RET is a transmembrane receptor that is implicated in a variety of processes such as cell proliferation, differentiation, migration, survival, and death. One of the proteins that is activated downstream of RET is the guanine nucleotide exchange factor (GEF), BetaPIX. BetaPIX removes GDP bound to inactive Cdc42/Rac, freeing a space for GTP-binding and transformation of Cdc42/Rac to the active GTPase state. BetaPIX is vital in cytoskeletal rearrangements and the regulation of focal adhesion complex assembly and disassembly. Although our lab has previously shown that RET and BetaPIX do not directly interact, the mechanism through which BetaPIX is activated downstream of RET is not yet known. In the current study, we confirmed that RET activation mediates BetaPIX phosphorylation. We showed that mutations in the SH3 and Dbl-homology domains of BetaPIX result in slower rates and in some cases impairment of wound healing and cell migration downstream of RET, indicating a role for BetaPIX in RET-mediated cell migration. In contrast, we have shown that GTPase mutants of Cdc42 and Rac were not sufficient to impair wound healing, but did result in less cell migration. This suggests that similar, yet distinct roles exist for Cdc42 and Rac in the regulation of cytoskeletal dynamics. We have shown that SH3 and Dbl-homology domain mutations in BetaPIX result in changes in cell morphology, suggesting a role of BetaPIX in the development of cell extensions downstream of RET. Alternatively, we have shown that GTPase mutants of Cdc42 or Rac alone do not induce cell morphology changes, further emphasizing the similar, yet distinct roles for Cdc42 and Rac in RET-mediated cell migration. Lastly, we have shown that BetaPIX is re-localized from the focal adhesion to the cytoplasm upon RET activation, suggesting that following focal adhesion complex formation, BetaPIX may be recycled. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2009-09-23 13:05:57.86

The aim of this thesis is to evaluate the expression of both TSG101 tumor susceptibility gene and ret oncogene in human thyroid carcinoma specimens. Functional inactivation of TSG101 in mouse fibroblast leads to cellular transformation and the ability to form metastatic tumors in nude mice. No genomic deletion of TSG101 gene has been reported in human cancer, casting a doubt on the role of TSG101 as a classical tumor suppressor. Subsequent studies reveal that TSG101 is a frequent target of spilicing defects, which is correlated with cellular stress and p53 status, and might reflect the cellular environment during the cancer development. Furthermore, recent reports demonstrate TSG101 as a part of the MDM2/p53 regulatory circuitry, a well recognized circuitry that upon deregulation results in tumorigenesis. In this study we have analyzed TSG101 gene expression in 85 specimens of thyroid carcinomas. The results indicated that 100% of papillary carcinomas (48/48), 85% of follicular carcinomas (18/21), 91% of medullary carcinomas (10/11) and 60% of undifferentiated carcinomas (3/5) showed strong to moderate cytoplasmic staining, whereas the staining was completely negative, or cytoplasmic dot-staining in the adjacent non-neoplastic follicular cells. Occasionally, the staining could be found in the nucleus. Subsequently, sequence analysis of 17 papillary carcinoma specimens revealed no mutation in steadiness box region, indicating that it might not be the cause of TSG101 protein overexpression. In summary, our results indicate strong correlation of TSG101 overexpression and thyroid carcinomas. Further experiments are urged to clarify the relationship of TSG101 overexpression and thyroid tumorigenesis. Rearrangement of ret proto-oncogene is unique to papillary thyroid carcinoma (PTC). These rearrangements consist of the fusion of ret tyrosine kinase domain to a variety of heterologous genes, thus generating chimeric transforming oncogenes termed, ret/PTC. The frequency of ret/PTC activation in non-radiation exposured adult populations has been reported to vary from 0-55% depending on the geographic distribution. To detect ret rearrangement and to identify candidate of novel ret/PTC in 62 specimens of PTC collected from southern Taiwan, a RT-multiplex PCR method was used to reveal the possible specimens that harbor ret rearrangements. Type specific-PCR amplification and subsequent sequence analysis of PCR product were performed to identify the known types of ret/PTC. We have identified two cases of ret/PTC1, two cases of ret/PTC3 and one case of ELKS-RET. Excitingly, four cases of unknown ret/PTC type were identified. Hence, 5¡¦-RACE strategy will be employed to identify novel ret/PTC in these four specimens.

