RET Protein Research Articles

Functional analysis of RET with multiple endocrine neoplasia type 2.

Multiple endocrine neoplastic type 2 (MEN2) is an endocrine carcinoma syndrome which is caused by a germline activation mutation that occurs during transfection (RET) proto-oncogene transmission. MEN2A patients are affected by RET (C634Y, C634R) mutation; MEN2B patients are affected by RET (M918T) mutation. We aim to identify RET mutations' (C634R and M918T) expression, location, and signaling activation during the disease's progression, which providing a theoretical basis for the study on etiology of multiple endocrine neoplasia. This study was conducted to determine whether RET dysfunction involves an induced mutation into SK-N-MC cells. Wildtype RET and mutant RET plasmids (M918T and C634R) were constructed and transfected into SK-N-MC cells, the protein level was detected by Western blot, the efficiency of the transfected cells was detected by real time PCR, and the location of RET protein in cells as-determined by immunofluorescence. SK-N-MC cells with different RET plasmids treated/untreated by GDNF, AKT and ERK1/2 phosphorylation detected by specific antibodies. We found that C634R mutation could enhance RET protein expression and change the location of the mutated protein and forced it into the nucleus, GDNF treatment alone can only enhance M918T RET phosphorylation level and not impact WT or C614R mutation, and AKT/ERK1/2 pathway can be affected by GDNF treatment. RET dysfunction involves an induced mutation into SK-N-MC cells.

ATF4 Targets RET for Degradation and Is a Candidate Tumor Suppressor Gene in Medullary Thyroid Cancer.

Medullary thyroid cancer (MTC) is an aggressive tumor that harbors activating mutations of the RET proto-oncogene. We previously reported that RET inhibits transcriptional activity of ATF4, the master regulator of the stress response pathway, to prevent cell death. We hypothesized that loss of function of ATF4 plays a role in initiation of MTC. Targeted deletion of Atf4 in mice was used to assess ATF4 function in the thyroid gland. ATF4 overexpression was achieved by adenoviral and lentiviral vectors. We used immunohistochemical analysis and western blotting of MTC tumors to determine protein levels of RET and ATF4 and the Kaplan-Meier method to determine their association with clinical outcome. Targeted deletion of Atf4 in mice causes C-cell hyperplasia, a precancerous lesion for MTC. Forced ATF4 expression decreased survival of MTC cells and blocked the activation of RET downstream signaling pathways (phosphorylated ERK, phosphorylated AKT, and p70S6K). ATF4 knockdown decreased sensitivity to tyrosine kinase inhibitor-induced apoptosis. Moreover, ATF4 expression decreased RET protein levels by promoting RET ubiquitination. We found decreased or loss of ATF4 in 52% of MTC tumors (n = 39) compared with normal thyroid follicle cells. A negative correlation was observed between RET and ATF4 protein levels in MTC tumors, and low ATF4 expression was associated with poor overall survival in patients with MTC. ATF4 was identified as a negative regulator of RET, a candidate tumor suppressor gene, and may be a molecular marker that distinguishes patients at high risk of MTC from those with a longer survival prognosis.

Advances in studies of the genes related to thyroid carcinoma

Thyroid carcinoma (TC) is the most common endocrine cancer and its incidence has been increasing globally over the past decades. With the development of the genetic technology, more and more evidences showed that many genes affect the biological behaviors of TC, making sense to early diagnosis, predicting the prognosis and targeted therapy for TC. BRAF mutation is specific to papillary thyroid carcinoma (PTC). It can not only predict the prognosis, but also have diagnosis value. RET rearrangements are identified as a specific genetic event in PTC. Though the preoperative detection of RET/PTC rearrangements has not proven useful in choosing the appropriate surgical management, new medications which are capable of inhibiting RET protein kinase activity may help to therapy the PTCs. RET mutation has specific meaning for detecting familial medullar thyroid carcinoma. Though RAS mutation can be discovered in follicular thyroid carcinoma (FTC), follicular variant of papillary thyroid carcinoma (FvPTC), poorly differentiated thyroid carcinoma (PDTC) and undifferentiated thyroid carcinoma (UTC), the relationship between RAS mutation and prognosis remains controversial. P53 can be detected in more invasive variants of PTC, PDTC and UTC. P53 can be used as a prognosis-predictor. Rescuing the function of mutant p53 (mutp53) protein is an attractive anticancer therapeutic strategy. 30%-35% FTC and 37.5% FvPTC have PAX8-PPARγ rearrangement, which can distinguish carcinomas from adenomas in follicular neoplasms of the thyroid. Pioglitazone may have therapeutic efficacy in patients with PPFP-positive TCs. FTC and PTC have TERT promoter mutation, usually predicting poor prognosis. Other genes also influence on the biological behavior of TC, having diagnosis value and prognostic significance. Though the gene study about TC develops rapidly, many problems remain unclear. Further studies on TC-related genes are needed.

