RET Receptor Tyrosine Kinase Research Articles

Vandetanib as a potential new treatment for estrogen receptor-negative breast cancers.

The receptor tyrosine kinase RET is implicated in the progression of luminal breast cancers (BC) but its role in estrogen receptor (ER) negative tumors is unknown. Here we investigated the expression of RET in breast cancer patients tumors and patient-derived xenografts (PDX) and evaluated the therapeutic potential of Vandetanib, a tyrosin kinase inhibitor with strong activity against RET, EGFR and VEGFR2, in ER negative breast cancer PDX. The RT-PCR analysis of RET expression in breast tumors of 446 patients and 57 PDX, showed elevated levels of RET in ER+ and HER2+ subtypes and in a small subgroup of triple-negative breast cancers (TNBC). The activity of Vandetanib was tested in vivo in three PDX models of TNBC and one model of HER2+ BC with different expression levels of RET and EGFR. Vandetanib induced tumor regression in PDX models with high expression of RET or EGFR. The effect was associated with inhibition of RET/EGFR phosphorylation and MAP kinase pathway and increased necrosis. In a PDX model with no expression of RET nor EGFR, Vandetanib slowed tumor growth without inducing tumor regression. In addition, treatment by Vandetanib decreased expression of murine Vegf receptors and the endothelial marker Cd31 in the four PDX models tested, suggesting inhibition of tumor vascularization. In summary, these preclinical results suggest that Vandetanib treatment could be useful for patients with ER negative breast cancers overexpressing Vandetanib's main targets.

Neurotrophic factors: promising candidates in tissue regeneration.

Neurotrophic factors: promising candidates in tissue regeneration.

Abstract B192: Identification and characterization of highly potent and selective RET kinase inhibitors for the treatment of RET-driven cancers

Abstract Background: Activating mutations and oncogenic fusions of the RET receptor tyrosine kinase have been identified in multiple tumor types, including thyroid, lung, breast and colon carcinoma. Furthermore, tyrosine kinase inhibitors (TKIs) with anti-RET activity have produced clinical responses in patients whose tumors harbor RET alterations. However, currently available RET inhibitors were initially developed to target kinases other than RET and are only moderately potent against RET, inhibit multiple kinases other than RET or poorly inhibit secondary resistance mutations (e.g. gatekeeper mutations) common to other TKIs. We have discovered novel, potent and selective RET inhibitors. The resulting compounds exhibit nanomolar potency against wild type RET and select RET mutants, including the KIF5B-RET fusion and V804M gatekeeper mutation, in both enzyme and cellular assays, with minimal activity against highly related kinases. AR025 is representative of this series; the activity of AR025 and related analogs in relevant in vitro and in vivo models will be presented here. Methods: In vitro and in vivo evaluations, including enzyme and cell-based assays, pharmacokinetic (PK)/pharmacodynamics (PD) correlations, drug metabolism characterization, and non-clinical safety evaluation, were conducted using standard methods. Tumor growth inhibition and PD studies were carried out using subcutaneous allografts of NIH-3T3 cells expressing KIF5B-RET in nude mice. Results: AR025 demonstrated nanomolar potency against both wild type and mutant RET proteins in enzyme and cellular assays. AR025 had minimal activity against an enzyme panel of >200 diverse kinases and demonstrated >50x cellular selectivity against VEGFR, with similar selectivity against other related kinases. AR025 possessed low intravenous clearance and high oral exposure in mice, rats and dogs. Finally, a single oral dose of 30mg/kg produced more than 90% inhibition of phospho-RET in NIH-3T3-KIF5B-RET mouse allografts, while twice-per-day continuous dosing resulted in greater than 90% tumor growth inhibition. Notably, AR025 was minimally toxic at doses up to 100mg/kg per day. Conclusions: We have identified a series of potent and selective RET inhibitors with high oral bioavailability and favorable PK properties in animals. One of these, AR025, demonstrated potent inhibition of RET in enzyme and cellular assays, with minimal activity against highly related kinases. In an NIH3T3-KIF5B-RET allograft model, AR025 effectively inhibited phospho-RET and caused dramatic tumor growth inhibition without significant toxicity. The identification of potent and selective RET inhibitors with significant in vivo activity and minimal toxicity may overcome the limitations of currently available inhibitors with anti-RET activity. Citation Format: Barbara J. Brandhuber, Nisha Nanda, Julia Haas, Karyn Bouhana, Lance Williams, Shannon Winski, Michael Burkard, Brian Tuch, Kevin Ebata, Jennifer Low, Francis Sullivan, Lauren Hanson, Tony Morales, Guy Vigers, Jessica Gaffney, Ross D. Wallace, James Blake, Yutong Jiang, S. Michael Rothenberg, Steven Andrews. Identification and characterization of highly potent and selective RET kinase inhibitors for the treatment of RET-driven cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B192.

