RET Expression Research Articles

Mitochondria-Targeted Nitroxide, Mito-CP, Suppresses Medullary Thyroid Carcinoma Cell Survival In Vitro and In Vivo

Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor mainly caused by mutations in the RET proto-oncogene. For MTC therapy, the U.S. Food and Drug Administration recently approved vandetanib and cabozantinib, multikinase inhibitors targeting RET and other tyrosine kinase receptors of vascular endothelial growth factor, epidermal growth factor, or hepatocyte growth factor. Nevertheless, not all patients with the progressive MTC respond to these drugs, requiring the development of additional therapeutic modalities that have distinct activity. We aimed to evaluate mitochondria-targeted carboxy-proxyl (Mito-CP), a mitochondria-targeted redox-sensitive agent, for its tumor-suppressive efficacy against MTC. In vitro cultures of 2 human MTC cell lines, TT and MZ-CRC-1, and TT xenografts in mice were treated with Mito-CP in comparison with vandetanib. The effects on cell survival/death, RET expression, mitochondrial integrity, and oxidative stress were determined. Contrary to vandetanib, Mito-CP induced RET downregulation and strong cytotoxic effects in both cell lines in vitro, including caspase-dependent apoptosis. These effects were accompanied by mitochondrial membrane depolarization, decreased oxygen consumption, and increased oxidative stress in cells. Intriguingly, Mito-CP-induced cell death, but not RET downregulation, was partially inhibited by the reactive oxygen species scavenger, N-acetyl-cysteine, indicating that Mito-CP mediates tumor-suppressive effects via redox-dependent as well as redox-independent mechanisms. Orally administered Mito-CP effectively suppressed TT xenografts in mice, with an efficacy comparable to vandetanib and relatively low toxicity to animals. Our results suggest that Mito-CP can effectively suppress MTC cell growth/survival via a mechanism distinct from vandetanib effects. Mitochondrial targeting may be a potential strategy for MTC therapy.

The isolectin IB4 binds RET receptor tyrosine kinase in microglia

Ret receptor tyrosine kinase is the signaling component of the receptor complex for the family ligands of the glial cell line-derived neurotrophic factor (GDNF). Ret is involved in the development of enteric nervous system, of sympathetic, parasympathetic, motor and sensory neurons, and it is necessary for the post-natal maintenance of dopaminergic neurons. Ret expression has been as well demonstrated on microglia and several evidence indicate that GDNF regulates not only neuronal survival and maturation but also certain functions of microglia in the brain. Here, we demonstrated that the plant lectin Griffonia (Bandeiraea) simplicifolia lectin I, isolectin B4 (IB4), commonly used as a microglial marker in the brain, binds to the glycosylated extracellular domain of Ret on the surface of living NIH3T3 fibroblasts cells stably transfected with Ret as well as in adult rat brain as revealed by immunoblotting. Furthermore, confocal immunofluorescence analysis demonstrated a clear overlap in staining between pRet and IB4 in primary microglia cultures as well as in adult rat sections obtained from control or post-ischemic brain after permanent middle artery occlusion (pMCAO). Interestingly, IB4 staining identified activated or ameboid Ret-expressing microglia under ischemic conditions. Collectively, our data indicate Ret receptor as one of the IB4-reactive glycoconjugate accounting for the IB4 stain in microglia under physiological and ischemic conditions.

Allele-Specific Expression at theRETLocus in Blood and Gut Tissue of Individuals Carrying Risk Alleles for Hirschsprung Disease

RET common variants are associated with Hirschsprung disease (HSCR; colon aganglionosis), a congenital defect of the enteric nervous system. We analyzed a well-known HSCR-associated RET haplotype that encompasses linked alleles in coding and noncoding/regulatory sequences. This risk haplotype correlates with reduced level of RET expression when compared with the wild-type counterpart. As allele-specific expression (ASE) contributes to phenotypic variability in health and disease, we investigated whether RET ASE could contribute to the overall reduction of RET mRNA detected in carriers. We tested heterozygous neuroblastoma cell lines, ganglionic gut tissues (18 HSCR and 14 non-HSCR individuals) and peripheral blood mononuclear cells (PBMCs; 16 HSCR and 14 non-HSCR individuals). Analysis of the data generated by SNaPshot and Pyrosequencing revealed that the RET risk haplotype is significantly more expressed in gut than in PBMCs (P = 0.0045). No ASE difference was detected between patients and controls, irrespective of the sample type. Comparison of total RET expression levels between gut samples with and without ASE, correlated reduced RET expression with preferential transcription from the RET risk haplotype. Nonrandom RET ASE occurs in ganglionic gut regardless of the disease status. RET ASE should not be excluded as a disease mechanism acting during development.

Abstract PD01-06: Inhibition of the Ret receptor tyrosine kinase in combination with endocrine therapy impacts on migration and metastatic potential of estrogen receptor positive breast cancer models.