Expressions of glial cell line-derived neurotrophic factor and its tyrosine kinase receptor RET in the terminal rectum of fetal rats with congenital anorectal malformations at different gestational ages

Objective To explore the expressions and distributions of glial cell line-derived neurotrophic factor (GDNF) and itstyrosine kinase receptor RET in the terminal rectums of fetal rats with congenital anorectal malformations (ARM) at different gestationalage, and to explore their effects on the enteric nervous system in the terminal rectum of ARM fetal rats. Methods Thirty-five SD pregnancy rats were divided into a saline group (n=10) and an ethylenethiourea experiment group (n=25) by simple randomized study.The fetal rats were removed from the pregnant rats at the gestational 16 d, 18 d and 20 d. The fetal rats were divided into the saline control group, the ethylenethiourea control group (fetal rats without ARM) and the ethylenethiourea malformation group (ARM fetal rats) by the naked eye and dissecting microscope.HE staining was used to observe the morphology and the intestinal ganglion cells in the terminal rectum were counted.The immunohistochemical staining and Western blot methods were used to observe the distributions of GDNF and RET in the rectum at the gestational 16 d, 18 d and 20 d. The quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of GDNF mRNA in the fetal rats in the terminal rectum at the gestational 16 d, 18 d and 20 d. Results HE staining: the development of anorectal terminal in 3 groups of fetal rats was unclear at the gestational 16 d. A small amount of scattered nerve plexuses were observed in the muscular layer.The nuclei were small and sparse.The axons and cytoplasms were less.The serosal layer, muscular layer, submucosa, mucosal layer and glands in the terminal rectum were gradually clear in the saline control group and the ethylenethiourea control group at the gestational 18 d and 20 d. The intermuscular submucosal nerve plexuses increased gra-dually (11.400±3.134 and 11.200±3.425 at the gestational 18 d; 66.100±4.954 and 67.600±5.481 at the gestational 20 d). While, the layer was unclear in the ethylenethiourea malformation group and the nerve plexus was less (7.800±1.989 at the gestational 18 d, and 25.200±3.048 at the gestational 20 d), and the difference was statistically significant compared with 2 control groups (F=7.591, 271.833, all P 0.05); the expressions of GDNF and RET protein were 103.624±27.533 and 105.184±19.634 at the gestational 18 d; 151.496±33.622 and 150.738±21.423 at the gestational 20 d in 2 control groups.Compared with the ethylenethiourea malformation group (79.169±11.697 at the gestational 18 d; 94.873±11.309 at the gestational 20 d), and the difference were statistically significant (all P<0.05). Conclusions The expressions of GDNF and its tyrosine kinase receptor RET had a certain temporal correlation in the terminal rectum of normal fetal rats at different gestational ages and ARM.Moreover, the abnormal expressions of GDNF and its tyrosine kinase receptor RET in the distal rectum of ARM fetal rats can affect the development of enteric nervous system. Key words: Glial cell line-derived neurotrophic factor; RET; Signal pathway; Enteric nervous system; Congenital anorectal malformation

RET/PTC Rearrangements Are Associated with Elevated Postoperative TSH Levels and Multifocal Lesions in Papillary Thyroid Cancer without Concomitant Thyroid Benign Disease.

