Rearranged During Transfection Expression Research Articles

Enhancer-activated RET confers protection against oxidative stress to KMT2A-rearranged acute myeloid leukemia.

Ectopic activation of rearranged during transfection (RET) has been reported to facilitate lineage differentiation and cell proliferation in different cytogenetic subtypes of acute myeloid leukemia (AML). Herein, we demonstrate that RET is significantly (p < 0.01) upregulated in AML subtypes containing rearrangements of the lysine methyltransferase 2A gene (KMT2A), commonly referred to as KMT2A-rearranged (KMT2A-r) AML. Integrating multi-epigenomics data, we show that the KMT2A-MLLT3 fusion induces the development of CCCTC-binding (CTCF)-guided de novo extrusion enhancer loop to upregulate RET expression in KMT2A-r AML. Based on the finding that RET expression is tightly correlated with the selective chromatin remodeler and mediator (MED) proteins, we used a small-molecule inhibitor having dual inhibition against RET and MED12-associated cyclin-dependent kinase 8 (CDK8) in KMT2A-r AML cells. Dual inhibition of RET and CDK8 restricted cell proliferation by producing multimodal oxidative stress responses in treated cells. Our data suggest that epigenetically enhanced RET protects KMT2A-r AML cells from oxidative stresses, which could be exploited as a potential therapeutic strategy.

Fulvestrant plus vandetanib versus placebo for the treatment of patients with metastatic breast cancer resistant to aromatase inhibitor therapy (FURVA): a multicentre, Phase 2, randomised controlled trial

BackgroundFURVA, a randomised, double-blind Phase II trial, investigated whether the addition of vandetanib to fulvestrant improved progression-free survival (PFS) in patients with an aromatase inhibitor(AI)-resistant advanced breast cancer.MethodsPostmenopausal women with oestrogen receptor-positive (ER+ve)/HER2-negative advanced breast cancer, who experienced disease progression on an AI, were randomised (1:1) to fulvestrant 500 mg (Q28) with vandetanib 300 mg od (f + v) or placebo (f + p) until disease progression or discontinuation. The primary endpoint was PFS; secondary endpoints included overall survival (OS) and the influence of REarranged during Transfection (RET) signalling on outcomes.ResultsIn total, 165 participants were randomised to f + v (n = 80) or f + p (n = 85). Median PFS was 5.5 months (m) for f + v compared to 5.5 m for f + p (hazard ratio (HR) 0.88; 95% CI: 0.62–1.23; P = 0.22). Unexpectedly, high total RET expression was associated with a PFS advantage of 8.87 m vs 3.94 with low RET (HR 0.493: 95% CI 0.32–0.77; P = 0.002) independent of the treatment arm, supported by an OS advantage 21.95 m vs 18.04 (HR 0.584; 95% CI 0.34–1.00; P = 0.051) in the high-RET group.ConclusionThe addition of vandetanib to fulvestrant does not improve PFS. However, high total RET expression was associated with improved PFS, suggesting RET may have a prognostic role in patients treated with fulvestrant.Clinical trial registrationClinicalTrials.gov, NCT02530411.

Lung adenocarcinoma with neuroendocrine differentiation: Molecular markers testing and treatment outcomes