Abstract The Ret receptor tyrosine kinase is overexpressed in approximately 30% of human breast cancers, many of which are estrogen receptor-alpha positive (ER+). Using the ER+ breast cancer models MCF7 and T47D, we have shown that RET is an ER target gene and that Ret signaling enhances estrogen-driven anchorage-dependent and -independent proliferation (Boulay et al 2008). In our current work we are investigating the potential role of Ret in endocrine therapy response using in vivo and in vitro models. Ret is activated by peptides of the glial derived neurotrophic factor (GDNF) family. GDNF treatment of ER+ T47D and MCF7 breast cancer cell lines stimulates activation of multiple intracellular pathways, increases anchorage-dependent and -independent growth, as well as migration. To test if Ret activation impacts on endocrine therapy response, we treated MCF7/Aromatase (Aro)-expressing cells for seven days with different endocrine agents in the presence or absence of GDNF to activate Ret. Activation of Ret signaling rescued the proliferative block that resulted from treatment of the MCF7/Aro cells with tamoxifen, fulvestrant or letrozole. A transcriptome analysis was performed and an Ingenuity Pathway Analysis of the transcripts altered in MCF7/Aro cells treated with fulvestrant or letrozole revealed that the highest pathways altered by addition of GDNF are related to interferon signaling and inflammation. Indeed, proinflammatory RNAs, including IL6, IL8 and CXCL10 are among the top transcripts upregulated in cultures treated with fulvestrant plus GDNF, or letrozole plus GDNF. Elevated IL6 serum levels in breast cancer patients correlate with poor prognosis. We have concentrated on IL6 in the work that will be presented based on this clinical data, as well as our finding that IL6 stimulates Ret expression and activation in ER+ breast cancer models. We verified that addition of GDNF to MDCF/Aro cultures treated with fulvestrant or with letrozole led to an increase of IL6 in the conditional medium of the cultures. Moreover, addition of GDNF to fulvestrant- or tamoxifen-treated tumor cells increased migration, which could be inhibited by an IL6 blocking antibody, showing the importance of this cytokine for tumor cell motility. Additional data will be presented showing there is a Ret-IL6 feed-forward loop present in ER+ tumor cell models and that this is important for migration. In order to examine the role of Ret in vivo, in a metastatic breast cancer model, we have used the ER+/Ret+ J110 tumor cell line, generated from mammary tumors of MMTV-AIB1 transgenic females. Ret is active in J110 mammary tumors and its activity can be blocked with a Ret selective kinase inhibitor. In vivo data will be presented showing the effect of blocking Ret alone, or in combination with endocrine agents, on metastatic spread of primary tumors to the lungs. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD01-06.

Endocrine Regulation of Spermatogonial Stem Cells in the Seminiferous Epithelium of Adult Mice

A balance between self-renewal and differentiation of spermatogonial stem cells (SSCs) is required to maintain sperm production throughout male life. The seminiferous epithelium is organized into stages of spermatogenesis based on the complement of germ cell types within a tubular section of the testis. The stages exist in close physical proximity and foster diverse phases of germ cell development despite exposure to a similar endocrine milieu that supports coordinated spermatogenesis. The objective of the current study was to identify the population dynamics of SSCs in vivo. We hypothesized that SSC populations and their niches are specifically distributed across the mature seminiferous epithelium in the mouse testis. To test this hypothesis, we conducted stem cell transplantation of germ cells obtained from stage-specific clusters of seminiferous tubules representing areas of high responsiveness to follicle-stimulating hormone (IX–I), androgen (II–IV), and retinoid (V–VIII) signaling. Similarly, we analyzed the expression of genes linked with SSC activity in these groups of stages. No stage-specific differences in the colonization efficiency or the colony number were detected after SSC transplantation, indicating that SSCs are equally distributed across all stages of the seminiferous tubule. In contrast, SSCs obtained from donor stages IX–IV established larger donor-derived colonies due to increased colony expansion. SSCs originating from different stages have varying degrees of stem cell activity in vivo, a notion consistent with Gdnf, Ret, and Bcl6b expression data. These results support the conclusion of a stage-specific, microenvironment-regulating SSC self-renewal and suggest the presence of a transit-amplifying population of undifferentiated spermatogonia in vivo.

Gdnf Activates Midline Repulsion by Semaphorin3B via NCAM during Commissural Axon Guidance

The Neurotrophic factor gdnf plays diverse developmental roles, supporting survival and also acting asachemoattractant for axon and cell migration. We report that in the developing spinal cord, a focal source of gdnf is present in the floor plate (FP) where commissural axons cross the midline. Gdnf has nodirect guidance properties but switches on the responsiveness of crossing commissural growth cones to the midline repellent Semaphorin3B by suppressing calpain-mediated processing of the Sema3B signaling coreceptor Plexin-A1. Analysis of single and double mutant mouse models indicates that although gdnf is the principal trigger of Sema3B midline repulsion, it acts with another FP cue, NrCAM. Finally, genetic and invitro experiments provide evidence that this gdnf effect is RET independent and mediated by NCAM/GFRα1 signaling. This study identifies a regulator of midline crossing and reveals interplays between Semaphorin and gdnf signaling during axon guidance.