The RET proto-oncogene encodes a receptor tyrosine kinase (RTK), with two major isoforms, RET9 and RET51, which differ in their C-termini, and therefore recruit different signaling complexes. RET plays an important role in cell growth, differentiation, migration and survival. Regulation of RET is critical for normal cellular functioning, however, the biochemical mechanisms underlying the downregulation of RET isoforms, are still not clear. Cbl (Casitas B-lineage Lymphoma) is an E3-ubiquitin ligase that plays an essential role in mediating the degradation of RET. Recently, a negative regulator of Cbl, ARHGEF7 (β-pix/ Cool-1) was found to prevent Cbl-catalyzed deregulation of the Epidermal Growth Factor Receptor (EGFR). In the current study, we characterized and further examined the association between RET and Cbl. We showed that RET can associate with the c-Cbl and Cbl-b homologues, in co-immunoprecipitations. Using far western assays and GST-pulldowns, with the purified tyrosine kinase binding (TKB) domain of c-Cbl, we detected a potential novel direct interaction between RET and c-Cbl. Previously, an indirect association between RET and Cbl had been established, indicating that a bimodal interaction may occur. Furthermore, we proposed that ARHGEF7 may interfere with RET-Cbl interactions, either by sequestring Cbl, so that it is unable to bind to RET, or by forming a complex with both RET and Cbl, thereby blocking Cbl activation. Here, we investigated the possibility of a complex formation using co-immunoprecipitations. We showed that ARHGEF7 and c-Cbl can co-immunoprecipitate, but we could not detect either of the RET isoforms in this complex. Further examination of a possible relationship between RET isoforms, and ARHGEF7, showed that ARHGEF7 phosphorylation was dependent on RET activation. However, in an in vitro kinase assay, we showed that this phosphorylation did not occur directly, but may occur indirectly through a pathway yet unknown. Our data predicts that ARHGEF7 may modulate Cbl-binding to RET, and subsequently inhibit its degradation, in a manner similar to that seen for EGFR. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-07-14 14:15:31.472

The RET proto-oncogene encodes a receptor tyrosine kinase (RTK) that is widely expressed in neuroendocrine tissues and is essential for embryonic development of the kidney and enteric nervous system. Mutations leading to constitutive activation of the RET protein underlie various tumours of endocrine tissues. Conversely, loss-of-function mutations of RET lead to Hirschsprung disease, a congenital disorder characterized by a loss of enteric neurons throughout the colon and small intestine. Intracellular trafficking of RTKs through multiple cellular compartments has been shown to impact on downstream signalling. To date, the intracellular trafficking of RET has not been investigated. Here, we show that RET is rapidly internalized after activation and that trafficking to cytoplasmic endosomes plays an important role in downstream signalling. RET is alternatively spliced into multiple isoforms that are co-expressed in cells; therefore, we further investigated RET internalization in an isoform-specific context. This study revealed a number of differences between RET isoforms including differences in sub-cellular localization pre-activation, rate of internalization, and ability to recycle to the plasma membrane. Differential trafficking of RET isoforms alter their downstream signalling properties, providing an additional mechanism to explain the distinct contributions of RET isoforms to cellular processes. Finally, we investigated the impact of altered sub-cellular localization in the context of thyroid carcinoma. Activation of RET has been implicated in a number of thyroid tumours that differ in their inherent oncogenicity. We observed that altered subcellular localization of oncogenic forms of RET, RET/PTCs, enhance their oncogenicity. Interestingly, RET/PTC tumours are indolent and rarely metastasize compared to other RET-mediated forms of cancer. Further investigation revealed that RET/PTC oncogenes are expressed off relatively weak promoters, resulting in quantitatively less RET/PTC oncoprotein expression in these tumours compared to mutant RET expression in more aggressive cancers. Together, our results represent the first in-depth study of the trafficking properties of RET and indicate the importance of proper sub-cellular localization and trafficking in the maintenance of normal cell metabolism. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2009-11-19 22:51:47.38

No description available.

