Oncogenic rearrangements of the NTRK1 gene (also designated TRKA), encoding one of the receptors for the nerve growth factor, are frequently detected in thyroid carcinomas. Such rearrangements fuse the NTRK1 tyrosine kinase domain to 5-end sequences belonging to different genes. In previously reported studies we have demonstrated that NTRK1 oncogenic activation involves two genes, TPM3 and TPR, both localized similarly to the receptor tyrosine kinase, on the q arm of chromosome 1. Here we report the characterization of a novel NTRK1-derived thyroid oncogene, named TRK-T3. A cDNA clone, capable of transforming activity, was isolated from a transformant cell line. Sequence analysis revealed that TRK-T3 contains 1,412 nucleotides of NTRK1 preceded by 598 nucleotides belonging to a novel gene that we have named TFG (TRK-fused gene). The TRK-T3 amino acid sequence displays, within the TFG region, a coiled-coil motif that could endow the oncoprotein with the capability to form complexes. The TRK-T3 oncogene encodes a 68-kDa cytoplasmic protein reacting with NTRK1-specific antibodies. By sedimentation gradient experiments the TRK-T3 oncoprotein was shown to form, in vivo, multimeric complexes, most likely trimers or tetramers. The TFG gene is ubiquitously expressed and is located on chromosome 3. The breakpoint producing the TRK-T3 oncogene occurs within exons of both the TFG gene and the NTRK1 gene and produces a chimeric exon that undergoes alternative splicing. Molecular analysis of the NTRK1 rearranged fragments indicated that the chromosomal rearrangement is reciprocal and balanced and involves loss of a few nucleotides of germ line sequences.Chromosomal rearrangements producing chimeric oncogenes are frequently associated with human cancer, and several lines of evidence suggest that they are involved in the pathogenesis of their respective tumors (31).Among solid tumors, papillary thyroid carcinoma provides a unique model of a frequent generation of chimeric oncogenes. In this type of neoplasia, in fact, two proto-oncogenes, RET and NTRK1 (also named TRKA), have been found rearranged in about 50% of the samples assayed (5). RET and NTRK1 both encode transmembrane receptor tyrosine kinases (RTKs), whose expression is highly tissue specific, being restricted to specific components of the peripheral nervous system (1, 38, 40). The common mechanism of activation is represented by somatic rearrangements juxtaposing their tyrosine kinase (TK) domain to 5Ј-end sequences derived from unrelated loci and producing chimeric oncogenes whose products display a constitutive and ectopic TK enzymatic activity (29).The NTRK1 gene encodes one of the receptors for the nerve growth factor (NGF) (15, 16) and is located on the q arm of chromosome 1 (24, 26). NTRK1 was originally detected as an oncogene in a human colon carcinoma; this activated version of the gene was generated by a chromosomal rearrangement fusing the NTRK1 TK domain with sequences of a tropomyosin gene, TPM3, and was designated the ''TRK oncogene'' (21).Afterwards, ...

Purpose: Gastrointestinal stromal tumors (GIST) are characterized by gain-of-function mutations in KIT/ PDGFRA genes leading to a constitutive receptor activation which is well counteracted by imatinib. However, cases in which imatinib as first-line treatment has no effects are reported (primary resistance). Our purpose is to investigate alterations in downstream effectors, not reported so far in mutated GIST, possibly explaining the primary resistance to targeted treatments.Experimental Design: Two independent naive GIST cohorts have been analyzed for KIT, PDGFRA, KRAS, and BRAF mutations by direct sequencing. Cell lines expressing a constitutively activated and imatinibresponding KIT, alone or in combination with activated KRAS and BRAF, were produced and treated with imatinib. KIT receptor and its downstream effectors were analyzed by direct Western blotting.Results: In naive GISTs carrying activating mutations in KIT or PDGFRA a concomitant activating mutation was detected in KRAS (5%) or BRAF (about 2%) genes. In vitro experiments showed that imatinib was able to switch off the mutated receptor KIT but not the downstream signaling triggered by RAS-RAF effectors.Conclusions: These data suggest the activation of mitogen-activated protein kinase pathway as a possible novel mechanism of primary resistance to imatinib in GISTs and could explain the survival curves obtained from several clinical studies where 2% to 4% of patients with GIST treated with imatinib, despite carrying KIT-sensitive mutations, do not respond to the treatment.