Expression of the RET Proto-oncogene Is Regulated by TFAP2C in Breast Cancer Independent of the Estrogen Receptor

The RET proto-oncogene is expressed as part of the estrogen receptor (ER) cluster in breast cancer. We sought to determine if TFAP2C regulates Ret expression directly or indirectly through ER. Chromatin immunoprecipitation sequencing (ChIP-Seq) and gel-shift assay were used to identify TFAP2C binding sites in the RET promoter in four breast cancer cell lines. Ret mRNA and protein levels were evaluated in ER-positive and ER-negative breast cancer cell lines after knockdown of TFAP2C. Luciferase expression assay was performed to assess expression from two of the identified sites. ChIP-Seq identified five main binding peaks for TFAP2C in the RET promoter at -101.5 kb, -50.7 kb, -32.5 kb, +5.0 kb, and +33.6 from the RET transcriptional start site. Binding at three of the AP-2 sites was conserved across all four cell lines, whereas the RET -101.5 and RET +33.6 sites were each found to be unbound by TFAP2C in one cell line. A TFAP2C consensus element was confirmed for all five sites. Knockdown of TFAP2C by siRNA in ER-positive MCF-7 cells resulted in significant down regulation of Ret mRNA compared to nontargeting (NT) siRNA (0.09 vs. 1.0, P < 0.001). Knockdown of TFAP2C in ER-negative MDA-MB-453 cells also led to a significant reduction in Ret mRNA compared to NT siRNA (0.16 vs. 1.0, P < 0.001). In MCF-7 cells, knockdown of TFAP2C abrogated Ret protein expression (0.02 vs. 1.0, P < 0.001) before reduction in ER. TFAP2C regulates expression of the RET proto-oncogene through five AP-2 regulatory sites in the RET promoter. Regulation of Ret by TFAP2C occurs independently of ER expression in breast carcinoma.

RET is a potential tumor suppressor gene in colorectal cancer

Cancer arises as the consequence of mutations and epigenetic alterations that activate oncogenes and inactivate tumor suppressor genes. Through a genome-wide screen for methylated genes in colon neoplasms, we identified aberrantly methylated RET in colorectal cancer. RET, a transmembrane receptor tyrosine kinase and a receptor for the GDNF-family ligands, was one of the first oncogenes to be identified and has been shown to be an oncogene in thyroid cancer and pheochromocytoma. However, unexpectedly, we found RET is methylated in 27% of colon adenomas and in 63% of colorectal cancers, and now provide evidence that RET has tumor suppressor activity in colon cancer. The aberrant methylation of RET correlates with decreased RET expression, whereas the restoration of RET in colorectal cancer cell lines results in apoptosis. Furthermore, in support of a tumor suppressor function of RET, mutant RET has also been found in primary colorectal cancer. We now show that these mutations inactivate RET, which is consistent with RET being a tumor suppressor gene in the colon. These findings suggest that the aberrant methylation of RET and the mutational inactivation of RET promote colorectal cancer formation and that RET can serve as a tumor suppressor gene in the colon. Moreover, the increased frequency of methylated RET in colon cancers compared to adenomas suggests RET inactivation is involved in the progression of colon adenomas to cancer.

Evaluation of expression and function of vascular endothelial growth factor receptor 2, platelet derived growth factor receptors-alpha and -beta, KIT, and RET in canine apocrine gland anal sac adenocarcinoma and thyroid carcinoma

BackgroundToceranib phosphate (Palladia) has a reported objective response rate of 25% in both canine apocrine gland anal sac adenocarcinoma (AGASACA) and thyroid carcinoma (TC), with stable disease occurring in an additional 50-60% of dogs. The basis for the observed responses to toceranib is not known. The purpose of this study was to evaluate AGASACA and TC samples for the expression and activation of VEGFR2, PDGFRα, PDGFRβ, KIT and RET to assess whether dysregulation of these receptor tyrosine kinases (RTKs) may contribute to the biologic activity of toceranib.ResultsmRNA for VEGFR2, PDGFRα/β, KIT and RET was detected in all AGASACA samples. mRNA for VEGFR2, PDGFRα/β, and KIT was detected in all TC samples, while mRNA for RET was amplified in 10/15 samples. No phosphorylation of VEGFR2, PDGFRα/β, or KIT was observed on the arrays. However, phosphorylation of RET was detected in 54% of the primary AGASACA and 20% of TC. VEGFR2 was expressed in 19/24 primary and 6/10 metastatic AGASACA and 6/15 TC samples. KIT was present in 8/24 primary and 3/10 metastatic AGASACA and 9/15 TC samples. PDGFRα expression was noted in all tumor samples. In contrast PDGFRβ expression was found in only a few tumor samples but was evident in the stroma of all tumor specimens.ConclusionsKnown targets of toceranib are expressed in both AGASAC and TC. Given the observed expression of VEGFR and PDGFRα/β and phosphorylation of RET, these RTKs merit investigation as to their roles in the biology of AGSACA and TC and their contribution to toceranib’s activity.

