Novel pathogenic mechanisms of congenital insensitivity to pain with anhidrosis genetic disorder unveiled by functional analysis of neurotrophic tyrosine receptor kinase type 1/nerve growth factor receptor mutations.

Congenital insensitivity to pain with anhidrosis (CIPA) is a rare genetic disease characterized by absence of reaction to noxious stimuli and anhidrosis. The genetic bases of CIPA have remained long unknown. A few years ago, point mutations affecting both coding and noncoding regions of the neurotrophic tyrosine receptor kinase type 1 (NTRK1)/nerve growth factor receptor gene have been detected in CIPA patients, demonstrating the implication of the nerve growth factor/NTRK1 pathway in the pathogenesis of the disease. We have previously shown that two CIPA mutations, the G571R and the R774P, inactivate the NTRK1 receptor by interfering with the autophosphorylation process. We have extended our functional analysis to seven additional NTRK1 mutations associated with CIPA recently reported by others. Through a combination of biochemical and biological assays, we have identified polymorphisms and pathogenic mutations. In addition to the identification of residues important for NTRK1 activity, our analysis suggests the existence of two novel pathogenic mechanisms in CIPA: one based on the NTRK1 receptor processing and the other acting through the reduction of the receptor activity

Role of STAT3 in In Vitro Transformation Triggered by TRK Oncogenes

TRK oncoproteins are chimeric versions of the NTRK1/NGF receptor and display constitutive tyrosine kinase activity leading to transformation of NIH3T3 cells and neuronal differentiation of PC12 cells. Signal Transducer and Activator of Transcription (STAT) 3 is activated in response to cytokines and growth factors and it has been recently identified as a novel signal transducer for TrkA, mediating the functions of NGF in nervous system. In this paper we have investigated STAT3 involvement in signalling induced by TRK oncogenes. We showed that TRK oncogenes trigger STAT3 phosphorylation both on Y705 and S727 residues and STAT3 transcriptional activity. MAPK pathway was involved in the induction of STAT3 phosphorylation. Interestingly, we have shown reduced STAT3 protein level in NIH3T3 transformed foci expressing TRK oncogenes. Overall, we have unveiled a dual role for STAT3 in TRK oncogenes-induced NIH3T3 transformation: i) decreased STAT3 protein levels, driven by TRK oncoproteins activity, are associated to morphological transformation; ii) residual STAT3 transcriptional activity is required for cell growth

Targeting COPZ1 non-oncogene addiction counteracts the viability of thyroid tumor cells

Thyroid carcinoma is generally associated with good prognosis, but no effective treatments are currently available for aggressive forms not cured by standard therapy. To find novel therapeutic targets for this tumor type, we had previously performed a siRNA-based functional screening to identify genes essential for sustaining the oncogenic phenotype of thyroid tumor cells, but not required to the same extent for the viability of normal cells (non-oncogene addiction paradigm). Among those, we found the coatomer protein complex ζ1 (COPZ1) gene, which is involved in intracellular traffic, autophagy and lipid homeostasis. In this paper, we investigated the mechanisms through which COPZ1 depletion leads to thyroid tumor cell death. We showed that siRNA-mediated COPZ1 depletion causes abortive autophagy, endoplasmic reticulum stress, unfolded protein response and apoptosis. Interestingly, we observed that mouse tumor xenografts, locally treated with siRNA targeting COPZ1, showed a significant reduction of tumor growth. On the whole, we demonstrated for the first time the crucial role of COPZ1 in the viability of thyroid tumor cells, suggesting that it may be considered an attractive target for novel therapeutic approaches for thyroid cancer

Genomic and transcriptomic analyses of thyroid cancers identify DICER1 somatic mutations in adult follicular-patterned RAS-like tumors

BackgroundPapillary 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 BRAFV600E, 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.MethodsHere, 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).ResultsThe occurrence of BRAFV600E (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.ConclusionEven 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

STAT3 activation by TRK oncogenes.

<p>(<b>A</b>) Western blot analysis of PC12 cells transfected with empty pRC/CMV vector, TRKA, TRK-T3, and TRK cDNAs. NGF treatment (50ng/ml, 10′) is indicated. Cell lysates and immunocomplexes were separated by SDS PAGE as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009446#s4" target="_blank">Material and Methods</a> and immunoblotted with indicated antibodies. In the bottom panel two distinct exposures of the same blot were used for documenting TRKA or TRK oncoproteins phosphorylation. (<b>B</b>) HeLa cells were co-transfected with pM67 and pRL-TK in combination with the indicated TRK oncogene cDNAs in the absence (left graph) or presence (right graph) of STAT3 cDNA and assayed for STAT3-dependent luciferase activity 48 hours later. Activity is expressed as the ratio of luciferase/renilla activity, and reported as fold-inductions over empty vector (left panel) or STAT3 (right panel) (RLA: Relative Luciferase Activity). The data represent the mean values ± SD of triplicate samples. Similar results were obtained in three independent experiments. (<b>C</b>) MAPK involvement in TRK-induced Stat3 phosphorylation. Western blot analysis of PC12 cells transfected with empty pRC/CMV vector, TRK-T3 or TRK cDNAs. NGF (10′, 50ng/ml) and UO126 (16 hr, 10 µM) treatments are indicated. Immunoblotting was performed with the indicated antibodies.</p

Role of Stat3 in TRK-induced cell growth of NIH3T3 derived foci.

<p>(<b>A</b>) Phase contrast images of NF861, NF797 and NIH3T3 cells treated or not with S3I-201 (100 µM, 48 hr). (<b>B</b>) Cell viability of NF861 and NF797 cells treated with DMSO (0.3%) or S3I-201 (100 µM) determined by the Alamar Blue cell viability assay. (<b>C</b>) Western blot analysis of NIH3T3, NF861 and NF797 cells treated with S3I-201 (100 µM, 48 hr). Samples were immunoblotted with the indicated antibodies.</p

Reduction of Stat3 level is associated to morphological transformation induced by TRK oncogenes activity.

<p>(<b>A</b>) NIH3T3, NF861 and NF797 cells were treated with K252a (200nM) for the indicated time; cell lysates were processed and immunoblotted with the indicated antibodies. Densitometric analysis of the bands is reported in the bottom panel. Images were acquired by Biorad ChemiDoc and analysed with Image Quant software. Data are reported as ratio of STAT3/vinculin and normalized over untreated samples for any cell line. (<b>B</b>) Cell extracts from NIH3T3, NF861 and NF797 cells treated with K252a (200nM, 16 hr) were analyzed by Western blot with the indicated antibodies. (<b>C</b>) Cell extracts from NIH3T3, NF861 and NF797 cells treated with UO126 (10 µM, 16 hr) were analyzed by Western blot with the indicated antibodies. (<b>D</b>) Phase contrast images (top) and α-tubulin immunostaining (bottom) of NIH3T3 cells, NF797 cells treated or not with K252a (200nM, overnight) or UO126 (10 µM).</p

Senescent Thyrocytes, Similarly to Thyroid Tumor Cells, Elicit M2-like Macrophage Polarization In Vivo

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