by exposure to soluble TIMP3 protein or by complementary DNA transfection had no effect on the growth rate of NIM1 cells. Instead, it affected the adhesive, migratory and invasive capabilities of NIM1 cells by modulating several proteins involved in these processes. A striking effect was observed in vivo, as TIMP3 reduced the tumorigenicity of NIM1 cells by repressing angiogenesis and macrophage infiltration. Our data indicate that the loss of TIMP3 expression exerts a functional role in the pathogenesis of PTC.

Background: Papillary thyroid carcinoma (PTCs), the most frequent thyroid cancer, is usually not life threatening, but may recur or progress to aggressive forms resistant to conventional therapies. A more detailed understanding of the signaling pathways activated in PTCs may help to identify novel therapeutic approaches against these tumors. The aim of this study is to identify signaling pathways activated in PTCs., Methodology/principal Findings: We examined coordinated gene expression patterns of ligand/receptor (L/R) pairs using the L/R database DRLP-rev1 and five publicly available thyroid cancer datasets of gene expression on a total of 41 paired PTC/normal thyroid tissues. We identified 26 (up) and 13 (down) L/R pairs coordinately and differentially expressed. The relevance of these L/R pairs was confirmed by performing the same analysis on REarranged during Transfection (RET)/PTC1-infected thyrocytes with respect to normal thyrocytes. TGFA/EGFR emerged as one of the most tightly regulated L/R pair. Furthermore, PTC clinical samples analyzed by real-time RT-PCR expressed EGFR transcript levels similar to those of 5 normal thyroid tissues from patients with pathologies other than thyroid cancer, whereas significantly elevated levels of TGFA transcripts were only present in PTCs. Biochemical analysis of PTC cell lines demonstrated the presence of EGFR on the cell membrane and TGFA in conditioned media. Moreover, conditioned medium of the PTC cell line NIM-1 activated EGFR expressed on HeLa cells, culminating in both ERK and AKT phosphorylation. In NIM-1 cells harboring BRAF mutation, TGFA stimulated proliferation, contributing to PI3K/AKT activation independent of MEK/ERK signaling., Conclusions/significance: We compiled a reliable list of L/R pairs associated with PTC and validated the biological role of one of the emerged L/R pair, the TGFA/EGFR, in this cancer, in vitro. These data provide a better understanding of the factors involved in the biology of PTCs and would be useful in developing combination therapeutic approaches against these cancers.

Objective: The recent concept that oncogenes responsible for thyroid neoplastic transformation are able to elicit an inflammatory protumourigenic microenvironment raises interest in further studies on papillary thyroid cancer (PTC) associated with thyroid autoimmunity., Patients: The clinical and molecular features, and the expression of inflammation-related genes, were investigated in a large series of PTCs with and without associated thyroiditis (groups A, n = 128 and B, n = 215)., Results: The two groups did not show significant differences in clinical and prognostic features, whereas they harboured a significantly different genetic background (P = 0.001), with RET/PTC1 being more represented in PTCs associated with autoimmunity, and BRAF(V600E) in patients with PTC alone. A RET/PTC rearrangement was also found in 41% of non-neoplastic thyroiditis tissues, contralateral to tumours harbouring either RET/PTC or BRAF mutations. The expression of genes encoding CCL20, CXCL8 and l-selectin was significantly higher in PTC specimens (either with RET/PTC, BRAF(V600E) or unknown genetic lesion) compared with normal thyroid samples. On the contrary, thyroiditis showed l-selectin expression levels even higher than PTCs, but CCL20 and CXCL8 levels comparable with normal tissues., Conclusions: The present data extend the knowledge about the tight relationships among oncogenes, thyroiditis and thyroid cancer. A different genetic background among PTCs with and without associated autoimmunity has been firstly demonstrated. The strong association between RET/PTC1 and thyroiditis points to a critical role of this oncoprotein in the modulation of the autoimmune response. Moreover, preliminary expression studies, indicating enhanced expression of inflammatory molecules in PTCs, suggest a proinflammatory, nonautoimmune relationship between thyroiditis and thyroid cancer.