Glial cell line–derived neurotrophic factor promotes invasive behaviour in testicular seminoma cells

The glial cell line-derived neurotrophic factor (GDNF) has multiple functions that promote cell survival, proliferation and migration in different cell types. The experimental over-expression of GDNF in mouse testis leads to infertility and promotes seminomatous germ cell tumours in older animals, which suggests that deregulation of the GDNF pathway may be implicated in germ cell carcinogenesis. GDNF activates downstream pathways upon binding to its specific co-receptor GDNF family receptor-a 1 (GFRA1). This complex then interacts with Ret and other co-receptors to activate several intracellular signalling cascades. To explore the involvement of the GDNF pathway in the onset and progression of testicular germ cell tumours, we analysed GFRA1 and Ret expression patterns in seminoma samples. We demonstrated, via immunohistochemistry, that GFRA1, but not Ret, is over-expressed in in situ carcinoma (CIS) and in intratubular and invasive seminoma cells compared with normal human germ cells. Functional analysis of the GDNF biological activity was performed on TCam-2 seminoma cell line. Reverse transcription-PCR (RT-PCR) and immunohistochemical analyses demonstrate that TCam-2 cells express both GFRA1 and Ret mRNA, but only GFRA1 was detected at the protein level. In TCam-2 cells, although GDNF is not mitogenic, it is able to induce migration, as demonstrated by a Boyden chamber assay, possibly through the Src and MEK pathways. Moreover, GDNF promotes invasive behaviour, an effect dependent on pericellular protease activity, possibly through the activity of matrix metalloproteinases. GFRA1 over-expression in CIS and seminoma cells, along with the functional analyses in TCam-2 cells, suggests an involvement of the GDNF pathway in the progression of testicular germ cell cancer.

Abstract LB-88: Identification of recurrent oncogenic KIF5B-RET rearrangements in non-small cell lung cancer

Abstract Background: A large number of non-small cell lung cancer (NSCLC) cases from never/former light smokers can be explained by driving oncogenic alterations in EGFR, KRAS, ERBB2, BRAF, ALK and ROS1. EGFR and ALK targeted tyrosine kinase inhibitors are effective clinical therapies for EGFR mutant and ALK rearranged NSCLC, respectively. We wished to study cancers that do not harbor a known genomic alteration in order to identify novel therapeutic targets that may increase treatment options for NSCLC patients. Methods and Results: We initially sequenced a NSCLC specimen from a 44 year old Caucasian never smoker in a CLIA certified lab (Foundation Medicine) using a next generation sequencing assay that captures and sequences 2574 coding exons representing 145 cancer relevant genes plus 37 introns from 14 genes frequently rearranged in cancer. We identified an 11,294,741 bp pericentric inversion on chromosome 10 generating a novel gene fusion joining exons 1-15 of KIF5B to exons 12-20 of RET (K15:R12). This fusion gene contains the kinesin motor and coiled-coil domains of KIF5B and the entire tyrosine kinase domain of RET. Expression of RET was confirmed by immunohistochemistry (IHC) and cDNA sequencing detected a 7.3-fold RET expression increase beginning at exon 12 relative to exons 1-11. Screening of additional 117 NSCLC patients using RET IHC identified 22 positive cases; sequencing of 15/22 of these identified 1 additional patient with a KIF5B-RET fusion. We also evaluated 526 tumors from never/former limited smokers (121 Caucasian and 405 Asian) and identified 10 additional (1/121 Caucasian (0.8%) and 9/405 Asian (2%)) positive patients. All fusion positive tumors were wild type for known oncogenes (frequency of 6.3 % (10/159) in WT patients). Four unique KIF5B-RET variants were identified: 8 K15:R12 (variant 1), 3 K16:R12 (variant 2), 1 K22:R12 (variant 3) and 1 K15:R11 (variant 4). We introduced K15:R12 (variant 1) into Ba/F3 cells and observed IL-3 independent growth consistent with oncogenic transformation. These KIF5B-RET Ba/F3 cells were sensitive to sunitinib, sorafenib and vandetinib, multi-targeted kinase inhibitors that inhibit RET, but not gefitinib, an EGFR kinase inhibitor. Sunitinib, but not gefitinib, inhibited RET phosphorylation in the KIF5B-RET Ba/F3 cells. Conclusions: We identify a novel oncogenic KIF5B-RET fusion gene in a subset of NSCLC patients lacking other known driver mutations. Our findings suggest that RET kinase inhibitors should be tested in prospective clinical trials for therapeutic benefit in NSCLC patients bearing KIF5B-RET rearrangements. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-88. doi:1538-7445.AM2012-LB-88