RET/PTC rearrangements, resulting in aberrant activity of the RET protein tyrosine kinase receptor, occur exclusively in papillary thyroid cancer (PTC). In this study, we examined the association between RET/PTC rearrangements and thyroid hormone homeostasis, and explored whether concomitant diseases such as nodular goiter and Hashimoto's thyroiditis influenced this association. A total of 114 patients diagnosed with PTC were enrolled in this study. Thyroid hormone levels, clinicopathological parameters and lifestyle were obtained through medical records and surgical pathology reports. RET/PTC rearrangements were detected using TaqMan RT-PCR and validated by direct sequencing. No RET/PTC rearrangements were detected in benign thyroid tissues. RET/PTC rearrangements were detected in 23.68% (27/114) of PTC tissues. No association between thyroid function, clinicopathological parameters and lifestyle was observed either in total thyroid cancer patients or the subgroup of patients with concomitant disease. In the subgroup of PTC patients without concomitant disease, RET/PTC rearrangement was associated with multifocal cancer (P = 0.018). RET/PTC rearrangement was also correlated with higher TSH levels at one month post-surgery (P = 0.037). Based on likelihood-ratio regression analysis, the RET/PTC-positive PTC cases showed an increased risk of multifocal cancers in the thyroid gland (OR = 5.57, 95% CI, 1.39–22.33). Our findings suggest that concomitant diseases such as nodular goiter and Hashimoto's thyroiditis in PTC may be a confounding factor when examining the effects of RET/PTC rearrangements. Excluding the potential effect of this confounding factor showed that RET/PTC may confer an increased risk for the development of multifocal cancers in the thyroid gland. Aberrantly increased post-operative levels of TSH were also associated with RET/PTC rearrangement. Together, our data provides useful information for the treatment of papillary thyroid cancer.

Identification of mutation in the RET gene in a patient with medullary thyroid cancer

Medullary thyroid carcinoma (MTC) is a type of tumors drived from thyroid parafollicular cells (C-cells), comprises 5–10% of all thyroid malignancies. About 25% of MTC cases occur as an autosomal dominant heredited disorder which is mostly caused by activating mutations of RET pro-oncogen and they occur as components of the multiple endocrine neoplasia type 2 syndromes (MEN 2A and 2B) or familial MTC. The guidelines for genetic testing in MTC of the European Thyroid Association recommend that exons 5, 8, 10, 11, 13, 14, 15 and 16 should always be screened starting in patients. This study aims to identify the genetic causes of a male patient diagnosed with MEN2 and pheochromocytoma. Genomic DNA was extracted from peripheral blood of patient. Eight recommened exons (5, 8, 10, 11, 13, 14, 15 and 16) of the RET gene were amplified by PCR and sequenced by Sanger method on the ABI 3500 genetic annalyzer. The results showed a heterozygous C to T transition at nucleotide 2753 in exon 16 of the RET gene. The c.2753 C&gt;T transition resulted in a missense mutation of methionine to threonine (p.M918T) in the RET protein. This mutation was located in the intracellular tyrosine kinase domain of protein and has been reported in 95% of MEN2B cases, therefore, it would be the causative mutant of our patient. Our patient harboring a pM918T germline mutation and there is a 50% probability that he will pass this mutation to a child. Thus, his six-year-old boy with normal health status was advised to have the genetic test for this mutation. The results showed that he had not inherited the mutation c.2753T&gt; C from his father.

Abstract 3660: ATF4 is a putative tumor suppressor gene in medullary thyroid cancer