Thyroid cancer is the most common endocrine malignancy; it accounts for approximately 1% of all new case of cancer each year, and its incidence has increased significantly over the last few decades. The majority of thyroid tumors originate from follicular epithelial cells. Among them, papillary (PTC) and follicular carcinomas (FTC) represent the most common forms of differentiated thyroid cancer and account for approximately 80% and 15% of all cases, respectively. Specific genetic lesions are associated to each thyroid tumor histotype: BRAF mutations and RET/PTC and TRK oncogenes have been detected in PTC, whereas FTC is characterized by PAX8/PPARgamma rearrangements and RAS mutations. In this review we summarize studies on the molecular biology of the differentiated thyroid tumors, with particular interest in the associated genetic lesions and their role in thyroid carcinogenesis. We also report recent findings on gene expression and miRNA profiles of PTC and FTC.

Activation of the RET gene by chromosomal rearrangements generating RET/PTC oncogenes is a frequent, early, and causative event in papillary thyroid carcinoma (PTC). We have previously shown that, in human primary thyrocytes, RET/PTC1 induces a transcriptional program including the MET proto-oncogene. In PTCs, beta-catenin is frequently mislocated to the cytoplasm nucleus. We investigated the interplay between Ret/ptc1 signaling and Met in regulating the proinvasive phenotype and beta-catenin localization in cellular models of human PTC. Here, we show that Met protein is expressed and is constitutively active in human thyrocytes exogenously expressing RET/PTC1 as well as a mutant (Y451F) devoid of the main Ret/ptc1 multidocking site. Both in transformed thyrocytes and in the human PTC cell line TPC-1, Ret/ptc1-Y451-dependent signaling and Met cooperated to promote a proinvasive phenotype. Accordingly, gene/functional silencing of either RET/PTC1 or MET abrogated early branching morphogenesis in TPC-1 cells. The same effect was obtained by blocking the common downstream effector Akt. Y451 of Ret/ptc1 was required to promote proliferation and nuclear translocation of beta-catenin, suggesting that these oncogene-driven effects are Met-independent. Pharmacologic inhibition of Ret/ptc1 and Met tyrosine kinases by the multitarget small molecule RPI-1 blocked cell proliferation and invasive ability and dislocated beta-catenin from the nucleus. Altogether, these results support that Ret/ptc1 cross talks with Met at transcriptional and signaling levels and promotes beta-catenin transcriptional activity to drive thyrocyte neoplastic transformation. Such molecular network, promoting disease initiation and acquisition of a proinvasive phenotype, highlights new options to design multitarget therapeutic strategies for PTCs.

Inflammation has long been suspected to contribute to tumor growth. However, the concept that oncogenes, known for decades as responsible for cell neoplastic transformation, build up an inflammatory pro-tumorigenic microenvironment is emerging only in the last few years. The well known oncogenes RAS and MYC have been causally linked to tumor angiogenesis through different ways. Moreover, in thyroid tumors, where many of the genetic tumor-initiating events have been identified, the oncogenes driving tumorigenesis were proved able to induce an inflammatory program. This minireview will focus on growing evidence implicating the role of intrinsic, oncogene-driven pathways leading to pro-tumoral inflammation.

Chronic and persistent inflammation contributes to cancer development and can predispose to carcinogenesis. Infection-driven inflammations are involved in the pathogenesis of approximately 15-20% of human tumors. However, even tumors that are not epidemiologically linked to pathogens are characterized by the presence of an inflammatory component in their microenvironment. Hallmarks of cancer-associated inflammation include the presence of infiltrating leukocytes, cytokines, chemokines, growth factors, lipid messengers, and matrix-degrading enzymes. Schematically, two interrelated pathways link inflammation and cancer: (1) genetic events leading to neoplastic transformation promote the construction of an inflammatory milieu; (2) tumor-infiltrating leukocytes, in particular macrophages, are prime regulators of cancer inflammation. Thus, an intrinsic pathway of inflammation (driven in tumor cells), as well as an extrinsic pathway (in tumor-infiltrating leukocytes) have been described and both contribute to tumor progression.