Abstract 1243: RET and integrin crosstalk provides functional plasticity

Abstract The RET receptor tyrosine kinase is an important mediator of cell growth, proliferation, and differentiation. RET becomes activated upon formation of a complex with the glial cell-line derived neurotrophic factor (GDNF) ligand and GDNF-family receptor (GFRa), which leads to activation of multiple downstream signalling pathways, including PI3K/AKT, MEK/ERK, STAT3, and SRC. Constitutive activation of RET, through translocations or missense mutations, leads to papillary thyroid carcinoma (PTC) and medullary thyroid carcinoma (MTC), respectively. Also, RET expression has been detected in pancreatic and breast cancers, and has been linked to increased metastatic potential. In order to clarify the underlying role of RET in tumour progression, we examined the relationship between RET and two integrin subunits, β1 (ITGB1) and β3 (ITGB3). Integrin proteins are important for cell attachment to the extracellular matrix and focal adhesion (FA) formation. Functionally, integrins are important for cell-adhesion and migration as they provide traction and leverage for cell movement. Previously, we have shown that RET is able to increase cell-migration and adhesion, and that both ITGB1 and ITGB3 are important for these processes. Here, we show that RET-mediated cell-migration is persistent over 24 hours, and that patterns of cell-adhesion fluctuate over this time, predictably representing different forces needed to move across the microenvironment. Cell-adhesion is increased upon 1 hour of GDNF treatment, but is lost between 3-12 hours of GDNF treatment. Interestingly, we observed that ITGB1 activation downstream of RET, detected by co-immunoprecipitation with paxillin, is transient, and lasts for 1 hour. Conversely, ITGB3 activation downstream of RET, detected using an active heterodimer-specific antibody, is sustained between 1-12 hours of GDNF treatment. These results demonstrate unique functional roles for ITGB1 and ITGB3 downstream of RET. We also examined the importance of signalling pathways downstream of RET for integrin activation, and found that, in a 2D collagen environment, PI3K/AKT and MEK/ERK are important for RET-mediated FA formation. However, in a 3D collagen environment where cells formed spheroids, PI3K/AKT and STAT3 are important for cell-outgrowth, downstream of RET activation. This likely represents different cellular responses needed to overcome environment-specific obstacles. Finally, we showed evidence for a relationship between RET, ITGB1 and ITGB3 in tumour progression using a cohort of various clinically annotated thyroid cancer samples. Ultimately, we have shown a role for in activation of two integrin subunits, ITGB1 and ITGB3, downstream of RET and how each of these proteins may contribute to metastatic events, particularly those involved in thyroid cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1243. doi:1538-7445.AM2012-1243

Abstract 5595: ALK mutant-driven cell lines develop crizotinib resistance by secondary mutations and ALK-independent means

Abstract The clinical success of crizotinib against mutant ALK-expressing cancers has generated great interest; however, the emergence of drug resistance is limiting its durability in some patients. In NSCLC, crizotinib resistance is linked to secondary mutations within EML4-ALK at amino acids L1196M, C1156Y, L1152R. A mutation at amino acid F1174L reduced the sensitivity to criozotinib in a patient with an inflammatory myofibroblastic tumor harboring a RANBP2-ALK translocation. The goal of our studies was to evaluate mechanisms of crizotinib resistance in a range of ALK-mutant expressing cell lines. Cells from ALCL (Karpas299), NSCLC (NCI-H3122 and NCI-H2228) or neuroblastoma (SKNSH and KELLY) developed resistance to crizotinib after being exposed to incremental increases in the compound over several weeks. NPM-ALK expressing Karpas299 cells independently acquired point mutations in ALK at amino acids N1178H and I1171N. As the concentration of drug was increased, cells that harbored the NPM-ALK N1178H mutation also expressed a mutation at G1269A. Each mutation enhanced the temperature dependent auto-phosphorylation activity of the kinase and diminished the inhibitory effect of crizotinib on downstream ALK signaling. Mutations in Karpas299 cells were also accompanied by up regulation of a drug efflux transporter, BCRP, whose heightened expression level was dependent on the presence of the compound. In contrast no additional mutations were discovered in ALK in crizotinib resistant NCI-H3122 and NCI-H2228 (EML4-ALK) or SKNSH and KELLY (ALK F1174L) cell lines. Instead both NCI-H3122 and NCI-H2228 resistant cell lines displayed increased levels of phospho-HER2 and HER3 and activation of AKT and ERK that may be due to autocrine secretion of a growth factor. NSCLC resistant lines also displayed an apparent change in phenotype with a mesenchymal-to-epithelial transition. SKNSH and KELLY are inherently less sensitive to crizotinib. We found that these cells may survive exposure to high levels of the compound by altering RET expression. Our observations represent potential mechanisms of resistance demonstrated in a small number of cell lines. It will be important to align these results with emerging data from drug-resistant patients to understand the multiple mechanisms by which resistance might develop. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5595. doi:1538-7445.AM2012-5595

Divergent fate and origin of neurosphere-like bodies from different layers of the gut