Abstract Medullary thyroid carcinoma (MTC) is derived from the calcitonin-producing C-cells of the thyroid gland. Oncogenic mutations of the RET proto-oncogene are found in all hereditary MTC and ∼45% of the sporadic cases. However, it is clear that additional genetic lesions contribute to the development of both RET-dependent and RET independent MTC. We analyzed 30 primary medullary thyroid cancer (MTC) tumors by comparative genomic hybridization (CGH) arrays (Agilent 244K) and a subset of those tumors by ultra-high-resolution SNP arrays (Affymetrix SNP6.0). A consistent finding with both arrays was a loss localized to 22q13.1 in 40% of tumors. This region contains the ATF4 gene and loss of this gene was validated by real time PCR. ATF4 is a central mediator of integrated stress response pathway and activates expression of a cohort of downstream target genes known to be involved in cell survival, apoptosis, autophagy and senescence. We hypothesize that the loss of ATF4 expression contributes to MTC progression through mechanisms involving aberrant proliferation and escape from apoptosis. We present several findings to support this. First, ATF4 protein levels were found downregulated in 50% MTC tumor tissues (n = 43) and low ATF4 expression was associated with poor overall survival in MTC patients (HR = 5.015, 95% CI: 1.38-10.64), P = 0.013). A negative correlation was observed between RET and ATF4 protein expression in MTC tumors (r = -0.89, R square = 0.799, p&amp;lt;0.0001). Second, we showed that targeted heterozygote and homozygous deletion of Atf4 in mice causes C cell hyperplasia, a precancerous lesion preceding MTC. Third, ATF4 overexpression inhibits proliferation and survival of MTC derived cell lines. ATF4 decreases RET protein levels by inducing ubiquitination and degradation of activated RET. In contrast, ATF4 depletion upregulates RET protein levels as well as RET autophosphorylation. Finally, combination treatment of tyrosine kinase inhibitor (sunitinib) and eeyarestatin that stimulates ATF4 transcription, induces apoptosis of the MTC cells in a synergistic manner through activation of ATF4 and its target genes NOXA and PUMA as well. These results suggest ATF4 as a potential tumor suppressor gene in MTC. We postulate that inactivation of ATF4 is a central impediment to induction of apoptosis in MTC tumors. Therefore, strategies to upregulate ATF4 during TKI therapy would render tumor cells exquisitely sensitive to stress induced apoptosis, offering a novel synergistic treatment. Citation Format: Rozita Bagheri-Yarmand, Gilbert J. Cote, Elizabeth G. Grubbs, Michelle D. Williams, Steven I. Sherman, Robert F. Gagel. ATF4 is a putative tumor suppressor gene in medullary thyroid cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3660.

A loss-of-function genetic screening identifies novel mediators of thyroid cancer cell viability.

RET, BRAF and other protein kinases have been identified as major molecular players in thyroid cancer. To identify novel kinases required for the viability of thyroid carcinoma cells, we performed a RNA interference screening in the RET/PTC1(CCDC6-RET)-positive papillary thyroid cancer cell line TPC1 using a library of synthetic small interfering RNAs (siRNAs) targeting the human kinome and related proteins. We identified 14 hits whose silencing was able to significantly reduce the viability and the proliferation of TPC1 cells; most of them were active also in BRAF-mutant BCPAP (papillary thyroid cancer) and 8505C (anaplastic thyroid cancer) and in RAS-mutant CAL62 (anaplastic thyroid cancer) cells. These included members of EPH receptor tyrosine kinase family as well as SRC and MAPK (mitogen activated protein kinases) families. Importantly, silencing of the identified hits did not affect significantly the viability of Nthy-ori 3-1 (hereafter referred to as NTHY) cells derived from normal thyroid tissue, suggesting cancer cell specificity. The identified proteins are worth exploring as potential novel druggable thyroid cancer targets.

Commentary to Gorelenkova Miller and Mieyal (2015): sulfhydryl-mediated redox signaling in inflammation: role in neurodegenerative diseases

Gorelenkova Miller and Mieyal (Arch Toxicol 89(9): 1439-1467, 2015) recently published a review paper suggesting that reversible cysteine plays a key role in redox-linked signal transduction via alteration of protein function, resulting in an association with many diseases including neurodegenerative disorders. Following their suggestions, we considered the correlation between sulfhydryl-mediated redox signaling and neurodegenerative diseases by focusing on RET proteins, a protein tyrosine kinases (PTKs) potentially sited upstream of the signal transduction cascade. c-RET is the receptor for glial cell line-derived neurotrophic factor family ligands. c-RET has been reported to be involved in not only Hirschsprung disease via development of the enteric nervous system but also neurodegenerative diseases including Parkinson's disease and amyotrophic lateral sclerosis. We also showed that c-RET might be associated with hearing loss via neurodegeneration of spiral ganglion neurons in the inner ear after birth in mice and humans. Moreover, we have reported that three kinds of oxidative stress, ultraviolet light-induced stress, osmotic stress and arsenic-induced stress, modulate kinase activity of RET-PTC1 without an extracellular domain as well as c-RET by conformational change of RET protein (dimerization) via disulfide bond formation. The oxidative stresses also modulate kinase activity of RET-PTC1 with cysteine 365 (C365) replaced by alanine with promotion of dimer formation, but not with cysteine 376 (C376) replaced by alanine. Since C376 of Ret-PTC-1 or its equivalent is most highly conserved and crucial for activity in PTKs, the cysteine could be one of major targets for oxidative stresses.

