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.      

Conservation of RET proto-oncogene splicing variants and implications for RET isoform function

The RET proto-oncogene encodes a receptor tyrosine kinase required for development of the kidney and neural crest-derived cell types. Alternative splicing of the 3' exons of human RET results in three protein isoforms with distinct C-termini: RET9, RET51, and RET43. These RET isoforms show differential binding to downstream adapter molecules, suggesting they may have distinct signaling functions. We have characterized Ret 3' sequences in mouse and investigated alternative splicing of this region. We found that the organization of Ret 3' sequences is very similar to human RET. The mouse locus also has alternatively spliced C-terminal coding regions, and the sequences corresponding to RET9 and RET51 are highly conserved in both position and sequence with the human locus. Further, we compared the predicted C-terminal amino acids of RET9 and RET51 in seven vertebrate species, and found that they are well conserved. We have identified sequence encoding a putative ret43 isoform in mouse, however the predicted amino acid sequence showed low homology to human RET43. Our data suggest that RET isoforms are evolutionarily highly conserved over a broad range of species, which may indicate that each isoform has a distinct role in normal RET function.     

RET rearrangements in non-small cell lung cancer: Evolving treatment landscape and future challenges

The Rearranged during Transfection (RET) oncogene has been extensively investigated in solid malignancies, particularly thyroid cancer and non-small cell lung cancer (NSCLC), and represents an attractive therapeutic target. RET rearrangements occur in 1–2% of lung adenocarcinomas, where they function as potent oncogenic drivers. Importantly, tumors harboring RET fusions are particularly sensitive to RET tyrosine kinase inhibitors. Results of the LIBRETTO-001 and ARROW clinical trials led to the approval of novel potent and selective RET inhibitors, selpercatinib and pralsetinib, able to overcome the limits of previously used multikinase inhibitors. Herein, we review the most relevant evidences about the role of RET signaling in NSCLC. In addition, we interrogated the Project GENIE database to investigate common clinical and molecular features of RET-fusion positive NSCLC. This analysis revealed that RET rearrangements occurred more frequently in younger and light smoker patients and were associated with a lower tumor mutational burden, compared to RET-fusion negative tumors. Moreover, we assessed and described the differences between RET genomic alterations in NSCLC and thyroid cancers. Finally, we summarized how the treatment landscape of RET-rearranged NSCLC has changed in the last few years, which are the available data about the recognized mechanisms of resistance to RET inhibitors and the challenges for future development of novel therapeutic strategies, aiming to improve management of patients with RET-fusion positive NSCLC.     

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.     

Ret isoform function and marker gene expression in the enteric nervous system is conserved across diverse vertebrate species

The enteric nervous system (ENS) derives from migratory neural crest cells that colonize the developing gut tube, giving rise to an integrated network of neurons and glial cells, which together regulate important aspects of gut function, including coordinating the smooth muscle contractions of the gut wall. The absence of enteric neurons in portions of the gut (aganglionosis) is the defining feature of Hirschsprung’s disease (HSCR) and has been replicated in a number of mouse models. Mutations in the RET tyrosine kinase account for over half of familial cases of HSCR and mice mutant for Ret exhibit aganglionosis. RET exists in two main isoforms, RET9 and RET51 and studies in mouse have shown that RET9 is sufficient to allow normal development of the ENS. In the last several years, zebrafish has emerged as a model of vertebrate ENS development, having been supported by a number of demonstrations of conservation of gene function between zebrafish, mouse and human. In this study we further analyse the potential similarities and differences between ENS development in zebrafish, mouse and human. We demonstrate that zebrafish Ret is required in a dose-dependent manner to regulate colonization of the gut by neural crest derivatives, as in human. Additionally, we show that as in mouse and human, zebrafish ret is produced as two isoforms, ret9 and ret51. Moreover, we show that, as in mouse, the Ret9 isoform is sufficient to support colonization of the gut by enteric neurons. Finally, we identify zebrafish orthologues of genes previously identified to be expressed in the mouse ENS and demonstrate that these genes are expressed in the developing zebrafish ENS, thereby identifying useful ENS markers in this model organism. These studies reveal that the similarities between gene expression and gene function across vertebrate species is more extensive than previously appreciated, thus supporting the use of zebrafish as a general model for vertebrate ENS development and the use of zebrafish genetic screens as a way to identify candidate genes mutated in HSCR cases.     