Cabozantinib for the treatment of progressive metastatic medullary thyroid cancer

Cabozantinib (XL-184) is a potent inhibitor of MET, VEGFR 2/KDR, RET and other receptor tyrosine kinases, such as KIT, AXL and FLT3. Its efficacy against MTC has been demonstrated in a prospective, randomized, placebo-controlled study (EXAM). Cabozantinib comparing to placebo significantly prolonged progression free survival both in hereditary and sporadic MTC, 11.2 vs 4.0 months, respectively. Final analysis showed no global differences in overall survival (OS) between cabozantinib and placebo. However, in a subgroup with RET M918T mutation the difference in OS was significant: 44.3 vs 18.9 months, respectively. Among the most frequent cabozantinib-related adverse events (AEs), observed in >30% of patients were diarrhea, palmar-plantar erythrodysesthesia, decreased weight, decreased appetite, nausea, fatigue, dysgeusia, hair color changes and hypertension. Expert Commentary: Cabozantinib constitutes an effective treatment option with acceptable toxicity in MTC patients showing either germinal or sporadic tumor RET M918T mutation as the drug prolonged OS in these subjects.

Lipid Rafts Are Physiologic Membrane Microdomains Necessary for the Morphogenic and Developmental Functions of Glial Cell Line-Derived Neurotrophic Factor In Vivo.

Glial cell line-derived neurotrophic factor (GDNF) promotes PNS development and kidney morphogenesis via a receptor complex consisting of the glycerophosphatidylinositol (GPI)-anchored, ligand binding receptor GDNF family receptor α1 (GFRα1) and the receptor tyrosine kinase Ret. Although Ret signal transduction in vitro is augmented by translocation into lipid rafts via GFRα1, the existence and importance of lipid rafts in GDNF-Ret signaling under physiologic conditions is unresolved. A knock-in mouse was produced that replaced GFRα1 with GFRα1-TM, which contains a transmembrane (TM) domain instead of the GPI anchor. GFRα1-TM still binds GDNF and promotes Ret activation but does not translocate into rafts. In Gfrα1(TM/TM) mice, GFRα1-TM is expressed, trafficked, and processed at levels identical to GFRα1. Although Gfrα1(+/TM) mice are viable, Gfrα1(TM/TM) mice display bilateral renal agenesis, lack enteric neurons in the intestines, and have motor axon guidance deficits, similar to Gfrα1(-/-) mice. Therefore, the recruitment of Ret into lipid rafts by GFRα1 is required for the physiologic functions of GDNF in vertebrates. Significance statement: Membrane microdomains known as lipid rafts have been proposed to be unique subdomains in the plasma membrane that are critical for the signaling functions of multiple receptor complexes. Their existence and physiologic relevance has been debated. Based on in vitro studies, lipid rafts have been reported to be necessary for the function of the Glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors. The receptor for GDNF comprises the lipid raft-resident, glycerophosphatidylinositol-anchored receptor GDNF family receptor α1 (GFRα1) and the receptor tyrosine kinase Ret. Here we demonstrate, using a knock-in mouse model in which GFRα1 is no longer located in lipid rafts, that the developmental functions of GDNF in the periphery require the translocation of the GDNF receptor complex into lipid rafts.

Distinct Temporal Regulation of RET Isoform Internalization: Roles of Clathrin and AP2.

The RET receptor tyrosine kinase (RTK) contributes to kidney and nervous system development, and is implicated in a number of human cancers. RET is expressed as two protein isoforms, RET9 and RET51, with distinct interactions and signaling properties that contribute to these processes. RET isoforms are internalized from the cell surface into endosomal compartments in response to glial cell line-derived neurotropic factor (GDNF) ligand stimulation but the specific mechanisms of RET trafficking remain to be elucidated. Here, we used total internal reflection fluorescence (TIRF) microscopy to demonstrate that RET internalization occurs primarily through clathrin coated pits (CCPs). Activated RET receptors colocalize with clathrin, but not caveolin. The RET51 isoform is rapidly and robustly recruited to CCPs upon GDNF stimulation, while RET9 recruitment occurs more slowly and is less pronounced. We showed that the clathrin-associated adaptor protein complex 2 (AP2) interacts directly with each RET isoform through its AP2 μ subunit, and is important for RET internalization. Our data establish that interactions with the AP2 complex promote RET receptor internalization via clathrin-mediated endocytosis but that RET9 and RET51 have distinct internalization kinetics that may contribute to differences in their biological functions.