Preclinical Efficacy and Safety Assessment of an Antibody-Drug Conjugate Targeting the c-RET Proto-Oncogene for Breast Carcinoma.

The RET proto-oncogene has been implicated in breast cancer, and the studies herein describe the preclinical and safety assessment of an anti-RET antibody-drug conjugate (ADC) being developed for the treatment of breast cancer. RET protein expression was analyzed in breast tumor samples using tissue microarrays. The fully human anti-RET antibody (Y078) was conjugated to the DM1 and DM4 derivatives of the potent cytotoxic agent maytansine using thioether and disulfide linkers, respectively. The resulting compounds, designated Y078-DM1 and Y078-DM4, were evaluated for antitumor activity using human breast cancer cell lines and established tumor xenograft models. A single-dose, 28-day, safety study of Y078-DM1 was performed in cynomolgus monkeys. By immunohistochemistry, RET expression was detected in 57% of tumors (1,596 of 2,800 tumor sections) and was most common in HER2-positive and basal breast cancer subtypes. Potent in vitro cytotoxicity was achieved in human breast cancer cell lines that have expression levels comparable with those observed in breast cancer tissue samples. Dose-response studies in xenograft models demonstrated antitumor activity with both weekly and every-3-weeks dosing regimens. In cynomolgus monkeys, a single injection of Y078-DM1 demonstrated dose-dependent, reversible drug-mediated alterations in blood chemistry with evidence of on-target neuropathy. RET is broadly expressed in breast cancer specimens and thus represents a potential therapeutic target; Y078-DM1 and Y078-DM4 demonstrated antitumor activity in preclinical models. Optimization of the dosing schedule or an alternate cytotoxic agent with a different mechanism of action may reduce the potential risk of neuropathy. Clin Cancer Res; 21(24); 5552-62. ©2015 AACR.

Selective repression of RET proto-oncogene in medullary thyroid carcinoma by a natural alkaloid berberine.

BackgroundThe gain-of-function mutation of the RET proto-oncogene, which encodes a receptor tyrosine kinase, is strongly associated with the development of several medullary thyroid carcinomas (MTCs). Thus, the RET protein has been explored as an excellent target for progressive and advanced MTC. In this study we have demonstrated a therapeutic strategy for MTC by suppressing the transcription of RET proto-oncogene though the stabilization of G-quadruplex structure formed on the promoter region of this gene using a natural product berberine.MethodsMedullary thyroid carcinoma (MTC) TT cell line has been used to evaluate the effects of berberine on RET expression and its downstream signaling pathways. The specificity of berberine was demonstrated by using the papillary thyroid carcinoma TPC1 cell line, which lacks the G-quadruplex forming sequence on the RET promoter region due to chromosomal rearrangement.ResultsBerberine suppressed the RET expression by more than 90 % in MTC TT cells at a concentration of 2.5 μg/ml with minimal effect on the TPC1 cells. Canadine, which is a structural analogue of berberine, showed little interaction with RET G-quadruplex and also had no effect on RET expression in MTC TT cells. The down-regulation of RET with berberine further inhibited the cell proliferation through cell cycle arrest and activation of apoptosis in TT cells, which was confirmed by a 2-fold increase in the caspase-3 activity and the down-regulation of cell-cycle regulatory proteins.ConclusionOur data strongly suggest that the G-quadruplex forming region and the stabilization of this structure play a critical role in mediating the repressive effect of berberine on RET transcription.

KIF5B-RET Fusion gene may coincide oncogenic mutations of EGFR or KRAS gene in lung adenocarcinomas.