Enteric neural stem cells (ENSCs) are a population of neural crest-derived multipotent stem cells present in postnatal gut that may play an important role in regeneration of the enteric nervous system. In most studies, these cells have been isolated from the layer of the gut containing the myenteric plexus. However, a recent report demonstrated that neurosphere-like bodies (NLBs) containing ENSCs could be isolated from mucosal biopsy specimens from children, suggesting that ENSCs are present in multiple layers of the gut. The aim of our study was to assess whether NLBs isolated from layers of gut containing either myenteric or submucosal plexus are equivalent. We divided the mouse small intestine into two layers, one containing myenteric plexus and the other submucosal plexus, and assessed for NLB formation. Differences in NLB density, proliferation, apoptosis, neural crest origin, and phenotype were investigated. NLBs isolated from the myenteric plexus layer were present at a higher density and demonstrated greater proliferation, lower apoptosis, and higher expression of nestin, p75, Sox10, and Ret than those from submucosal plexus. Additionally, they contained a higher percentage of neural crest-derived cells (99.4 ± 1.5 vs. 0.7 ± 1.19% of Wnt1-cre:tdTomato cells; P < 0.0001) and produced more neurons and glial cells than those from submucosal plexus. NLBs from the submucosal plexus layer expressed higher CD34 and produced more smooth muscle-like cells. NLBs from the myenteric plexus layer contain more neural crest-derived ENSCs while those from submucosal plexus appear more heterogeneous, likely containing a population of mesenchymal stem cells.

Ureteric Morphogenesis Requires Fgfr1 and Fgfr2/Frs2α Signaling in the Metanephric Mesenchyme

Conditional deletion of fibroblast growth factor receptors (Fgfrs) 1 and 2 in the metanephric mesenchyme (MM) of mice leads to a virtual absence of MM and unbranched ureteric buds that are occasionally duplex. Deletion of Fgfr2 in the MM leads to kidneys with cranially displaced ureteric buds along the Wolffian duct or duplex ureters. Mice with point mutations in Fgfr2's binding site for the docking protein Frs2α (Fgfr2(LR/LR)), however, have normal kidneys; the roles of the Fgfr2/Frs2α signaling axis in MM development and regulating the ureteric bud induction site are incompletely understood. Here, we generated mice with both Fgfr1 deleted in the MM and Fgfr2(LR/LR) point mutations (Fgfr1(Mes-/-)Fgfrf2(LR/LR)). Unlike mice lacking both Fgfr1 and Fgfr2 in the MM, these mice had no obvious MM defects but had cranially displaced or duplex ureteric buds, probably as a result of decreased Bmp4 expression. Fgfr1(Mes-/-)Fgfr2(LR/LR) mice also had subsequent defects in ureteric morphogenesis, including dilated, hyperproliferative tips and decreased branching. Ultimately, they developed progressive renal cystic dysplasia associated with abnormally oriented cell division. Furthermore, mutants had increased and ectopic expression of Ret and its downstream targets in ureteric trunks, and exhibited upregulation of Ret/Etv4/5 signaling effectors, including Met, Myb, Cxcr4, and Crlf1. These defects were associated with reduced expression of Bmp4 in mesenchymal cells near mutant ureteric bud tips. Taken together, these results demonstrate that Fgfr2/Frs2α signaling in the MM promotes Bmp4 expression, which represses Ret levels and signaling in the ureteric bud to ensure normal ureteric morphogenesis.

RET: A Multi-Faceted Gene in Human Cancer

Receptor tyrosine kinases (RTK) are transmembrane (TM) proteins featuring an intracellular domain containing the tyrosine kinase (TK) enzyme. RTKs are often involved in cancer formation [13]. Notable examples are epidermal growth factor receptor (EGFR/ HER1) and anaplastic lymphoma kinase (ALK) in non-small cell lung carcinoma (NSCLC) [4], KIT in gastrointestinal stromal tumors (GIST) [5], FLT3 in acute myeloid leukemia (AML) [6], and HER2/ ERBB2/neu in breast cancer [7]. In some cases, cancer cells up-regulate expression of the RTK (as an example HER2 in breast cancer), its cognate growth factor or both, in other cases, structural alterations such as chromosomal rearrangements leading to the RTK recombination to heterologous genes (as an example EML4-ALK in lung adenocarcinoma) or point mutations (as EGFR, KIT or FLT3 mutations in NSCLC, GIST, or AML, respectively), lead to unchecked kinase and oncogenic activity [1-3]. This notion has stimulated the search for agents, such as monoclonal antibodies against the RTK extracellular domain (like trastuzumab for HER2 or cetuximab for EGFR) or ATP-competitive small molecule protein kinase inhibitors (PKIs) (like gefitinib and erlotinib for EGFR or crizotinib for ALK), to combat cancers driven by oncogenic RTKs [1-3]. The RET RTK was originally identified as an oncogene activated by a rearrangement occurred in vitro during transfection of NIH3T3 cells with human lymphoma DNA [8]. RET protein belongs to a cell-surface complex able to bind glial-derived neurotrophic factor (GDNF) ligands (GDNF, neurturin, artemin, and persephin) in conjunction with co-receptors of the GDNF receptor α family, designated GFRα 1-4 [9]. Binding to the ligand-co-receptor complex leads to RET dimerization and kinase activation. RET expression is tightly regulated during development and in the adulthood is limited to specific tissues, including neural crest-derived cells. RET is essential for the development of the enteric neurvous system and kidney, and germline loss-of-function mutations in RET cause Hirschsprung disease (aganglionic megacolon) and congenital anomalies of the kidney or lower urinary tract [10,11]. RET gene maps to chromosome 10q11.2. Fig. 1 shows that it is splitted in 21 coding exons. Exons 1-10 code for the extracellular region; exon 11 codes for the COOH-terminal part of the extracellular region, the TM domain, and the intracellular juxtamembrane domain. Finally, exons 12-21 code for the intracellular domain. An alternative splicing at exon 19 determine the synthesis of three RET protein isoforms with different C-terminal tails. In RET9 (1072 aa), exon 19 is unspliced; in RET51 (1114 aa), exon 19 is spliced to exon 20; in RET43 (1106 aa), exon 19 is spliced to to exon 21 [12-15]. RET9 and RET51 are the most abundant and well characterized isoforms (Fig. 1). RET protein features an extracellular portion (RET-EC) that