The REarranged in Transfection (RET) tyrosine kinase is an important signalling protein for the development of neural crest-derived tissues such as the enteric and sympathetic nervous systems. RET is constitutively activated in multiple human tumour types, such as thyroid carcinomas and some non-small cell lung cancers. RET has 3 distinct isoforms, RET9, RET43 and RET51, which are named after the lengths of their unique C-terminal tails. Here, we investigate the role of RET in the TT thyroid carcinoma cell line, where it is a driver of tumourigenesis, and in the MiaPaCa-2 pancreatic carcinoma cell line, where RET is not driving tumour initiation, but may nonetheless have a profound effect on tumour progression. We generated lentiviral constructs for shRNAs that target either RET9 or RET51 specifically, or a common region shared by all RET isoforms. TT and MiaPaCa-2 cells were transduced using these lentiviral particles to create stable cell lines containing knockdowns of total RET, RET9, or RET51. Using a variety of morphological and biochemical assays, we found that RET expression is critical for TT cell survival, and that both RET9 and RET51 play significant roles in driving cell proliferation in TT cells. Conversely, RET is not critical for MiaPaCa-2 cell survival, and RET knockdown had no effect on MiaPaCa-2 proliferation. MiaPaCa-2 cells instead underwent dramatic morphological changes, from their normal spindle-like mesenchymal appearance to an increasingly flattened and epithelioid character, in response to RET9, RET51 or total-RET knockdown. The observed morphological changes were coupled with significantly reduced invasiveness through matrigel towards a source of chemoattractant, suggesting a critical role for RET in mediating cell invasiveness. These results suggest that RET may not only drive tumourigenesis, but can also enhance disease progression when expressed in other tumour types. We predict that RET may play critical roles in perineural invasion in pancreatic cancers, a process where cells invade along peripheral nerve fibers by following an increasing concentration of chemoattractants secreted by nerve and glial cells. Thus, RET may be a valuable target to slow, or stop this process, which would have significant clinical implications in a wide variety of cancers. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2013-08-30 11:45:41.969