BackgroundThe KIF5B-RET rearrangement is detected with the frequency of 1 ~ 2 % in ‘triple marker’-negative lung adenocarcinomas, i.e., EGFR, KRAS and EML4-ALK wild type. These mutational changes are known to be mutually exclusive, but the co-existence of ALK rearrangement with activating mutations of EGFR is rarely found.MethodsWe examined the KIF5B-RET fusion gene in frozen tissues from 154 surgically resected lung tumors using RT-PCR with direct sequencing and the mutation status of EGFR and KRAS genes using PNA clamping. We tested KIF5B-RET translocation in Formalin Fixed Paraffin Embedded using fluorescence in situ hybridization. We also measured RET mRNA and protein expression by RT-PCR and immunohistochemistry, respectively.ResultsThe existence of KIF5B-RET fusion gene was identified in 9 patients. The mean age was 67.2 and M: F ratio 4:5. Of 9 patients, 3 patients harbored wild type of EGFR and KRAS gene. However, KIF5B-RET fusion gene coincided with EGFR or KRAS mutation in 6 patients. These six pts were also positive for both RET break-apart probes (23.9 %) and KIF5B-RET fusion (44.4 %). However, there were no correlations between RET mRNA and protein expression in the KIF5B-RET-positive patients. The median disease free survival and overall survival were 23.9 months and 29.5 months, respectively.ConclusionsTaken together, our data suggest one-step screening platform for KIF5B-RET as well as EGFR, K-RAS, ALK oncogenic mutations be necessary for lung adenocarcinoma patients because EGFR or KRAS mutation are not infrequently found in KIF5B-RET-positive patients.

Abstract 5546: A natural alkaloid, berberine, as a novel transcriptional repressor of RET proto-oncogene in medullary thyroid carcinoma

Abstract The development of several medullary thyroid carcinomas is strongly associated with the gain-of-function mutation of the RET proto-oncogene, which encodes a receptor tyrosine kinase, so that the RET protein has been regarded as a potential therapeutic target for the treatment of progressive and advanced MTC. In our study we have explored a new strategy to repress the transcription of the RET proto-oncogene in MTC using small molecules. Interestingly, we found that a natural product berberine inhibits the RET gene transcription by specifically targeting the promoter region of this gene, thereby reducing the RET protein expression in MTC TT cells. The down-regulation of RET with berberine also results in the suppression of cell proliferation through cell cycle arrest and activation of apoptosis in TT cells. Overall, our results strongly suggest that berberine stabilizes G-quadruplexes formed from the G-rich sequence present on the promoter region of this gene and the stabilization of this structure play a critical role in mediating the repressive effect of berberine on RET transcription. Citation Format: Daekyu Sun, Vishnu Kumarasamy. A natural alkaloid, berberine, as a novel transcriptional repressor of RET proto-oncogene in medullary thyroid carcinoma. [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 5546. doi:10.1158/1538-7445.AM2015-5546

RET gene and tumor

RET gene is an important oncogene, which is closely associated with the development of various types of human tumors. The mainly mechanisms of RET gene associated tumor are mutation and over-expression of wild type. Activated RET protein participates in the proliferation, apoptosis, metastasis through some signal pathways and influences the tumorigenesis and development. Key words: Neoplasms; Therapy; RET gene

SLIT2/ROBO1-miR-218-1-RET/PLAG1: a new disease pathway involved in Hirschsprung's disease.

Hirschsprung's disease (HSCR) is a rare congenital disease caused by impaired proliferation and migration of neural crest cells. We investigated changes in expression of microRNAs (miRNAs) and the genes they regulate in tissues of patients with HSCR. Quantitative real-time PCR and immunoblot analyses were used to measure levels of miRNA, mRNAs, and proteins in colon tissues from 69 patients with HSCR and 49 individuals without HSCR (controls). Direct interactions between miRNAs and specific mRNAs were indentified in vitro, while the function role of miR-218-1 was investigated by using miR-218 transgenic mice. An increased level of miR-218-1 correlated with increased levels of SLIT2 and decreased levels of RET and PLAG1mRNA and protein. The reductions in RET and PLAG1 by miR-218-1 reduced proliferation and migration of SH-SY5Y cells. Overexpression of the secreted form of SLIT2 inhibited cell migration via binding to its receptor ROBO1. Bowel tissues from miR-218-1 transgenic mice had nerve fibre hyperplasia and reduced numbers of gangliocytes, compared with wild-type mice. Altered miR-218-1 regulation of SLIT2, RET and PLAG1 might be involved in the pathogenesis of HSCR.