The Distinctive MicroRNA Signature of Acute Myeloid Leukemia with Translocation t(8;16)(p11;p13)/MYST3-CREBBP Is Responsible for RET Overexpression and Is Regulated by Epigenetic Mechanisms

Abstract 2434 IntroductionAcute myeloid leukemia (AML) with t(8;16)(p11;p13) and MYST3-CREBBP rearrangement [t(8;16) AML] is an infrequent leukemia subtype with specific clinical and biological characteristics including a specific gene expression profile with overexpression of RET protooncogene. Our group has previously described a specific signature of microRNAs (miRNAs), most of which were downregulated, and found an inverse correlation between RET mRNA expression and the expression of several of these miRNAs. Since MYST3 and CREBBP are chromatin-modifying proteins, we have now investigated the role of epigenetic mechanisms in the downregulation of the miRNAs in our signature and examined RET as a potential target of some of these miRNAs. MethodsTo analyze the possible epigenetic regulation of microRNA signature of t(8;16) AML, we first selected those miRNA genes with CpG islands in the promoter regions according to UCSC Genome Browser (http://genome.ucsc.edu). Changes in the expression of selected miRNA levels were studied in cryopreserved cells from a t(8;16) AML patient and in the K562 cell line after treatment for 72 hours with demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC, decitabine) and histone deacetylase inhibitor trichostatin A (TSA). Expression of selected miRNAs after treatment with 5-aza-dC and TSA was analyzed using TaqMan MicroRNA Assays (Applied Biosystems). In addition, the degree of quantitative methylation in the promoter region of these miRNA genes was measured with Human Cancer miRNA Genes, Signature Panel Methyl-Profiler DNA Methylation PCR Array (Qiagen). For the validation of RET as the target of specific miRNAs, we performed a Renilla/Luciferase assay with a modified expression vector psiCHECK-2 containing the 3'UTR region of RET cloned in the 3'UTR region of Renilla gene. Modulation of RET expression by selected miRNAs was analyzed after transfection of the corresponding pre-miRNAs in the K-562 cell line, and luminescence was read at 24 hours. ResultsSince methylation is a common mechanism of the regulation of miRNA expression, we performed a bioinformatic analysis which showed that 40% of the miRNAs in our t(8;16) AML signature harbored CpG islands in their promoter gene region, making them susceptible to silencing by methylation. After treatment with 5-AZA-dc, 60% of these miRNAs were re-expressed more than two fold-change. Interestingly, TSA induced a greater re-expression in 75% of these miRNAs. The degree of methylation status was analyzed in 8 of these miRNAs using the DNA methylation PCR Array. CpG hypermethylation was present to a certain degree in three miRNAs (22% in let-7g, 29% in miR-1, and 56% in miR-126) and to a greater extent (99%) in the miR-23b cluster, which comprises miR-23b, miR-24 and miR-27b.Due to the strong inverse correlation of several miRNAs of the t(8;16) AML signature and RET mRNA levels, we then focused on miRNA target validation of RET. The analysis of the 3’UTR region of RET with TargetScan v5.1 revealed the presence of target sites for 9 miRNAs of the signature (miR-15b, miR-195, miR-218, miR-15a, miR-34a, miR-424, miR-128, and miR-27a/b), including 2 putative binding sites for miR-218. The Renilla/Luciferase assay, performed with the modified psicheck2 vector containing the 3’UTR of the RET gene, showed that 6 of these 9 miRNAs (miR-218, miR-128, miR-27b, miR-15a, miR-195, and miR-424) produced a significant downregulation of Renilla translation (41–49%), confirming our hypothesis that RET is a target of some of the miRNAs of our t(8;16) AML signature. Interestingly, 4 of the miRNAs targeting RET (miR-218, miR-27b, miR-15a and miR-424) were re-expressed after treatment with 5-AZA-dc and TSA. ConclusionsOur findings provide greater insight into the genetic and epigenetic abnormalities in t(8;16) AML. Here we have shown that our t(8,16) AML-specific miRNA signature could be a functional consequence of impaired epigenetic mechanisms, including hypermethylation and abnormal histone acetylation. In addition, we provide the first validation of RET as a target of several miRNAs in this t(8;16) AML miRNA signature. Disclosures:No relevant conflicts of interest to declare.