Mutational analysis of the intramembranous H10 loop of yeast Nhx1 reveals a critical role in ion homoeostasis and vesicle trafficking

Yeast Nhx1 [Na+(K+)/H+ exchanger 1] is an intracellular Na+(K+)/H+ exchanger, localizing to the late endosome where it is important for ion homoeostasis and vesicle trafficking. Phylogenetic analysis of NHE (Na+/H+ exchanger) sequences has identified orthologous proteins, including HsNHE6 (human NHE6), HsNHE7 and HsNHE9 of unknown physiological role. These appear distinct from well-studied mammalian plasma membrane isoforms (NHE1-NHE5). To explore the differences between plasma membrane and intracellular NHEs and understand the link between ion homoeostasis and vesicle trafficking, we examined the consequence of replacing residues in the intramembranous H10 loop of Nhx1 between transmembrane segments 9 and 10. The critical role for the carboxy group of Glu355 in ion transport is consistent with the invariance of this residue in all NHEs. Surprisingly, residues specifically conserved in the intracellular isoforms (such as Phe357 and Tyr361) could not be replaced with closely similar residues (leucine and phenylalanine) found in the plasma membrane isoforms without loss of function, revealing unexpected side chain specificity. The trafficking phenotypes of all Nhx1 mutants, including hygromycin-sensitivity and missorting of carboxypeptidase Y, were found to directly correlate with pH homoeostasis defects and could be proportionately corrected by titration with weak base. The present study demonstrates the importance of the H10 loop of the NHE family, highlights the differences between plasma membrane and intracellular isoforms and shows that trafficking defects are tightly coupled with pH homoeostasis.     

Phosphoinositide-dependent phosphorylation of PDK1 regulates nuclear translocation

3-phosphoinositide-dependent kinase 1 (PDK1) phosphorylates the activation loop of a number of protein serine/threonine kinases of the AGC kinase superfamily, including protein kinase B (PKB; also called Akt), serum and glucocorticoid-induced kinase, protein kinase C isoforms, and the p70 ribosomal S6 kinase. PDK1 contains a carboxyl-terminal pleckstrin homology domain, which targets phosphoinositide lipids at the plasma membrane and is central to the activation of PKB. However, PDK1 subcellular trafficking to other compartments is not well understood. We monitored the posttranslational modifications of PDK1 following insulin-like growth factor 1 stimulation. PDK1 underwent rapid and transient phosphorylation on S396, which was dependent upon plasma membrane localization. Phosphorylation of S396 was necessary for nuclear shuttling of PDK1, possibly through its influence on an adjacent nuclear export sequence. Thus, mitogen-stimulated phosphorylation of PDK1 provides a means for directed PDK1 subcellular trafficking, with potential implications for PDK1 signaling.     

Traditional and targeted exome sequencing reveals common, rare and novel functional deleterious variants in RET-signaling complex in a cohort of living US patients with urinary tract malformations