Abstract 3567: Identification of potential biomarkers and novel therapeutic candidates for neuroendocrine cancer

Abstract Neuroendocrine (NE) cancers originate from secretory cells of the body's various endocrine structures. Surgical resection is the primary treatment for these tumors. However, the majority of NE cancers are metastatic at the time of detection and no effective adjuvant therapies exist for primary NE tumors or their metastases. Patients often develop two or more NE cancers, a syndrome termed multiple endocrine neoplasia (MEN). DNA sequencing has lead to breakthroughs in the identification of the causes of familial forms of the four types of MEN syndromes. Mutations in a protein of unknown function, menin, are associated with MEN1. Mutations in the receptor tyrosine kinase RET are linked to MEN2 and MEN3. The cyclin-dependent kinase inhibitor, CDNK1B, is mutated in MEN4. Unfortunately, a large percentage of NE cancers are sporadic, resulting from somatic mutations, and a high percentage of these do not harbor menin, RET, or CDNK1B mutations. Due to the lack of development of successful adjuvant therapies there has been little improvement in survival rates of NE cancer patients in the past several decades. Thus, there is a great need for the elucidation of signaling molecules that promote NE tumorigenesis in order to guide the development of novel and effective treatments for this family of cancers. Our laboratory has found that cyclin-dependent kinase 5 (Cdk5) is an important tumorigenic signaling molecule in sporadic medullary thyroid carcinoma (MTC), a type of NE cancer that originates from calcitonin-producing C cells in the thyroid. New data supports a role for Cdk5 in multiple types of NE cancer and provides a new prospective from which to explore their causes. Cdk5 is expressed in many NE cancers and inhibition of Cdk5 activity blocks growth of NE cancer cell lines. Phosphoproteomic analysis of growing versus arrested MTC tumors, induced by expression of the Cdk5 activator p25 in our transgenic mouse model of MTC, revealed elevation of over 200 phosphorylation sites. We have used short interfering peptides (SIPs) to selectively target a set of these phosphorylation sites and found that 15 of these SIPs blocked growth of NE cancer cell lines. Phosphorylation state specific antibodies were generated to several of these sites and phosphorylation levels were confirmed to be decreased in arrested MTC tumors. In conclusion, 1) multiple types of NE cancer cells are dependent on Cdk5 activity for growth, 2) phosphorylation of multiple signaling proteins is elevated downstream of Cdk5, and 3) select inhibition of these pathways blocks growth of NE cancer cells. Future studies will focus on analysis of these phosphoproteins in human NE tumors to validate of them as biomarkers for targeted cancer therapies as well as exploration of these phosphoproteins as direct targets for developing new therapeutics that are effective against NE cancers. Citation Format: Angela Carter, Karine Pozo, Chun-Feng Tan, Fiemu Nwariaku, James Bibb. Identification of potential biomarkers and novel therapeutic candidates for neuroendocrine cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3567. doi:10.1158/1538-7445.AM2015-3567

Abstract 1687: Vandetanib as a potential new treatment for ER negative breast cancers