Beyond ALK-RET, ROS1 and other oncogene fusions in lung cancer.

Fusions of the RET and ROS1 protein tyrosine kinase oncogenes with several partner genes were recently identified as new targetable genetic aberrations in cases of non-small cell lung cancer (NSCLC) lacking activating EGFR, KRAS, ALK, BRAF, or HER2 oncogene aberrations. RET and ROS1 fusion-positive tumors are mainly observed in young, female, and/or never smoking patients. Studies based on in vitro and in vivo (i.e., mouse) models and studies of several fusion-positive patients indicate that inhibiting the kinase activity of the RET and ROS1 fusion proteins is a promising therapeutic strategy. Accordingly, there are several ongoing clinical trials aimed at examining the efficacy of tyrosine kinase inhibitors (TKIs) against RET and ROS1 proteins in patients with fusion-positive lung cancer. Other gene fusions (NTRK1, NRG1, and FGFR1/2/3) that are targetable by existing TKIs have also been identified in NSCLCs. Options for personalized lung cancer therapy will be increased with the help of multiplex diagnosis systems able to detect multiple druggable gene fusions.

Abstract 5262: Activating transcription factor 4 regulates RET autophosphorylation and ubiquitin-dependent degradation

Abstract RET proto-oncogene, a receptor tyrosine kinase (RTK), activates a complex network of signal transduction pathways that when unregulated contributes to cell transformation. Germ line missense mutations, which give rise to constitutively active oncogenic RET, were found to cause multiple endocrine neoplasia type 2 (MEN2), a dominant inherited cancer syndrome that affects multiple neuroendocrine organs. Recent findings also suggest that impairment of RTK down-regulation or deactivation may be involved in tumorigenesis. The molecular mechanism that functions to attenuate RET signaling remain elusive. ATF4 is a central mediator of integrated stress response pathway. Activation of ATF4 results in tumor cell death under stress conditions thereby suggesting that ATF4 agonists constitute a potential therapeutic strategy for inhibiting tumor growth. Here, we report that ATF4 overexpression in medullary thyroid cancer cells harboring RET C634W mutation decreases RET protein expression but not the mRNA levels. In addition, ATF4 accelerates RET turnover and promotes its ubiquitination. Moreover, a RET kinase dead mutant is more stable than wild type or M918T RET suggesting that kinase activity is required for ATF4-mediated degradation. Finally, ATF4 depletion upregulates RET protein levels and RET autophosphorylation. Our findings unravel ATF4 as a feedback negative attenuator of RET signaling that could be used as a potential therapeutic target for medullary thyroid cancer or other malignancy with RET abnormalities. Citation Format: Rozita Bagheri-Yarmand, Gilbert J. Cote, Robert F. Gagel. Activating transcription factor 4 regulates RET autophosphorylation and ubiquitin-dependent degradation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5262. doi:10.1158/1538-7445.AM2014-5262

Abstract 5251: ASCL1 and RET expression define a clinically relevant subgroup of lung adenocarcinoma characterized by neuroendocrine differentiation