Signaling by the glial cell line-derived neurotrophic factor (GDNF)-RET receptor tyrosine kinase and SPRY1, a RET repressor, is essential for early urinary tract development. Individual or a combination of GDNF, RET and SPRY1 mutant alleles in mice cause renal malformations reminiscent of congenital anomalies of the kidney or urinary tract (CAKUT) in humans and distinct from renal agenesis phenotype in complete GDNF or RET-null mice. We sequenced GDNF, SPRY1 and RET in 122 unrelated living CAKUT patients to discover deleterious mutations that cause CAKUT. Novel or rare deleterious mutations in GDNF or RET were found in six unrelated patients. A family with duplicated collecting system had a novel mutation, RET-R831Q, which showed markedly decreased GDNF-dependent MAPK activity. Two patients with RET-G691S polymorphism harbored additional rare non-synonymous variants GDNF-R93W and RET-R982C. The patient with double RET-G691S/R982C genotype had multiple defects including renal dysplasia, megaureters and cryptorchidism. Presence of both mutations was necessary to affect RET activity. Targeted whole-exome and next-generation sequencing revealed a novel deleterious mutation G443D in GFRα1, the co-receptor for RET, in this patient. Pedigree analysis indicated that the GFRα1 mutation was inherited from the unaffected mother and the RET mutations from the unaffected father. Our studies indicate that 5?% of living CAKUT patients harbor deleterious rare variants or novel mutations in GDNF-GFRα1-RET pathway. We provide evidence for the coexistence of deleterious rare and common variants in genes in the same pathway as a cause of CAKUT and discovered novel phenotypes associated with the RET pathway.     