Abstract Introduction: Recent studies have shown that the receptor tyrosine kinase RET is involved in the biology of ER positive breast cancers and in the response to endocrine treatment, but its role in ER negative tumors is unknown. Here we investigated the expression of RET in BC patients tumors and patient-derived xenografts (PDX) and evaluated the therapeutic potential of Vandetanib in ER negative BC PDX. Methods: RET mRNA expression was analyzed in BC of 446 patients and 57 PDX by RT-PCR analysis. The activity of Vandetanib, a tyrosine kinase inhibitor targeting RET, EGFR and VEGFR2, was tested in three PDX of triple-negative breast cancer (TNBC) and one PDX of HER2+ BC with different levels of RET expression. Protein expression of P-RET, RET, EGFR, P-EGFR and c-KIT were determined by immunohistochemistry (IHC). Analyses of PI3K and MAPK pathways and angiogenesis were performed by IHC and RT-PCR in both untreated and Vandetanib-treated tumors. Results: In both clinical samples and PDX, elevated levels of RET were found in ER+ and HER2+ tumors, and in a subgroup of TNBC tumors. In the HBCx5 (HER2+) and HBCx24 (TNBC) PDX, both with RET over-expression, treatment by Vandetanib resulted in tumor growth inhibition (TGI) of 90% and 98%, respectively. In both models, tumor regressions were observed in 50% of xenografts. The effect of Vandetanib was associated to a marked inhibition of RET phosphorylation. To determine whether the lack of RET over-expression was associate to Vandetanib resistance, we treated two additional TNBC PDX with low and no expression of RET: HBCx4B and HBCx14. In these models, treatment by Vandetanib still inhibited tumor growth with a TGI of 85%. Tumor regressions were registered in 42% of animals in the PDX model with low expression of RET (HBCx4B), while no tumor regression were observed in HBCx14. IHC analyses showed an over-expression of EGFR in the HBCx4B xenograft and inhibition of EGFR phosphorylation in treated tumors, suggesting that tumor response to Vandetanib could depend on EGFR inhibition in this tumor. Further analyses of treated tumors revealed a decreased expression of phospho-ERK in the 4 PDX models, indicating inhibition of MAPK pathway, while the phosphorylation status of the PI3K pathway markers S6 and 4EBP1 was unchanged. Finally, treatment by Vandetanib decreased expression of murine Vegf receptors and the endothelial marker Cd31 in the 4 PDX tested, indicating angiogenesis inhibition. Conclusions: Treatment by Vandetanib resulted in strong tumor growth inhibition in ER negative PDX with over-expression of RET. This effect was associated to inhibition of RET phosphorylation and MAPK pathway and decreased tumor vascularization. The lack of RET over-expression did not predict Vandetanib resistance, and over-expression of EGFR was also associated to a marked tumor response. These preclinical results suggest that Vandetanib treatment could be useful for patients with ER negative breast cancers expressing Vandetanib's targets. Citation Format: Elisabetta Marangoni, Rana Hatem, Dalila Labiod, Sophie Chateau-Joubert, Rania El Botty, Jean-Luc Servely, Ludmilla De Plater, Ivan Bièche. Vandetanib as a potential new treatment for ER negative breast cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1687. doi:10.1158/1538-7445.AM2015-1687

Abstract 4991: RET and integrins cooperate in cell-microenvironment response

Abstract The RET receptor tyrosine kinase is expressed in neural crest-derived cell types, where it is activated by soluble ligands of the Glial Cell-derived Neurotrophic Factor (GDNF) family and signals through multiple downstream pathways, contributing to cell growth, survival, and migration during embryonic development. In addition to its normal developmental roles, oncogenic mutations or aberrant expression of RET are also linked to tumour spread and metastasis in multiple human tumour types. Thus, a consistent feature of RET involvement in these disease processes is its contribution to directional cell motility. We have previously shown that RET-mediated cell-adhesion and migration requires multiple members of the integrin family of cell adhesion molecules, including integrin β1 (ITGB1) and β3 (ITGB3) subunits, however the regulation of cell migration and invasion by RET and integrins has not been explored. In this study, we showed that GDNF-mediated RET activation induced rapid ITGB1 and ITGB3 activation with transiently increased cell adhesion, followed by enhanced cell protrusion formation and reduced adhesion typical of more migratory cells. In addition, we showed that directional cell migration in response to GDNF requires the presence of both extracellular matrix and a GDNF concentration gradient. Our data also suggest that ITGB1 and ITGB3 play complementary but non-compensatory roles in the initiation of RET-mediated cell migration and invasion, and that the inhibition of either ITGB1 or ITGB3 resulted in impaired migration and invasion in both 2D and 3D culture conditions. We have also shown that multiple signaling pathways downstream of RET are involved in these processes and that these pathways contribute differently to migration in 2D vs 3D microenvironments. These data highlight the ability of tumour cells to leverage different signaling pathways to mediate cell migration and invasion downstream of RET, and reveal ways in which cooperation between RET and integrins might affect aspects of tumour progression. Our data, together with other studies which have shown combination therapy targeting integrins and RTKs to be more effective than monotherapy, suggest that adjuvant treatment with agents targeting integrin function may further enhance therapeutic options for tumours where RET is expressed. Citation Format: Eric Lian, Mathieu J.F. Crupi, Jessica G. Cockburn, Simona M. Wagner, Anirudh Goel, Lois M. Mulligan. RET and integrins cooperate in cell-microenvironment response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4991. doi:10.1158/1538-7445.AM2015-4991