La gènesi i manteniment del sistema nerviós estan regulats per una sèrie de factors neurotròfics, d' entre els quals destaquen les neurotrofines (NGF, BDNF, NT-3 i NT-4/5) i els lligands de la família del GDNF o GFLs (GDNF, NRTN, PSPN i ARTN). Durant els darrers anys, s'han anat caracterizant les cascades de senyalització intracel·lular responsables dels efectes biològics exercits per aquests factors, amb especial atenció a l'NGF i les vies MEK/MAPK i PI 3-K/Akt, dues de les millors caracteritzades. En aquest treballs'han analitzat les vies de traducció de senyal engegades per TrkB (el receptor de BDNF i NT-4/5) i Ret el component comú del receptor per a tots els GFLs) i la seva participació en els processos de supervivència i creixement neurític en diversos paradigmes experimentals. En el cas de TrkB, i utilizant com a modelcèl·lules de neuroblastoma humà SH-SY5Y pre-tractades amb àcid retinoïc (RA), s'ha determinat que la via PI3-K/Akt és el principal mitjançador de la supervivència cel·lular, mentre que la via MEK/MAPK és necessària per als processos de creixement neurític. A més, s'ha caracteritzat el fenotip de les cèl·lules H-SY5Y pre-tractades amb RA i exposades durant temps llargs a BDNF, tot observant-se que aquestes es diferencien cap a un fenotip neuronal, expressant diversos marcadors neuronals específics i restant aturades en la fase G1 del cicle cel·lular. En el cas de Ret, s'ha observat que l'activitat PI 3-K és necessària per a la supervivència de motoneurones espinals de pollastre, neurones granulars del cerebel i neurones del gangli cervical superior, mentre que la via MEK/MAPK no sembla ésser important en els processos de supervivència. A més, c-Src sembla ser un mitjançador important en la senyalització per GFLs, estant implicat tant en la supervivència (actuant per sobre de PI 3-K) i el creixement neurític. La implicació de c-Src en aquests procesos, a més, podria ser específica dels GFLs, donat que aquesta proteïna no sembla estar implicada en la supervivència induïda per NGF. / La génesis y mantenimiento del sistema nervioso están regulados por una serie de factores neurotróficos, de entre los que destacan las neurotrofinas (NGF, BDNF, NT-3 y NT-4/5) y los ligandos de la familia del GDNF o GFLs (GDNF, NRTN, PSPN y ARTN). Durante los últimos años, se han ido caracterizando lascascadas de señalización intracelular responsables de los efectos biológicos ejercidos por estos factores, con especial atención al NGF y las vías MEK/MAPK y PI 3-K/Akt, dos de las mejores caracterizadas. En el presente trabajo se han analizado las vías de traducción de señal iniciadas por TrkB (el receptor de BDNF y NT-4/5) y Ret (el componente común del receptor para todos los GFLs) y su participación en los procesos de supervivencia y crecimiento neurítico en diversos paradigmas experimentales. En el caso deTrkB, y utilizando como modelo células de neuroblastoma humano SH-SY5Y pretratadas con ácido retinoico (RA), se ha determinado que la vía PI3-K/Akt es el principal mediador de la supervivencia celular, mientras que la vía MEK/MAPK es necesaria para los procesos de crecimiento neurítico. Además, se ha caracterizado el fenotipo de las células SH-SY5Y pretratadas con RA y expuestas durante tiempos largos a BDNF, observándose que éstas se diferencian hacia un fenotipo neuronal, expresando varios marcadores neuronales específicos y quedando detenidas en la fase G1 del ciclo celular. En el caso de Ret, se ha observado que la actividad PI 3-K es necesaria para la supervivencia de motoneuronas espinales de pollo, neuronas granulares del cerebelo y neuronas del ganglio cervical superior, mientras que la vía MEK/MAPK no parece ser importante en los procesos de supervivencia.Además, c-Src parece ser un mediador importante en la señalización por GFLs, estando implicado tanto en la supervivencia (actuando por encima de PI 3-K) y el crecimiento neurítico. La implicación de c-Src en estos procesos, además, podría ser específica de los GFLs, dado que esta proteína no parece estarimplicada en la supervivencia mediada por NGF. / The generation and maintenance of the nervous system is regulated by a series of neurotrophic factors that include the neurotrophins (NGF, BDNF, NT-3, and NT-4/5) and the GDNF family ligands (GFLs; GDNF, NRTN, PSPN, and ARTN). During the last years, the signaling cascades responsible for the biologic effects exerted by these factors are being characterized, with special attention to NGFand MEK/MAPK and PI 3-K/Akt pathways. In this work we have analyzed the transduction pathways engaged by TrkB (the receptor for BDNF and NT-4/5) and Ret (the common component of the receptor for GFLs) and their involvement in the processes of survival and neurite outgrowth in several experimental paradigms. Using SH-SY5Y human neuroblastoma cells pretreated with retinoic acid (RA) as a model system, we have determined that PI 3-K/Akt pathway is necessary for TrkB-induced survival, whereas MEK/MAPK pathway is necessary for neurite outgrowth. Moreover, we have characterizedthe phenotype of these cells after sequential RA pre-treatment and long-term BDNF exposure. Cells treated in that way differentiate towards a neuronal phenotype, express several neuronal markers, and are arrested at the G1 phase of the cell cycle. We have also observed that PI 3-K/Akt, but not MEK/MAPK pathway, is necessary for Ret-mediated survival in chicken spinal cord motor neurons, as well as in rat cerebellar and sympathetic neurons. Moreover, c-Src appears to mediate GFLs signaling, being involved in both neuronal survival (acting upstream of PI 3-K) and neurite outgrowth. The involvement of c-Src in such processes seems to be specific for GFLs, since this kinase is not needed for NGF-mediated survival.