IntroductionAngiogenesis has a critical role in the pathophysiology and progression of multiple myeloma (MM) supporting the growth and survival of MM cells. [1][2][3][4][5] The angiogenic process in MM is sustained mainly by the overexpression of proangiogenic factors directly by MM cells including VEGF, 6 angiopoietin-1 (ANG-1), 7 osteopontin (OPN), . 9 Nevertheless, the molecular mechanisms underlying the regulation of angiogenesis in MM have not been completely elucidated.The new candidate tumor-suppressor gene inhibitor of growth family member 4 (ING4) has been recently implicated in solid tumors as a repressor of tumor growth and angiogenesis through the association with NF-B. ING4 is a nuclear factor expressed in all normal tissues and markedly reduced in glioblastoma cells and head and neck squamous cell carcinoma, with levels inversely correlated with tumor grade. 10,11 Inhibition of ING4 expression strongly promotes the growth of glioma cells in vivo, whereas its overexpression leads to growth inhibition through ING4's capability to interact with p65 subunit of NF-B. 10 Interestingly, it has been also shown that tumors lacking ING4 showed increased vascularization compared with ING4-expressing tumors. 12 Moreover ING4 down-regulated the angiogenic-related molecules including IL-8 and the hypoxia inducible factor-1␣ (HIF-1 ␣) activity in hypoxic condition through the involvement of HIF prolyl hydroxylase 2 (HPH-2) 10,13 In turn, the role of hypoxia has been recently highlighted in the promotion of angiogenesis. 14 The expression of ING4 by MM cells, as well as its potential role in MM-induced angiogenesis, has never been investigated. In this study, we evaluated the expression of ING4 in malignant MM cells and the potential relationship between ING4 and the production of proangiogenic molecules by MM cells in normoxic and hypoxic conditions, as well as its relationship with the "in vitro and in vivo" angiogenesis. Submitted February 15, 2007; accepted September 4, 2007. Prepublished online as Blood First Edition paper, September 11, 2007; DOI 10.1182 DOI 10. /blood-2007 The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on August 29, 2018. by guest www.bloodjournal.org From Patients, materials, and methods Cells and cell culture conditionsCell lines. Human myeloma cell lines (HMCLs) XG-6, XG-1, and JJN3 were obtained from Dr Bataille (Nantes, France). U266 was obtained from the American Type Culture Collection (Rockville, MD). OPM2 and RPMI-8226 were purchased from DSM (Braunschweig, Germany). ARP-1 and H929 were generously received from Dr Shaughnessy's laboratory (Little Rock, AR).Cell cultures. HMCLs were incubated in RPMI medium at 10% FCS (Invitrogen Life Technologies, Milan, Italy) and maintained with or without IL-6 (3 ng/mL; Endogen Woburn, MA). In a series of experiments, HMCLs were incubated with the HPH-2 in...

No abstract

We have introduced three Hirschsprung (HSCR) mutations localized in the tyrosine kinase domain of RET into the RET/PTC2 chimaeric oncogene which is capable of transforming NIH3T3 mouse fibroblasts and of differentiating pC12 rat pheochromocytoma cells. The three HSCR mutations abolished the biological activity of RET/PTC2 in both cell types and significantly decreased its tyrosine phosphorylation. By contrast, a rare polymorphism in exon 18 does not alter the transforming capability of RET/PTC2 or its tyrosine phosphorylation. These data suggest a loss of function effect of HSCR mutations which might act through a dominant negative mechanism. Our model system is therefore capable of discriminating between causative HSCR mutations and rare polymorphisms in the tyrosine kinase domain of RET.

RET and NTRK1 are receptor tyrosine kinase (RTK) proteins which play a role in the development and maturation of specific component of the nervous system. Their alterations have been associated to several human diseases, including some forms of cancer and developmental abnormalities. These features have contributed to the concept that one gene can be responsible for more than one disease. Moreover, both genes encoding for the two RTKs show genetic alterations that belong to either "gain of function" or "loss of function" class of mutations. In fact, receptor rearrangements or point mutations convert RET and NTRK1 in dominantly acting transforming genes leading to thyroid tumors, whereas inactivating mutations, associated with Hirschsprung's disease (HSCR) and congenital insensitivity to pain with anhidrosis (CIPA), impair RET and NTRK1 functions, respectively. In this review we have summarized the main features of the two receptors, their physiological and pathological roles. In addition, we attempted to identify the correlations between the different genetic alterations and the related pathogenetic mechanisms.