Abstract 4990: The RET receptor Y791F variant activates the kinase but diminishes ligand responsiveness

Abstract The RET receptor tyrosine kinase is essential for normal development of the kidneys and enteric nervous system, and is also implicated in several human pathologies. Gain-of-function mutations in RET are associated with the familial cancer syndrome multiple endocrine neoplasia type 2 (MEN 2), where single amino acid substitutions lead to constitutive RET activation in the absence of its ligands of the glial cell line-derived neurotrophic factor (GDNF) family. Conversely, loss-of-function RET mutations are associated with Hirschsprung disease, a congenital abnormality of the enteric nervous system. Previous studies have identified a specific substitution variant of a phenylalanine for tyrosine at amino acid 791 in the RET kinase domain in both cancer and Hirschsprung disease patients. Thus, the functional implications of this variant and its contributions to these diverse phenotypes are not clear. Here, we have explored the role of MEN 2-associated RET mutations in RET-mediated cell invasion and migration. We showed that GDNF-stimulation promotes RET-mediated cell migration and that a GDNF-chemotactic gradient significantly increased invasion of cells expressing wild type RET or a MEN2B (M918T) mutant RET form. However, we found that a GDNF gradient did not enhance invasion of cells expressing RET-Y791F. Consistent with this, we showed that MEN 2-associated RET mutants M918T and Y791F were phosphorylated in the absence of GDNF and stimulated downstream signaling pathways. GDNF treatment further increased phosphorylation of the RET-M918T but not RET-Y791F proteins, suggesting that RET-Y791F has reduced responsiveness to GDNF ligand. Our results suggest that despite constitutive activation of RET signaling, cells expressing the Y791F variant may possess a diminished capacity to recognize and respond to GDNF in the cell microenvironment. This may be linked to a disturbance in the directional migration of cells expressing the mutant receptor thus contributing to the phenotypic variability observed. Citation Format: Andrew Fetz, Mathieu J.F. Crupi, Eric Lian, Brandy D. Hyndman, Lois M. Mulligan. The RET receptor Y791F variant activates the kinase but diminishes ligand responsiveness. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4990. doi:10.1158/1538-7445.AM2015-4990

Abstract 4986: Distinct temporal regulation of RET isoform internalization: Roles of clathrin and AP2

Abstract The RET receptor tyrosine kinase is an important contributor to kidney and enteric nervous system development. In normal development, RET signaling regulates cell proliferation, migration, survival and differentiation, but RET is also implicated in growth and spread of several human cancers. Activating mutations of RET are drivers of inherited and sporadic thyroid cancers, while expression of wildtype RET receptors in breast and pancreatic cancers has been associated with enhanced tumor invasion, metastasis and poor disease prognosis. RET is expressed as two major isoforms, RET9 and RET51, that have unique 9 or 51 amino acid C-terminal tails, respectively. These isoforms induce unique phosphorylation patterns on intracellular tyrosine residues, differentially bind downstream signalling proteins, and possess different intrinsic abilities to cause cellular transformation. We have previously shown that RET isoforms are internalized from the cell surface into endosomal compartments in response to glial cell line derived neurotrophic factor (GDNF) ligand stimulation, but the specific mechanisms of RET trafficking remain to be elucidated. Here, we used total internal reflection fluorescence microscopy to demonstrate that RET internalization occurs through clathrin coated pits (CCPs). Activated RET receptors colocalize with clathrin, but not caveolin, at sites of CCPs. The RET51 isoform is rapidly and robustly recruited to CCPs upon GDNF stimulation, while RET9 recruitment occurs more slowly and is less pronounced. We showed that the clathrin-associated adaptor protein complex 2 (AP2) is important for RET internalization. Our data establish that interactions with the AP2 complex promote RET receptor internalization via clathrin-mediated pathways but that RET9 and RET51 have distinct internalization kinetics that may contribute to the functional differences between RET isoforms which are critical to regulating RET's contribution to tumorigenesis and normal development. These data may also have clinical importance, as they characterize key regulatory events for an important oncogene that may in future indicate biologically relevant targets for therapeutic interventions in RET-associated tumors. Citation Format: Mathieu Joseph François Crupi, Piriya Yoganathan, Leslie N. Bone, Eric Lian, Andrew Fetz, Costin N. Antonescu, Lois M. Mulligan. Distinct temporal regulation of RET isoform internalization: Roles of clathrin and AP2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4986. doi:10.1158/1538-7445.AM2015-4986

Identification of Novel Small Molecule Inhibitors of Oncogenic RET Kinase.

Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI), ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the ‘DFG-out’ inactive conformation of RET activation loop. They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes. They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes. At a concentration of 10 nM, ALW-II-41-27, XMD15-44 and HG-6-63-01 inhibited RET kinase and signaling in human thyroid cancer cell lines carrying oncogenic RET alleles; they also inhibited proliferation of cancer, but not non-tumoral Nthy-ori-3-1, thyroid cells, with an IC50 in the nM range. The three compounds were capable of inhibiting the ‘gatekeeper’ V804M mutant which confers substantial resistance to established RET inhibitors. In conclusion, we have identified a type II TKI scaffold, shared by ALW-II-41-27, XMD15-44 and HG-6-63-01, that may be used as novel lead for the development of novel agents for the treatment of cancers harboring oncogenic activation of RET.

Molecular pathology of hereditary and sporadic medullary thyroid carcinomas.

Medullary thyroid carcinoma (MTC) is a relatively uncommon type of thyroid malignancy, with unique histologic features and molecular pathology. It is important to recognize, because its management, which is in part driven by the genetic basis of this disease, is different from follicular-derived thyroid tumors. The aim of this article is to briefly review the histopathologic features of MTC and then explore its molecular pathology, including the role of molecular diagnostic testing and the use of targeted therapy for advanced disease. A review of published literature was performed. A subset of MTC cases is hereditary and due to germline mutations in the RET tyrosine kinase receptor gene. Somatic mutations in either RET or RAS are also present in most sporadic tumors. Molecular genetic testing is routinely performed to identify hereditary cases. In addition, understanding the molecular basis of both hereditary and sporadic MTC has led to the development of targeted therapy with tyrosine kinase inhibitors. Although additional data are needed, tumor mutation status may affect response to targeted therapy. Therefore, it is possible that genetic testing of tumor tissue to predict treatment response, as is currently done for other cancer types, may come into practice in the future.

Activation of the RET Receptor Tyrosine Kinase by Agonist Antibodies Results in Distinct Signaling Effects

Cytokine and growth factor receptors are activated when the soluble growth factor binds and alters the protein‐protein interactions between receptor components on the cell surface, triggering intracellular receptor phosphorylation and downstream signaling. However, many receptors involve three or more cell surface components, and the effect of this additional complexity on how ligand binding is quantitatively coupled to receptor activation and signaling is not well understood. The RET receptor tyrosine kinase is an example of such a multi‐component receptor. RET is activated when the binding of a growth factor such as artemin (ART) brings about assembly of a pentameric complex that includes two copies of RET plus two copies of the GPI‐linked co‐receptor, GFRα3. We show here that RET can be directly activated by agonist antibodies, bypassing its GFRα co‐receptors. The signaling profiles of two agonist antibodies differ from each other, and from the natural ligand ART, in the level of downstream signal amplification. This result suggests that the activated receptor complexes that are formed by these different agonists engage with downstream signaling pathways in distinct ways, presumably reflecting different geometries within each complex, and thus show that activated RET can exist in multiple forms with different signaling properties. We also show that we can achieve higher signal amplitudes and a biological response similar to that seen with ART by using the agonist antibodies, but only if we cluster the activated receptor complexes using either monoclonal or polyclonal secondary anti‐Fc antibodies. This finding suggests that, while RET dimerization represents a minimum requirement for activation, higher‐order clustering might be required for a full biological response.