The RET gene encodes two main isoforms of a receptor tyrosine kinase (RTK) implicated in various human diseases. Activating germ-line point mutations are responsible for multiple endocrine neoplasia type 2-associated medullary thyroid carcinomas, inactivating germ-line mutations for Hirschsprung's disease, while somatic rearrangements (RET/PTCs) are specific to papillary thyroid carcinomas. SH2B1beta, a member of the SH2B adaptors family, and binding partner for several RTKs, has been recently described to interact with proto-RET. Here, we show that both RET isoforms and its oncogenic derivatives bind to SH2B1beta through the SRC homology 2 (SH2) domain and a kinase activity-dependent mechanism. As a result, RET phosphorylates SH2B1beta, which in turn enhances its autophosphorylation, kinase activity, and downstream signaling. RET tyrosine residues 905 and 981 are important determinants for functional binding of the adaptor, as removal of both autophosphorylation sites displaces its recruitment. Binding of SH2B1beta appears to protect RET from dephosphorylation by protein tyrosine phosphatases, and might represent a likely mechanism contributing to its upregulation. Thus, overexpression of SH2B1beta, by enhancing phosphorylation/activation of RET transducers, potentiates the cellular differentiation and the neoplastic transformation thereby induced, and counteracts the action of RET inhibitors. Overall, our results identify SH2B1beta as a key enhancer of RET physiologic and pathologic activities.

Somatotrophs are the only pituitary cells that express Ret, GFRalpha1 and GDNF. This study investigated the effects of Ret in a somatotroph cell line, in primary pituitary cultures and in Ret KO mice. Ret regulates somatotroph numbers by inducing Pit-1 overexpression, leading to increased p53 expression and apoptosis, both of which can be prevented with Ret or Pit-1 siRNA. The Pit-1 overexpression is mediated by sustained activation of PKCdelta, JNK, c/EBPalpha and CREB induced by a complex of Ret, caspase 3 and PKCdelta. In the presence of GDNF, Akt is activated, and the Pit-1 overexpression and resulting apoptosis are blocked. The adenopituitary of Ret KO mice is larger than normal, showing Pit-1 and somatotroph hyperplasia. In normal animals, activation of the Ret/Pit-1/p53 pathway by retroviral introduction of Ret blocked tumor growth in vivo. Thus, somatotrophs have an intrinsic mechanism for controlling Pit-1/GH production through an apoptotic/survival pathway. Ret might be of value for treatment of pituitary adenomas.

Rearrangements of the RET receptor tyrosine kinase gene generating RET/PTC oncogenes are specific to papillary thyroid carcinoma (PTC), the most frequent thyroid tumor. Here, we show that the RET/PTC1 oncogene, when exogenously expressed in primary normal human thyrocytes, induces the expression of a large set of genes involved in inflammation and tumor invasion, including those encoding chemokines (CCL2, CCL20, CXCL8, and CXCL12), chemokine receptors (CXCR4), cytokines (IL1B, CSF-1, GM-CSF, and G-CSF), matrix-degrading enzymes (metalloproteases and urokinase-type plasminogen activator and its receptor), and adhesion molecules (L-selectin). This effect is strictly dependent on the presence of the RET/PTC1 Tyr-451 (corresponding to RET Tyr-1062 multidocking site). Selected relevant genes (CCL20, CCL2, CXCL8, CXCR4, L-selectin, GM-CSF, IL1B, MMP9, UPA, and SPP1/OPN) were found up-regulated also in clinical samples of PTC, particularly those characterized by RET/PTC activation, local extrathyroid spread, and lymph node metastases, when compared with normal thyroid tissue or follicular thyroid carcinoma. These results, demonstrating that the RET/PTC1 oncogene activates a proinflammatory program, provide a direct link between a transforming human oncogene, inflammation, and malignant behavior.

The variety of diseases caused by mutations in RET receptor tyrosine kinase provides a classic example of phenotypic heterogeneity. Gain-of-function mutations of RET are associated with human cancer. Gene rearrangements juxtaposing the tyrosine kinase domain to heterologous gene partners have been found in sporadic papillary carcinomas of the thyroid (PTC). These rearrangements generate chimeric RET/PTC oncogenes. In the germline, point mutations of RET are responsible for multiple endocrine neoplasia type 2 (MEN 2A and 2B) and familial medullary thyroid carcinoma (FMTC). Both MEN 2 mutations and PTC gene rearrangements potentiate the intrinsic tyrosine kinase activity of RET and, ultimately, activate the RET downstream targets. Loss-of-function mutations of RET cause Hirschsprung's disease (HSCR) or colonic aganglionosis. A deeper understanding of the molecular signaling of normal versus abnormal RET activity in cancer will enable the development of potential new treatments for patients with sporadic and inherited thyroid cancer or MEN 2 syndrome. We now review the role and mechanisms of RET signaling in development and carcinogenesis.