The RET receptor tyrosine kinase is important during development of neural crest-derived tissues, particularly the enteric and sympathetic nervous systems, and in kidney morphogenesis. Activation of RET requires complex formation between a member of the Glial cell line-Derived Neurotrophic Factor family of ligands and a member of the GDNF-Family Receptor α co-receptors. Upon complex formation, RET becomes phosphorylated and subsequently activates multiple downstream signaling pathways, including those for cell-survival, differentiation, and migration. Mutations in RET can either interfere with or enhance normal RET signaling. Inhibiting RET mutations are associated with development of Hirschsprung disease, which is characterized by a lack of mature ganglia in the gut. Conversely, activating RET mutations are associated with several thyroid cancers. Papillary thyroid carcinoma is frequently associated with sporadic translocations between RET and other genes, known collectively as RET/PTC. A variety of heritable RET missense mutations lead to Multiple Endocrine Neoplasia type 2, which is associated with development of medullary thyroid carcinoma. Two cellular processes disrupted downstream of RET in these diseases are gene-expression and cell-migration. In order to clarify the effects of oncogenic mutations on gene-expression downstream of RET, we analyzed expression microarrays in a model using single mutant and isoform RET expression. We also examined the molecular mechanisms of cell-migration, using both functional cell-based assays and examination of integrins, cell-adhesion molecules important for cell-migration. Finally, we used a large cohort of thyroid tissues to examine RET and integrin expression. We showed that different forms of oncogenic RET do not affect transcription of different target genes, but rather target-gene transcription is proportional to phosphorylatability of mutant RET. We were also able to show that RET leads to activation of at least two integrin subunits (ITGB1 and ITGB3), and that they have unique activation patterns downstream of RET that correlate with cell-adhesion and migration. Finally, we showed that co-expression between RET, ITGB1, and ITGB3 is more frequent in malignant subtypes of thyroid tissues and that their co-expression is correlated to more aggressive thyroid cancer subtypes. Together, we have clarified how RET is able to mediate two important processes, gene-expression and cell-migration. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2011-11-08 13:53:32.474

Dominant-activating mutations in the RET (rear-ranged during transfection) proto-oncogene, which encodes a receptor tyrosine kinase, is often associated with the development of medullary thyroid carcinoma (MTC). The proximal promoter region of the RET gene consists of a guanine-rich sequence containing five runs of three consecutive guanine residues that serve as the binding site for transcriptional factors. As we have recently shown, this stretch of nucleotides in the promoter region is highly dynamic in nature and tend to form non-B DNA secondary structures called G-quadruplexes, which suppress the transcription of the RET gene. In the present study, ellipticine and its derivatives were identified as excellent RET G-quadruplex stabilizing agents. Circular dichroism (CD) spectroscopic studies revealed that the incorporation of a piperidine ring in an ellipticine derivative, NSC311153 improves its binding with the G-quadruplex structure and the stability induced by this compound is more potent than ellipticine. Furthermore, this compound also interfered with the transcriptional mechanism of the RET gene in an MTC derived cell line, TT cells and significantly decreased the endogenous RET protein expression. We demonstrated the specificity of NSC311153 by using papillary thyroid carcinoma (PTC) cells, the TPC1 cell line which lacks the G-quadruplex forming sequence in the promoter region due to chromosomal rearrangement. The RET downregulation selectively suppresses cell proliferation by inhibiting the intracellular Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways in the TT cells. In the present study, we also showed that the systemic administration of a water soluble NSC311153 analog in a mouse MTC xenograft model inhibited the tumor growth through RET downregulation.

O presente trabalho avalia as alterações de propriedades dos asfaltos pela modificação por polímero do tipo RET (Reactive Elastomeric Terpolymer) e de comportamento de misturas asfálticas densas usinadas com estes asfaltos modificados. Para atingir este objetivo, são apresentados os resultados do monitoramento realizado no trecho experimental executado na SP-330, Rodovia Anhanguera, que empregou este tipo de asfalto modificado e estudo que compreende a modificação de ligantes do tipo CAP20 e CAP40 com 1,0%, 1,5% e 2%, de polímero RET, com posterior dosagem de uma mistura na Faixa III do DERSA no teor considerado como o mais adequado. Para verificação das propriedades mecânicas desta mistura, foram realizados ensaios de Módulo se Resiliência, Resistência à Tração por Compressão Diametral e Resistência à Deformação Permanente em simulador do tipo LPC. / The present document presents the alterations of the properties of the polymer modified asphalts using the polymer RET (Reactive Elastomeric Terpolymer) and the behavior of dense asphaltic mixtures using these modified asphalts. To reach this objective, the monitoring results accomplished in the experimental tram executed in SP-330, Rodovia Anhanguera, that used this type of modified asphalt are presented. The study comprehends the modification of the CAP20 and CAP40 asphalts with 1,0%, 1,5% and 2,0% of RET polymer, with subsequent dosage of a mixture in the Grade III of DERSA applying the RET polymeric proportion considered more appropriate. For the verification of the mechanical properties of this mixtures, Resilience Module, Traction Resistance for Diametrical Compression and Permanent Deformation Resistance in a LPC type simulator were accomplished.