Quantitative Analysis of Receptor Tyrosine Kinase-Effector Coupling at Functionally Relevant Stimulus Levels

A major goal of current signaling research is to develop a quantitative understanding of how receptor activation is coupled to downstream signaling events and to functional cellular responses. Here, we measure how activation of the RET receptor tyrosine kinase on mouse neuroblastoma cells by the neurotrophin artemin (ART) is quantitatively coupled to key downstream effectors. We show that the efficiency of RET coupling to ERK and Akt depends strongly on ART concentration, and it is highest at the low (∼100 pM) ART levels required for neurite outgrowth. Quantitative discrimination between ERK and Akt pathway signaling similarly is highest at this low ART concentration. Stimulation of the cells with 100 pM ART activated RET at the rate of ∼10 molecules/cell/min, leading at 5-10 min to a transient peak of ∼150 phospho-ERK (pERK) molecules and ∼50 pAkt molecules per pRET, after which time the levels of these two signaling effectors fell by 25-50% while the pRET levels continued to slowly rise. Kinetic experiments showed that signaling effectors in different pathways respond to RET activation with different lag times, such that the balance of signal flux among the different pathways evolves over time. Our results illustrate that measurements using high, super-physiological growth factor levels can be misleading about quantitative features of receptor signaling. We propose a quantitative model describing how receptor-effector coupling efficiency links signal amplification to signal sensitization between receptor and effector, thereby providing insight into design principles underlying how receptors and their associated signaling machinery decode an extracellular signal to trigger a functional cellular outcome.

Activation and Decay Kinetics of the RET Receptor Tyrosine Kinase

We use ZD6474, an inhibitor of the kinase domain of the RET receptor tyrosine kinase, as a chemical probe to obtain quantitative information on the molecular kinetics of RET activation, autophosphorylation, dephosphorylation and trafficking in mouse neuroblastoma cells. Stimulation with RET's cognate ligand artemin (ART) resulted in up to 65% of cellular RET becoming phosphorylated over several minutes, followed by a slow decay. Addition of ZD6474 6 min after stimulation resulted in loss of phosphoRET (pRET) with t1/2 蠄 1 min, indicating that overall pRET dynamics reflects an evolving steady state between rapid RET phosphorylation and its comparably rapid dephosphorylation. Reconciling the observed rate of pRET formation with the rapid rate of pRET dephosphorylation and the high level of pRET at the steady state required invoking a rate-limiting activation of RET by ART, prior to receptor phosphorylation which occurs rapidly (rate constant 1.2 min-1). Dephosphorylation of pRET regenerates activated but unphosphorylated RET, which autophosphorylates again rapidly rather than reverting to the resting state. We additionally show that this rapidly cycling pRET is rapidly internalized through clathrin-coated pits, where it continues to activate ERK and Akt signaling. A fraction of the internalized pRET slowly converts to a longer-lived form (t1/2 ~ 10 min) that resides in late endosomes where it is protected from phosphatases and loses its signaling ability. Our results kinetically distinguish ART-dependent assembly of the activated RET receptor complex from its subsequent autophosphorylation, and provide a quantitative picture of RET receptor activation and trafficking.

Parkin cooperates with GDNF/RET signaling to prevent dopaminergic neuron degeneration.

Parkin and the glial cell line-derived neurotrophic factor (GDNF) receptor RET have both been independently linked to the dopaminergic neuron degeneration that underlies Parkinson's disease (PD). In the present study, we demonstrate that there is genetic crosstalk between parkin and the receptor tyrosine kinase RET in two different mouse models of PD. Mice lacking both parkin and RET exhibited accelerated dopaminergic cell and axonal loss compared with parkin-deficient animals, which showed none, and RET-deficient mice, in which we found moderate degeneration. Transgenic expression of parkin protected the dopaminergic systems of aged RET-deficient mice. Downregulation of either parkin or RET in neuronal cells impaired mitochondrial function and morphology. Parkin expression restored mitochondrial function in GDNF/RET-deficient cells, while GDNF stimulation rescued mitochondrial defects in parkin-deficient cells. In both cases, improved mitochondrial function was the result of activation of the prosurvival NF-κB pathway, which was mediated by RET through the phosphoinositide-3-kinase (PI3K) pathway. Taken together, these observations indicate that parkin and the RET signaling cascade converge to control mitochondrial integrity and thereby properly maintain substantia nigra pars compacta dopaminergic neurons and their innervation in the striatum. The demonstration of crosstalk between parkin and RET highlights the interplay in the protein network that is altered in PD and suggests potential therapeutic targets and strategies to treat PD.

Brain ischemia downregulates the neuroprotective GDNF-Ret signaling by a calpain-dependent mechanism in cultured hippocampal neurons.