The receptor tyrosine kinase RET is alternatively spliced to yield two main isoforms, RET9 and RET51, which differ in their carboxyl terminal. Activated RET induces different biological responses such as morphological transformation, neurite outgrowth, proliferation, cell migration and branching. The two isoforms have been suggested to have separate intracellular signaling pathways and different roles in mouse development. Here we show that both isoforms are able to induce cell scattering of SK-N-MC neuroepithelioma cell line and branching tubule formation in MDCK cell line. However, the Y1062F mutation, which abrogates the transforming activity of both activated RET isoforms in NIH3T3 cells, does not abolish scattering and branching morphogenesis of RET51, whereas impairs these biological effects of RET9. The GDNF-induced biological effects of RET51 are inhibited by the simultaneous abrogation of both Tyr1062 and Tyr1096 docking sites. Thus, Tyr1096 may substitute the functions of Tyr1062. GRB2 is the only known adaptor protein binding to Tyr1096. Dominant-negative GRB2 expressed in MDCK cells together with RET9 or RET51 significantly reduces branching. Therefore, GRB2 is necessary for RET-mediated branching of MDCK cells.

Gain-of-function mutations of ret receptor tyrosine kinase, the signaling receptor for glial cell line-derived neurotrophic factor, cause sporadic thyroid and adrenal malignancies as well as endocrine cancer syndromes, such as multiple endocrine neoplasia types 2A and 2B (MEN 2A and MEN 2B) and familial medullary thyroid carcinoma. Loss-of-function mutations of ret cause Hirschsprung's disease (HSCR) or colonic aganglionosis. In 20-30% of families with a mutation at residues 609, 611, 618, or 620 of RET, MEN 2A and familial medullary thyroid carcinoma cosegregate with HSCR. These mutations constitutively activate RET due to aberrant disulfide homodimerization and diminish the level of RET at the plasma membrane. It is not known how these mutations simultaneously lead to both gain- and loss-of-function RET-associated diseases. We provide an explanation for the dual phenotypic Janus mutation at Cys620 of RET. In Madin-Darby canine kidney (MDCK) cells, the Janus mutation impairs the glial cell line-derived neurotrophic factor-induced effects of RET on cell migration, differentiation, and survival but simultaneously promotes rapid cell proliferation.

The receptor tyrosine kinase RET, with a known role in embryonic development and in human pathologies, is alternatively spliced to yield at least two functional isoforms, which differ only in their carboxyl terminal. Enigma protein is a member of the PDZ-LIM family and is known to interact with the short isoform of RET/PTC2, a chimeric oncoprotein isolated from papillary thyroid carcinoma. Here, we show that Enigma also interacts in intact cells with the short isoform of RET-wt and of its pathologic mutants associated to MEN2 syndromes, RET-C634R and RET-M918T. In contrast, Enigma binds all the corresponding RET long isoforms very poorly and colocalizes with short but not long RET/PTC2 isoforms. The RET docking tyrosine for Enigma is the last but one before the divergence between the two isoforms and we demonstrated that short-isoform-specific amino acid residues +2 to +4 to this tyrosine are required for the interaction of RET/PTC2 with Enigma.