Central Role of RET in Thyroid Cancer

RET (rearranged during transfection) is a receptor tyrosine kinase involved in the development of neural crest derived cell lineages, kidney, and male germ cells. Different human cancers, including papillary and medullary thyroid carcinomas, lung adenocarcinomas, and myeloproliferative disorders display gain-of-function mutations in RET. Accordingly, RET protein has become a promising molecular target for cancer treatment.The human RET (rearranged during transfection) gene maps on 10q11.2 and is composed of 21 exons spanning a region of 55,000 bp. It encodes a single-pass trans-membrane protein, RET, that belongs to the receptor tyrosine kinase (RTK) family (Pasini et al. 1995). The RET extracellular segment contains four cadherin-like domains, followed by a domain containing cysteine residues involved in the formation of intramolecular disulfide bonds (Fig. 1A) (Anders et al. 2001; Airaksinen and Saarma 2002). RET protein is highly glycosylated and N-glycosylation is necessary for its transport to the cell surface. Only the fully mature glycosylated 170 kDa RET protein isoform is exposed to the extracellular compartment, whereas the mannose-rich 150 kDa isoform is confined to the Golgi (Takahashi et al. 1993; Carlomagno et al. 1996). The transmembrane segment is composed of 22 amino acids, among which S649 and S653 mediate self-association and dimerization of RET, possibly via formation of inter-molecular hydrogen bonding (Kjaer et al. 2006). The intracellular portion of RET contains the tyrosine kinase domain split into two subdomains by the insertion of 27 amino acids. The RET COOH-terminal tail varies in length as a result of alternative splicing of the 3′ end (carboxy terminal with respect to glycine 1063), generating three different isoforms that contain 9 (RET9), 43 (RET43), or 51 (RET51) amino acids (Myers et al. 1995). RET9 and RET51 are the most abundant isoforms, and they activate similar signaling pathways through interaction with diverse protein complexes, and may exert a differential role in development (Fig. 1A) (de Graaff et al. 2001).Open in a separate windowFigure 1.Illustration of the mechanisms of activation of wild-type (wt) RET and RET-derived oncoproteins. (A) Wild-type RET activation is mediated by ligand (GFL)-induced dimerization; ligand binding to RET is not direct and mediated by GFR-α coreceptors (not shown); major RET autophosphorylation sites and downstream signaling pathways are indicated. RET extracellular cadherin-like domains are represented in red. The split intracellular RET tyrosine kinase domain, as well as the three alternative carboxy-terminal RET tails, are also depicted. (B) RET/PTC activation is mediated by coiled-coil-induced dimerization (left); activation of RET cysteine mutants associated with MEN2A or FMTC is mediated by disulfide bonds-mediated dimerization (right).RET shows several autophosphorylation sites (Fig. 1A) (Liu et al. 1996; Kawamoto et al. 2004). RET tyrosine 1062 (Y1062) functions as a multidocking site for signaling molecules containing a phosphotyrosine-binding (PTB) domain (Asai et al. 1996). Phospho-Y1062 binding proteins include SHC, N-SHC (RAI), FRS2, IRS1/2, DOK1, and DOK4/5 that, in turn, contribute to the activation of RAS-MAPK (mitogen-activated protein kinases) and PI3K (phosphatidyl inositol 3 kinase)-AKT pathways. Y1096, specific to the RET51 splicing variant, couples to the PI3K-AKT and RAS-MAPK pathways, as well. These signaling cascades mediate RET-dependent cell survival, proliferation, and motility (Alberti et al. 1998; Murakami et al. 1999; Segouffin-Cariou and Billaud 2000; Melillo et al. 2001a,b; Schuetz et al. 2004). Y905 is located in the activation loop of the RET kinase and its phosphorylation is associated with RET kinase activation (Knowles et al. 2006). Finally, Y981 and Y1015 have been shown to be coupled to important signaling molecules such as SRC and PLC-γ, respectively (Borrello et al. 1996; Encinas et al. 2004).RET is the receptor for a group of neurotrophic growth factors that belong to the glial cell line-derived neurotrophic factor (GDNF) family (GFLs, GDNF family ligands), namely, GDNF, Neurturin (NRT), Artemin (ART), and Persephin (PSF) (Airaksinen and Saarma 2002). GFLs mediate RET protein dimerization and activation (Fig. 1A). GFLs are presented to RET by GPI (glycosylphosphatidylinositol)-anchored coreceptors, called GFR-α (GDNF family receptor α 1-4). Differential tissue expression dictates the specificity of action displayed by alternative GLF-GFR-α pairs during development and adult life (Baloh et al. 2000; Airaksinen and Saarma 2002).Together with other membrane (DCC and p75NTR) or nuclear (androgen receptor, AR) receptors, RET belongs to the family of so-called “dependence” receptors (Mehlen and Bredesen 2011). In the absence of ligand, RET exerts a proapoptotic activity, that is blocked on ligand stimulation (Bordeaux et al. 2000). Such pro-apoptotic activity is RET kinase-independent and mediated by cleavage of RET cytosolic portion by caspase-3, which, in turn, releases a carboxy-terminal RET peptide that is able to induce cell death (Bordeaux et al. 2000). It is feasible that such activity is important for RET developmental function, because it may control migration of RET-expressing cells by limiting survival of cells that move beyond ligand availability (Bordeaux et al. 2000; Cañibano et al. 2007). Whether modulation of this function is also important for RET-associated diseases is still unknown. However, it is interesting to note that a cancer-associated RET mutant (RET-C634R, see below) does not exert cleavage-dependent proapoptotic effects, whereas RET mutants associated with defective development (Hirschsprung disease, see below) exert strong proapoptotic activity that is refractory to modulation by ligand (Bordeaux et al. 2000).RET is expressed in enteric ganglia, adrenal medulla chromaffin cells, thyroid C cells, sensory and autonomic ganglia of the peripheral nervous system, a subset of central nervous system nuclei, developing kidney and testis germ cells (Manié et al. 2001; de Graaff et al. 2001). RET null mice display impaired development of superior cervical ganglia and enteric nervous system, kidney agenesia, reduction of thyroid C cells, and impaired spermatogenesis (Manié et al. 2001). Accordingly, individuals with germline loss-of-function mutations of RET are affected by intestinal aganglionosis causing congenital megacolon (Hirschsprung disease) (Brooks et al. 2005). RET loss-of-function mutations have also been identified in congenital anomalies of kidney and urinary tract (CAKUT), either isolated or in combination with Hirschsprung disease (Jain 2009).Several genetic alterations convert RET into a dominantly transforming oncogene. This review will describe RET-derived oncogenes that are associated with different types of human neoplasia (Fig. 1B).     