The glial cell line-derived neurotrophic factor (GDNF) has an important role in neuronal survival through binding to the GFRα1 (GDNF family receptor alpha-1) receptor and activation of the receptor tyrosine kinase Ret. Transient brain ischemia alters the expression of the GDNF signaling machinery but whether the GDNF receptor proteins are also affected, and the functional consequences, have not been investigated. We found that excitotoxic stimulation of cultured hippocampal neurons leads to a calpain-dependent downregulation of the long isoform of Ret (Ret51), but no changes were observed for Ret9 or GFRα1 under the same conditions. Cleavage of Ret51 by calpains was selectively mediated by activation of the extrasynaptic pool of N-methyl-d-aspartate receptors and leads to the formation of a stable cleavage product. Calpain-mediated cleavage of Ret51 was also observed in hippocampal neurons subjected to transient oxygen and glucose deprivation (OGD), a model of global brain ischemia, as well as in the ischemic region in the cerebral cortex of mice exposed to transient middle cerebral artery occlusion. Although the reduction of Ret51 protein levels decreased the total GDNF-induced receptor activity (as determined by assessing total phospho-Ret51 protein levels) and their downstream signaling activity, the remaining receptors still showed an increase in phosphorylation after incubation of hippocampal neurons with GDNF. Furthermore, GDNF protected hippocampal neurons when present before, during or after OGD, and the effects under the latter conditions were more significant in neurons transfected with human Ret51. These results indicate that the loss of Ret51 in brain ischemia partially impairs the neuroprotective effects of GDNF.

Glial cell line-derived neurotrophic factor induces cell proliferation in the mouse urogenital sinus.

Glial cell line-derived neurotrophic factor (GDNF) is a TGFβ family member, and GDNF signals through a glycosyl-phosphatidylinositol-linked cell surface receptor (GFRα1) and RET receptor tyrosine kinase. GDNF signaling plays crucial roles in urogenital processes, ranging from cell fate decisions in germline progenitors to ureteric bud outgrowth and renal branching morphogenesis. Gene ablation studies in mice have revealed essential roles for GDNF signaling in urogenital development, although its role in prostate development is unclear. We investigated the functional role of GDNF signaling in the urogenital sinus (UGS) and the developing prostate of mice. GDNF, GFRα1, and RET show time-specific and cell-specific expression during prostate development in vivo. In the UGS, GDNF and GFRα1 are expressed in the urethral mesenchyme (UrM) and epithelium (UrE), whereas RET is restricted to the UrM. In each lobe of the developing prostate, GDNF and GFRα1 expression declines in the epithelium and becomes restricted to the stroma. Using a well-established organ culture system, we determined that exogenous GDNF increases proliferation of UrM and UrE cells, altering UGS morphology. With regard to mechanism, GDNF signaling in the UrM increased RET expression and phosphorylation of ERK1/2. Furthermore, inhibition of RET kinase activity or ERK kinases suppressed GDNF-induced proliferation of UrM cells but not UrE cells. We therefore propose that GDNF signaling in the UGS increases proliferation of UrM and UrE cells by different mechanisms, which are distinguished by the role of RET receptor tyrosine kinase and ERK kinase signaling, thus implicating GDNF signaling in prostate development and growth.

When Does Active Management Add Value?

The receptor tyrosine kinase (RTK) Ret is activated by the formation of a complex consisting of ligands such as glial cell line-derived neurotrophic factor (GDNF) and glycerophosphatidylinositol-anchored coreceptors termed GFRαs. During activation, Ret translocates into lipid rafts, which is critical for functional responses to GDNF. We found that Ret was rapidly ubiquitinated and degraded in sympathetic neurons when activated with GDNF, but, unlike other RTKs that are trafficked to lysosomes for degradation, Ret was degraded predominantly by the proteasome. After GDNF stimulation, the majority of ubiquitinated Ret was located outside of lipid rafts and Ret was lost predominantly from nonraft membrane domains. Consistent with the predominance of Ret degradation outside of rafts, disruption of lipid rafts in neurons did not alter either the GDNF-dependent ubiquitination or degradation of Ret. GDNF-mediated survival of sympathetic neurons was inhibited by lipid raft depletion, and this inhibitory effect of raft disruption on GDNF-mediated survival was reversed if Ret degradation was blocked via proteasome inhibition. Therefore, lipid rafts sequester Ret away from the degradation machinery located in nonraft membrane domains, such as Cbl family E3 ligases, thereby sustaining Ret signaling.