The RET/PTC oncogenes, generated by chromosomal rearrangements in papillary thyroid carcinomas, are constitutively activated versions of protoRET, a gene encoding two protein isoforms of a transmembrane tyrosine kinase receptor. By using Ret/ptc2 short isoform (iso9), we have previously demonstrated that Tyr586 (Tyr1062 of protoRet) is the docking site for both the PTB and the SH2 domains of Shc. To determine the relevance of this interaction for the transforming activity of Ret/ptc oncogenes, we have generated and characterized novel Ret/ptc mutants unable to activate Shc: Ret/ptc2 long isoform (iso51)-Y586F and both isoforms of Ret/ptc2-N583A. These mutants neither activate Shc nor transform NIH3T3 cells. Since Tyr1062 shows features of a multifunctional docking site, we have used a Shc mutant (Shc Y317F) to directly assess Shc role. We have demonstrated that in our cell system Shc Y317F behaves like a dominant interfering mutant on the activation of the Grb2-Sos pathway by endogenous Shc triggered by Ret/ptc2. A strong reduction of the transforming activity of Ret/ptc2 in presence of this mutant was also demonstrated. Our data suggest that Shc activation play a key role in the transforming pathways triggered by Ret/ptc oncoproteins. Moreover, we have shown that coexpression of the Shc-Y317F mutant with Ret/ptc2 specifically causes apoptosis, and that the surviving cells lose the long-term expression of one of the two genes.

ret-derived oncogenes are frequently and specifically expressed in thyroid tumors. In contrast to the ret receptor, ret oncoproteins are characterized by ligand-independent tyrosine-kinase activity and tyrosine phosphorylation. In this study, novel synthetic arylidene 2-indolinone compounds were evaluated as inhibitors of the ret/ptc1 tyrosine kinase. Four compounds inhibited ret/ptc1 activity in immunokinase assay (IC50 27-42 microM) including one (1,3-dihydro-5,6-dimethoxy-3-[(4-hydroxyphenyl) methylene)-2H-indol-2-one) (Cpd 1) that selectively inhibited the anchorage-independent growth of NIH3T3 transformants expressing the ret/ptc1 gene (NIH3T3ptc1 cells). Following exposure to Cpd 1, the transformed phenotype of NIH3T3ptc1 cells was reverted, within 24 hr, to a normal fibroblast-like morphology in adherent-cell culture. In these cells, the constitutive tyrosine phosphorylation of ret/ptc1, of the transducing adaptor protein shc and of a series of co-immunoprecipitated peptides became much reduced, as demonstrated by immunoprecipitation/Western-blot analyses. Data presented provide additional evidence that ret/ptc1 is directly implicated in malignant transformation, and demonstrate the ability of Cpd 1 to interfere in the signal transduction pathway constitutively activated by the ret/ptc1 oncoprotein. These results confirm the interest of the arylidene 2-indolinone class of tyrosine-kinase inhibitors as tools for the study of ret signaling and the control of cell proliferation in ret- and ret/ptcs-associated diseases.

Mutations of the RET gene, encoding a receptor tyrosine kinase, have been associated with the inherited cancer syndromes MEN 2A and MEN 2B. They have also further been associated with both familial and sporadic medullary thyroid carcinomas. Missense mutations affecting cysteine residues within the extracellular domain of the receptor causes constitutive tyrosine kinase activation through the formation of disulfide-bonded homodimers. We have recently reported that a somatic 6 bp in-frame deletion, originally coding for Glu632-Leu633, potently activates the RET gene. This activation is increased with respect to the frequent MEN 2A-associated missense mutation Cys634Arg. This finding specifically correlated to the clinic behavior of the corresponding tumor, which was characterized by an unusually aggressive progression with both multiple and recurrent metastases. By examining the possibility that this deletion acts in a manner similar to cysteine substitution, we have analysed the molecular mechanism by which this oncogenic activation occurs. Phosphorylated dimers of the deleted Ret receptor were detected in immunoprecipitates separated under non-reducing conditions. Like other Cys point mutations, this 6 bp deletion affecting two amino acid residues between two adjacent Cys, is capable of activating the transforming ability of Ret by promoting receptor dimerization. These results suggest that alteration to cysteine residue position or pairing is capable of inducing ligand independent dimerization. Furthermore, we present data demonstrating that the processing and sorting of the Ret membrane receptor to the cell surface is affected by mutation type.