SNARE-mediated membrane traffic is required for focal adhesion kinase signaling and Src-regulated focal adhesion turnover

Integrin signaling is central to cell growth and differentiation, and critical for the processes of apoptosis, cell migration and wound repair. Previous research has demonstrated a requirement for SNARE-dependent membrane traffic in integrin trafficking, as well as cell adhesion and migration. The goal of the present research was to ascertain whether SNARE-dependent membrane trafficking is required specifically for integrin-mediated signaling. Membrane traffic was inhibited in Chinese hamster ovary cells by expression of dominant-negative (E329Q) N-ethylmaleimide-sensitive fusion protein (NSF) or a truncated form of the SNARE SNAP23. Integrin signaling was monitored as cells were plated on fibronectin under serum-free conditions. E329Q-NSF expression inhibited phosphorylation of focal adhesion kinase (FAK) on Tyr397 at early time points of adhesion. Phosphorylation of FAK on Tyr576, Tyr861 and Tyr925 was also impaired by expression of E329Q-NSF or truncated SNAP23, as was trafficking, localization and activation of Src and its interaction with FAK. Decreased FAK-Src interaction coincided with reduced Rac activation, decreased focal adhesion turnover, reduced Akt phosphorylation and lower phosphatidylinositol 3,4,5-trisphosphate levels in the cell periphery. Over-expression of plasma membrane-targeted Src or phosphatidylinositol 3-kinase (PI3K) rescued cell spreading and focal adhesion turnover. The results suggest that SNARE-dependent trafficking is required for integrin signaling through a FAK/Src/PI3K-dependent pathway.     

Mutations in the Extracellular Domain Cause RET Loss of Function by a Dominant Negative Mechanism

The RET proto-oncogene encodes a tyrosine kinase receptor expressed in neuroectoderm-derived cells. Mutations in specific regions of the gene are responsible for the tumor syndromes multiple endocrine neoplasia types 2A and 2B (MEN 2A and 2B), while mutations along the entire gene are involved in a developmental disorder of the gastrointestinal tract, Hirschsprung’s disease (HSCR disease). Two mutants in the extracellular domain of RET, one associated with HSCR disease and one carrying a flag epitope, were analyzed to investigate the impact of the mutations on RET function. Both mutants were impeded in their maturation, resulting in the lack of the 170-kDa mature form and the accumulation of the 150-kDa immature form in the endoplasmic reticulum. Although not exposed on the cell surface, the 150-kDa species formed dimers and aggregates; this was more pronounced in a double mutant bearing a MEN 2A mutation. Tyrosine phosphorylation and the transactivation potential were drastically reduced in single and double mutants. Finally, in cotransfection experiments both mutants exerted a dominant negative effect over protoRET and RET2A through the formation of a heteromeric complex that prevents their maturation and function. These results suggest that HSCR mutations in the extracellular region cause RET loss of function through a dominant negative mechanism.     

The many faces of RET dysfunction in kidney

Signaling pathways that are activated upon interaction of glial cell-line derived neurotrophic factor (Gdnf), its coreceptor Gfra1, and receptor tyrosine kinase Ret are critical for kidney development and ureter maturation. Outside the kidney, this pathway is implicated in a number of congenital diseases including Hirschsprung disease (intestinal aganglionosis, HSCR) and hereditary cancer syndromes (MEN 2). Total lack of Gdnf, Gfra1 or Ret in mice results in perinatal lethality due to bilateral renal agenesis or aplasia. In humans, RET mutations have been identified in a spectrum of congenital malformations involving the RET axis including isolated HSCR, isolated congenital anomalies of kidney or urinary tract (CAKUT), or CAKUT and HSCR together. The molecular basis for these pleiotropic effects of RET has just begun to be unraveled. In an effort to delineate the pathogenetic mechanisms that underlie these congenital malformations, we and others have characterized Ret''s role in early kidney and urinary system development. Here we present a brief overview of the “many faces” of Ret dysfunction in kidney with particular emphasis on Ret''s signaling specificity and intergenic interactions that confer normal urinary system development.Key words: RET, GDNF, kidney, RTK, CAKUT, branching morphogenesis, ureter     

Regulation of TGFβ receptor trafficking and signaling by atypical protein kinase C