Abstract ASCL1 is an important regulatory transcription factor in pulmonary neuroendocrine cell development, but its value as a biomarker of neuroendocrine differentiation in lung adenocarcinoma (AD) and as a potential prognostic biomarker remains unclear. We examined ASCL1expression in lung cancer samples of varied histologic subtype, clinical outcome, and smoking status and compared to expression of traditional neuroendocrine markers. ASCL1 mRNA expression was found almost exclusively in smokers with AD, in contrast to non-smokers. ASCL1 protein expression by immunohistochemistry (IHC) correlated best with synaptophysin compared to chromogranin and CD56/NCAM. Analysis of a compendium of 367 microarray-based gene expression profiles in stage I lung adenocarcinomas identified significantly higher expression levels of the RET oncogene in ASCL1 positive tumors (ASCL1+) compared to ASCL1- tumors (q-value &amp;lt; 10 - 9). High levels of RET expression in ASCL1+ but not in ASCL1- tumors was associated with significantly shorter overall survival in stage 1 (p = 0.007) and in all AD (p = 0.037). RET protein expression by IHC had an association with overall survival in the context of ASCL1 expression. In silico gene set analysis and in vitro experiments by ASCL1 shRNA in AD cells with high endogenous expression of ASCL1 and RET implicated ASCL1 as a potential upstream regulator of the RET oncogene. Also, silencing ASCL1 in AD cells markedly reduced cell growth and motility. These results suggest that ASCL1 and RET expression define a clinically relevant subgroup of approximately 10% of AD characterized by neuroendocrine differentiation. Citation Format: Farhad Kosari, Cristiane M. Ida, Marie Christine Aubry, Lin Yang, Irina V. Kovtun, Janet L. Schaefer Klein, Sandra C. Tomaszek, Stephen J. Murphy, Ping Yang, Dennis Wigle, George Vasmatzis. ASCL1 and RET expression define a clinically relevant subgroup of lung adenocarcinoma characterized by neuroendocrine differentiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5251. doi:10.1158/1538-7445.AM2014-5251

SRY interference of normal regulation of the RET gene suggests a potential role of the Y-chromosome gene in sexual dimorphism in Hirschsprung disease.

The Hirschsprung disease (HSCR) is a complex congenital disorder, arising from abnormalities in enteric nervous system (ENS) development. There is a gender disparity among the patients, with the male to female ratio as high as 5 : 1. Loss-of-function mutations of HSCR genes and haploinsufficiency of their gene products are the primary pathogenic mechanisms for disease development. Recent studies identified over half of the HSCR disease susceptibility genes as targets for the sex-determining factor SRY, suggesting that this Y-encoded transcription factor could be involved in sexual dimorphism in HSCR. Among the SRY targets, the tyrosine kinase receptor RET represents the most important disease gene, whose mutations account for half of the familial and up to one-third of the sporadic forms of HSCR. RET is regulated by a distal and a proximal enhancer at its promoter, in which PAX3 and NKX2-1 are the resident transcription factors respectively. We show that the SRY-box 10 (SOX10) co-activator interacts and forms transcriptional complexes with PAX3 and NKX2-1 in a sequence-independent manner and exacerbates their respective transactivation activities on the RET promoter. SRY competitively displaces SOX10 in such transcription complexes and represses their regulatory functions on RET. Hence SRY could be a Y-located negative modifier of RET expression; and if it is ectopically expressed during ENS development, such SRY repression could result in RET protein haploinsufficiency and promotion of HSCR development, thereby contributing to sexual dimorphism in HSCR.

Characterization of Wild-Type and Mutated RET Proto-Oncogene Associated with Familial Medullary Thyroid Cancer

We aimed to assess RET proto-oncogene polymorphisms in three different Iranian families with medullary thyroid cancer (MTC), and performed molecular dynamics simulations and free energy stability analysis of these mutations. This study consisted of 48 patients and their first-degree relatives with MTC confirmed by pathologic diagnosis and surgery. We performed molecular dynamics simulations and free energy stability analysis of mutations, and docking evaluation of known RET proto-oncogene inhibitors, including ZD-6474 and ponatinib, with wild-type and mutant forms. The first family consisted of 27 people from four generations, in which nine had the C.G2901A (P.C634Y) mutation; the second family consisted of six people, of whom three had the C.G2901T (P.C634F) mutation, and the third family, who included 12 individuals from three generations, three having the C.G2251A (P.G691S) mutation. The automated 3D structure of RET protein was predicted using I-TASSER, and validated by various protein model verification programs that showed more than 96.3% of the residues in favored and allowed regions. The predicted instability indices of the mutated structures were greater than 40, which reveals that mutated RET protein is less thermo-stable compared to the wild-type form (35.4). Simultaneous study of the cancer mutations using both in silico and medical genetic procedures, as well as onco-protein inhibitor binding considering mutation-induced drug resistance, may help in better overcoming chemotherapy resistance and designing innovative drugs.