The high-affinity nerve growth factor receptor corresponds to the tyrosine protein kinase encoded by the proto-oncogene trkA. Different findings suggest that nerve growth factor (NGF) can be operative in the growth modulation of tumor cell lines possessing high-affinity binding sites for this molecule. Using as immunizing material the SKNBE neuroblastoma cell line transfected with proto-trkA we produced a monoclonal antibody (MAb) able to recognize the high-affinity nerve growth factor receptor. The selected MAb, designated MGR12, is directed against an epitope present on the extracellular domain of the receptor since it showed reactivity on living trkA-expressing cells and was able to immunoprecipitate the proto-trkA molecule. The MGR12 MAb is directed against a non-functional epitope since it neither inhibited NGF binding nor induced receptor internalization. This new reagent appears to be an appropriate tool for analyzing the expression of high-affinity nerve growth factor receptor in tumors of different origin and for elucidating its involvement in tumor progression.

The RET proto-oncogene encodes two isoforms of a receptor tyrosine kinase which plays a role in neural crest and kidney development. Ret ligands have been recently identified as the neuron survival factor GDNF (Glial-Derived Neurotrophic Factor) and Neurturin. Somatic rearrangements of RET, designated RET/PTCs, have been frequently detected in papillary thyroid carcinomas. In addition, distinct germ-line mutations of RET gene have been associated with the inherited cancer syndromes MEN (Multiple Endocrine Neoplasia) 2A, 2B and FMTC (Familial Medullar Thyroid Carcinomas) as well as with the congenital megacolon or Hirschsprung's disease, thus enlightening a significant role of this receptor gene in diverse human pathologic conditions. In this study, by performing classical inhibition experiments using synthetic phosphopeptides and by site-directed mutagenesis of the putative docking site, we have determined that for Grb2 the latter is provided by the tyrosine 620 of Ret/ptc2 long isoform (corresponding to Tyr 1096 on proto-Ret). However, in intact cells, the interaction of Grb2 with the two short and long Ret isoforms expressed separately is of similar strength, thus suggesting that Ret short isoform interaction with Grb2 could be mediated not only by Shc but also by a molecule that binds preferentially to this isoform. This possibility is supported by the evidence that the mutant Ret/ptc2Y620F long isoform displays a weak coimmunoprecipitation with Grb2 and that this mutant, lacking the docking site for Grb2 but owing all the others phosphotyrosines, surprisingly displays a reduced transforming activity compared to that of the two WTs oncogenes. We thus conclude that in intact cells both Ret isoforms bind to Grb2, although with different modalities. In addition, the present results are in agreement with the possibility that different signal transduction pathways are associated with the two isoforms of Ret.

Germline mutations of RET gene, encoding a receptor tyrosine kinase, have been associated with the MEN2A and MEN2B inherited cancer syndromes. In MEN2A mutations affecting cysteine residues in the extracellular domain of the receptor cause constitutive activation of the tyrosine kinase by the formation of disulfide-bonded homodimers. In MEN2B a single mutation in the tyrosine kinase domain (Met918Thr) has been identified. This mutation does not lead to dimer formation, but has been shown (both biologically and biochemically) to cause ligand-independent activation of the Ret protein, but to a lesser extent than MEN2A mutations. Intramolecular activation by cis-autophosphorylation of RetMEN2B monomers has been proposed as a model for activation, although alternative mechanisms can be envisaged. Here we show that the activity of RetMEN2B can be increased by stable dimerization of the receptor. Dimerization was achieved experimentally by constructing a double mutant receptor with a MEN2A mutation (Cys634Arg) in addition to the MEN2B mutation, and by chronic exposure of RetMEN2B-expressing cells to the Ret ligand GDNF. In both cases full activation of RetMEN2B, measured by 'in vitro' transfection assays and biochemical parameters, was seen. These results indicate that the MEN2B phenotype could be influenced by the tissue distribution or concentration of Ret ligand(s).