Transforming growth factor beta (TGFβ) signaling is linked to the membrane trafficking of TGFβ receptors. The Protein Kinase C (PKC) family of serine/threonine kinases have been implicated in modulating the endocytic processes of various receptors. The present study investigated whether PKC activity plays a role in the trafficking, and signaling of TGFβ receptors, and further explored which PKC isoforms may be responsible for altered TGFβ signaling patterns. Using immunofluorescence microscopy and ¹²⁵I-TGFβ internalization assays, we show that the pharmacological inhibition of PKC activity alters TGFβ receptor trafficking and delays TGFβ receptor degradation. Consistent with these findings, we demonstrate that PKC inhibition extends TGFβ-dependent Smad2 phosphorylation. Previous studies have shown that PKCζ associates with TGFβ receptors to modulate cell plasticity. We therefore used siRNA directed at the atypical PKC isoforms to investigate if reducing PKCι and PKCζ protein levels would delay TGFβ receptor degradation and extend TGFβ signaling. Our findings suggest that atypical PKC isoforms regulate TGFβ signaling by altering cell surface TGFβ receptor trafficking and degradation.     

Trafficking of Lyn through the Golgi caveolin involves the charged residues on alphaE and alphaI helices in the kinase domain

Src-family kinases, known to participate in signaling pathways of a variety of surface receptors, are localized to the cytoplasmic side of the plasma membrane through lipid modification. We show here that Lyn, a member of the Src-family kinases, is biosynthetically transported to the plasma membrane via the Golgi pool of caveolin along the secretory pathway. The trafficking of Lyn from the Golgi apparatus to the plasma membrane is inhibited by deletion of the kinase domain or Csk-induced "closed conformation" but not by kinase inactivation. Four residues (Asp346 and Glu353 on alphaE helix, and Asp498 and Asp499 on alphaI helix) present in the C-lobe of the kinase domain, which can be exposed to the molecular surface through an "open conformation," are identified as being involved in export of Lyn from the Golgi apparatus toward the plasma membrane but not targeting to the Golgi apparatus. Thus, the kinase domain of Lyn plays a role in Lyn trafficking besides catalysis of substrate phosphorylation.     

AZD1480 Blocks Growth and Tumorigenesis of RET- Activated Thyroid Cancer Cell Lines

Persistent RET activation is a frequent event in papillary thyroid carcinoma (PTC) and medullary thyroid carcinoma (MTC). In these cancers, RET activates the ERK/MAPK, the PI3K/AKT/mTOR and the JAK/STAT3 pathways. Here, we tested the efficacy of a JAK1/2- inhibitor, AZD1480, in the in vitro and in vivo growth of thyroid cancer cell lines expressing oncogenic RET. Thyroid cancer cell lines harboring RET/PTC1 (TPC-1), RET M918T (MZ-CRC1) and RET C634W (TT) alterations, as well as TPC-1 xenografts, were treated with JAK inhibitor, AZD1480. This inhibitor led to growth inhibition and/or apoptosis of the thyroid cancer cell lines in vitro, as well as to tumor regression of TPC-1 xenografts, where it efficiently blocked STAT3 activation in tumor and stromal cells. This inhibition was associated with decreased proliferation, decreased blood vessel density, coupled with increased necrosis. However, AZD1480 repressed the growth of STAT3- deficient TPC-1 cells in vitro and in vivo, demonstrating that its effects in this cell line were independent of STAT3 in the tumor cells. In all cell lines, the JAK inhibitor reduced phospho-Y1062 RET levels, and mTOR effector phospho-S6, while JAK1/2 downregulation by siRNA did not affect cell growth nor RET and S6 activation. In conclusion, AZD1480 effectively blocks proliferation and tumor growth of activated RET- thyroid cancer cell lines, likely through direct RET inhibition in cancer cells as well as by modulation of the microenvironment (e.g. via JAK/phospho-STAT3 inhibition in endothelial cells). Thus, AZD1480 should be considered as a therapeutic agent for the treatment of RET- activated thyroid cancers.