Craniopharyngiomas Express Embryonic Stem Cell Markers (SOX2, OCT4, KLF4, and SOX9) as Pituitary Stem Cells but Do Not Coexpress RET/GFRA3 Receptors

Adult stem cells maintain some markers expressed by embryonic stem cells and express other specific markers depending on the organ where they reside. Recently, stem/progenitor cells in the rodent and human pituitary have been characterized as expressing GFRA2/RET, PROP1, and stem cell markers such as SOX2 and OCT4 (GPS cells). Our objective was to detect other specific markers of the pituitary stem cells and to investigate whether craniopharyngiomas (CRF), a tumor potentially derived from Rathke's pouch remnants, express similar markers as normal pituitary stem cells. We conducted mRNA and Western blot studies in pituitary extracts, and immunohistochemistry and immunofluorescence on sections from normal rat and human pituitaries and 20 CRF (18 adamantinomatous and two papillary). Normal pituitary GPS stem cells localized in the marginal zone (MZ) express three key embryonic stem cell markers, SOX2, OCT4, and KLF4, in addition to SOX9 and PROP1 and β-catenin overexpression. They express the RET receptor and its GFRA2 coreceptor but also express the coreceptor GFRA3 that could be detected in the MZ of paraffin pituitary sections. CRF maintain the expression of SOX2, OCT4, KLF4, SOX9, and β-catenin. However, RET and GFRA3 expression was altered in CRF. In 25% (five of 20), both RET and GFRA3 were detected but not colocalized in the same cells. The other 75% (15 of 20) lose the expression of RET, GFRA3, or both proteins simultaneously. Human pituitary adult stem/progenitor cells (GPS) located in the MZ are characterized by expression of embryonic stem cell markers SOX2, OCT4, and KLF4 plus the specific pituitary embryonic factor PROP1 and the RET system. Redundancy in RET coreceptor expression (GFRA2 and GFRA3) suggest an important systematic function in their physiological behavior. CRF share the stem cell markers suggesting a common origin with GPS. However, the lack of expression of the RET/GFRA system could be related to the cell mislocation and deregulated growth of CRF.

Runx1 promotes neuronal differentiation in dorsal root ganglion

Transcription factor Runx1 controls the cell type specification of peptidergic and nonpeptidergic nociceptive dorsal root ganglion (DRG) neurons by repressing TrkA and calcitonin gene-related peptide (CGRP) expression and activating Ret expression during late embryonic and early postnatal periods (Chen et al., 2006b; Kramer et al., 2006; Yoshikawa et al., 2007). Because Runx1 is expressed in DRG from early developmental stages, we examined the roles of Runx1 in the proliferation and the neuronal differentiation of DRG cells. We used transgenic Runx1-deficient (Runx1−/−::Tg) mice which are rescued from early embryonic lethality by selective expression of Runx1 in hematopoietic cells under the control of GATA-1 promoter. We found that TrkA-expressing (TrkA+) DRG neurons were decreased at embryonic day (E) 12.5 in contrast to the previous study showing that TrkA+ DRG neurons were increased at E17.5 in Runx1−/−::Tg mice (Yoshikawa et al., 2007). The number of DRG neurons which express neuronal markers Hu, NeuN and Islet1 was also reduced in Runx1−/−::Tg mice at E12.5, suggesting that the neuronal differentiation was suppressed in these mice. The cell cycle analysis using BrdU/IDU revealed that the number of DRG cells in S-phase and G2/M-phase was increased in Runx1−/−::Tg mice at E12.5, while the length of S-phase was not changed between Runx1+/+::Tg and Runx1−/−::Tg mice, suggesting that Runx1 negatively controls the proliferation of DRG progenitor cell subpopulation in early embryonic period. Hes1 is a negative regulator of neuronal differentiation (Ishibashi et al., 1995; Tomita et al., 1996), and we found that the number of Hes1+ DRG cells was increased in Runx1−/−::Tg mice at E12.5. In summary, the present study suggests a novel function that Runx1 activates the neuronal differentiation of DRG cell subpopulation through the repression of Hes1 expression in early embryonic period.

GDNF family ligand RET receptor in the brain of adult zebrafish

RET is a tyrosine kinase receptor, and transduces signaling by family of glial cell line-derived neurotrophic factor ligands (GFLs). RET is involved in the development of enteric nervous system, of sympathetic, parasympathetic, motor and sensory neurons. RET exists in two main isoforms originated by differential splicing, RET9 and RET51; phylogenetic studies have shown that the RET gene is conserved across vertebrates. The aim of this study was to investigate the RET expression within the brain of zebrafish, using immunohistochemistry, western blotting and RT-PCR. In homogenate brains both RET protein and mRNA were observed. RET immunoreactivity was widespread in neurons and neural processes of all the major regions of the brain. These results demonstrate the occurrence of RET and suggest an involvement of GDNF family ligands in the brain of adult zebrafish.