The RET proto-oncogene encodes a Tyrosine Kinase Receptor (RTK) which plays an important function in the proliferation and/or differentiation of neuroectodermic cells. Germline mutation of a methionine to a threonine within the RET TK domain predisposes to the Multiple Endocrine Neoplasia type 2B (MEN 2B). It has been demonstrated that, unlike c-Ret, the MEN 2B mutated Ret displays constitutive TK activity, tyrosine autophosphorylation and transforms fibroblasts. However, this oncoprotein is more than a fully activated wild-type (WT) Ret TK since it also displays modified substrate specificity. Change in substrate specificity leads to the tyrosine autophosphorylation of MEN 2B Ret on new sites as well as the phosphorylation of several novel downstream targets. But, none of these substrates have been identified and the ability of MEN 2B Ret phosphoprotein to interact with Src Homology 2 (SH2) domain containing molecules has been poorly investigated. In this report, using a constitutively activated Ret TK form, Ret-ptc 2, we demonstrate that the MEN 2B as the activated WT Ret TK binds to several SH2 signalling proteins such as Shc, Grb-2, Phospholipase Cgamma, Crk and Nck. However, in contrast to the activated WT form, expression of the MEN 2B mutated Ret-ptc 2 results in the tyrosine phosphorylation of a panel of proteins which interestingly interact with Crk and Nck. We identified Paxillin, a cytoskeletal protein as one of the Crk associated proteins that is dramatically phosphorylated in MEN 2B but not in WT Ret expressing cells. These data suggest that MEN 2B mutated Ret triggers distinct signalling pathways that might be related to its transforming power.

The RET proto-oncogene encodes two isoforms of a receptor type tyrosine kinase which plays a role in neural crest and kidney development. Distinct germ-line mutations of RET have been associated with the inherited cancer syndromes MEN2A, MEN2B and FMTC as well as with the congenital disorder Hirschsprung disease (HSCR), whereas somatic rearrangements (RET/PTCs) have been frequently detected in the papillary thyroid carcinoma. Despite these findings, suggesting a relevant role for RET product in development and neoplastic processes, little is known about the signalling triggered by this receptor. In this study, we have demonstrated that the transducing adaptor molecule Shc is recruited and activated by both Ret isoforms and by the rearranged cytoplasmatic Ret/ptc2 oncoproteins as well as by the membrane bound receptor activated by MEN2A or by MEN2B associated mutations. Moreover, our analysis has identified the Ret tyrosine residue and the Shc domains involved in the interaction. In fact, here we show that both the phosphotyrosine binding domains of Shc, PTB and SH2, interact with Ret/ptc2 in vitro. However, PTB domain binds 20 folds higher amount of Ret/ptc2 than SH2. The putative binding site for either SH2 and PTB domains has been identified as Tyr586 of Ret/ptc2 (Tyr1062 on proto-Ret). In keeping with this finding, by using RET/PTC2-Y586F mutant, we have demonstrated that this tyrosine residue, the last amino acid but one before the divergence of the two Ret isoforms, is the docking site for Shc.

RET/PTC oncogenes, generated by chromosomal rearrangements in papillary thyroid carcinomas, are constitutively activated versions of proto-RET, a gene coding for a receptor-type tyrosine kinase (TK) whose ligand is still unknown. RET/PTCs encode fusion proteins in which proto-RET TK and C-terminal domains are fused to different donor genes. The respective Ret/ptc oncoproteins display constitutive TK activity and tyrosine phosphorylation. We found that Ret/ptcs associate with and phosphorylate the SH2-containing transducer phospholipase Cgamma (PLCgamma). Two putative PLCgamma docking sites, Tyr-505 and Tyr-539, have been identified on Ret/ptc2 by competition experiments using phosphorylated peptides modelled on Ret sequence. Transfection experiments and biochemical analysis using Tyr-->Phe mutants of Ret/ptc2 allowed us to rule out Tyr-505 and to identify Tyr-539 as a functional PLCgamma docking site in vivo. Moreover, kinetic measurements showed that Tyr-539 is able to mediate high-affinity interaction with PLCgamma. Mutation of Tyr-539 resulted in a drastically reduced oncogenic activity of Ret/ptc2 on NIH 3T3 cells (75 to 90% reduction) both in vitro and in vivo, which correlates with impaired ability of Ret/ptc2 to activate PLCgamma. In conclusion, this paper demonstrates that Tyr-539 of Ret/ptc2 (Tyr-761 on the proto-RET product) is an essential docking site for the full transforming potential of the oncogene. In addition, the present data identify PLCgamma as a downstream effector of Ret/ptcs and suggest that this transducing molecule could play a crucial role in neoplastic signalling triggered by Ret/ptc oncoproteins.