The molecular pathogenesis of tumors arising from the thyroid follicular epithelial cells, including papillary (PTC) and follicular thyroid carcinoma (FTC), is only partially understood, and the role of tumor suppressor genes has not yet been assessed. The metallothionein (MT) gene family encodes a class of metal-binding proteins involved in several cellular processes, and their expression is often deregulated in human tumors. Recently, downregulation of MT gene expression in PTC has been reported, suggesting a possible oncosuppressor role of this gene family in the pathogenesis of thyroid tumors. To further explore this possibility, we performed expression and functional studies. Analysis of microarray data of thyroid tumors of different histologic types showed that several MT genes were downregulated with respect to normal tissue. The microarray data were corroborated by quantitative PCR experiments, showing downregulation of MTs in PTC and FTC, but to a greater extent in papillary carcinoma. The expression of MTs was also investigated at the protein level by immunohistochemistry; the results were consistent with the microarray data, showing general downregulation in tumor samples, which was more evident in PTC. The functional consequence of MT downregulation was addressed employing an experimental model made of the PTC-derived K1 cell line in which MT1G expression is repressed by promoter methylation. Restoration of MT1G expression by cDNA transfection affected growth rate and in vivo tumorigenicity of K1 cells, indicating an oncosuppressor role for MT1G in thyroid papillary tumorigenesis. Several tumor types, differing in biological and clinical behavior, originate from the thyroid epithelial follicular cells. They include well-differentiated, indolent papillary thyroid and follicular thyroid carcinomas (PTC and FTC), as well as extremely aggressive anaplastic carcinoma. 1 Studies performed in several laboratories, including ours, have demonstrated that distinct molecular events are associated with specific tumor types. 2 FTC is characterized by the PAX8/ PPARg rearrangement and activating mutations of RAS genes. 1 PTC is associated with rearrangements involving the RET and NTRK1 tyrosine kinase receptors, 2 and the V600E BRAF-activating mutation. [3][4][5] More recently, microarray studies have identified several genes that might be important in the molecular pathogenesis and the malignant progression of thyroid cancer and could be used as diagnostic or prognostic molecular markers. These candidate genes are involved in several different processes, such as cell adhesion, cell cycle progression, mitogenic control and tumorigenesis. [6][7][8][9][10] In spite of that, the molecular pathogenesis of thyroid cancer is still incomplete; in particular, a role of tumor suppressor genes has not yet been assessed. Metallothioneins (MTs) are low-molecular weight proteins of 6-7 kDa, with high content of cysteine (30%) and complete absence of aromatic amino acids and histidine 11 capable of binding heavy metals with hig...

The thyroid TRK-T3 oncogene results from the fusion of the tyrosine kinase (TK) domain of NTRK1 (one of the receptors for the Nerve Growth Factor) on chromosome 1 to sequences of a novel gene, TFG, on chromosome 3. The 68 kDa TRK-T3 fusion oncoprotein displays a constitutive tyrosine kinase activity resulting in its capability to transform mouse NIH3T3 cells. The TFG portion of TRK-T3 contains a coiled-coil domain most likely responsible for the constitutive, ligand-independent activation of the receptor tyrosine kinase activity. We have previously shown that TRK-T3 oncoprotein forms, in vivo, complexes of three or four molecules. By mean of di erent experimental approaches, we show here that TRK-T3 activity depends on oligomers formation. In addition, the analysis of di erent TRK-T3 mutants indicates that the TFG coiled-coil domain and its Nterminal region are both required for the activation and the fully transforming activity of the TRK-T3 oncoprotein, although, most likely, they play a role in di erent steps of the transforming process. The deletion of the coiled-coil domain abrogates the oligomers formation leading to a constitutive activation; the deletion of the Nterminal region, although not a ecting phosphorylation and complexes formation, abrogates transformation, thus suggesting a role in cellular localization and/or interaction with substrata.