Among the histologic types of lung cancer, adenocarcinoma is the most common. Moreover, lung adenocarcinoma with neuroendocrine differentiation (LANED) is a rare histologic character. So far, the clinical significance remains unclear. We searched for the patients diagnosed with LANED from the electronic pathology database between January 2000 and June 2020 in a tertiary hospital. The tumor specimens were reviewed by a pathologist to confirm the diagnosis. EGFR mutation, ALK translocation, as well as programmed death ligand 1 (PD-L1) and rearranged during transfection (RET) expression were tested in the specimens of LANED. The clinical data were also collected and analyzed. A total of 10 patients diagnosed with LANED were included. Most were male (80%) and ever smokers (70%). The median age was 71.5 years old. At diagnosis, most had tumors harboring no EGFR mutation (70%), negative ALK translocation (88.9%), and without PD-L1 expression (90%). All specimens tested by immunohistochemical staining for RET expression (n=9) showed positive results. Among the 10 patients, five underwent operation (stage I, n=4; stage II, n=1). The patient with stage II disease had recurrence 11 months later. For patients with advanced stages (stage III, n=1; stage IV, n=4), the treatment modalities varied and the overall survival ranged from 11.0 to 46.7 months. LANED might be associated with a high proportion of RET expression, whereas EGFR mutation, ALK alteration, and PD-L1 expression were uncommon. Further large-scale prospective studies on molecular testing profile and clinical significance of LANED are warranted.

An integrative pan cancer analysis of RET aberrations and their potential clinical implications

RET (rearranged during transfection), encoding a tyrosine kinase receptor, is a novel therapeutic target for cancers. The aberrations of RET are commonly found in cancers. Here, we profiled a comprehensive genomic landscape of RET mutations, copy number variants (CNVs), co-occurrence of RET and its mRNA expression and methylation levels in pan cancer, paving the way to the development of new RET-targeted therapies in clinic. Analysis of RET somatic mutations, CNVs, co-occurrence, mRNA expression and methylation were performed among 32 cancer types from The Cancer Genome Atlas (TCGA) dataset covering a total of 10,953 patients with 10,967 samples. RET aberrations were found in 3.0% of diverse cancers. The top two RET-altered tumors were skin cutaneous melanoma (SKCM) and uterine corpus endometrial carcinoma (UCEC) with dominant mutations in the other and PKinase_Tyr domains. RET-G823E and RET-S891L were most commonly found in SKCM and UCEC. Thyroid carcinoma (THCA) demonstrated the highest rate of coiled-coil domain containing 6 (CCDC6)-RET fusions, which constitutively activate RET kinase. Two FDA-approved RET inhibitors—pralsetinib and selpercatinib have been implied for the treatment of patients with RET S891L mutant UCEC and the treatment of patients with metastatic RET-fusion positive THCA and non-small cell lung cancer (NSCLC) at therapeutic level 1. We also identified four RET M918T-altered cases in patients with pheochromocytoma and paraganglioma (PCPG), which may induce drug resistance against multikinase inhibitors. Next, 273 co-occurring aberrations, most frequently in Notch signaling, TGF-β pathway, cell cycle, and Ras-Raf-MEK-Erk/JNK signaling, were uncovered among 311 RET altered cases. TP53 mutations (162 patients) leads to the most significant co-occurrence associated with RET aberrations. Furthermore, the RET expression was found most significantly increased in breast invasive carcinoma (BRCA) and neck squamous cell carcinoma (HNSC), as compared to their corresponding normal tissues. At last, patients with higher expression and sequence variant frequency have a worse prognosis, such as sarcoma patients. This work provided a profound and comprehensive analysis of RET and co-occurred alterations, RET mRNA expression and the clinical significance in pan cancer, offering new insights into targeted therapy for patients with RET anomalies.

Higher RET Gene Expression Levels Do Not Represent anAlternative RET Activation Mechanism in Medullary Thyroid Carcinoma.

This study was designed to investigate whether RET (rearranged during transfection) mRNA over-expression could be considered an alternative driver event for the development of medullary thyroid carcinoma (MTC), and if different RET isoforms could play a role in MTC tumorigenesis. Eighty-three MTC patients, whose mutational profile was previously identified by next-generation sequencing (NGS) IONS5, were included in this study. Expression analysis was performed by the quantitative reverse transcription-polymerase chain reaction technique. RET expression levels were found to be significantly higher in cases with RET somatic mutations than in cases that were negative for RET somatic mutations (p = 0.003) as well as in cases with a somatic mutation, either in RET or RAS than in cases negative for both these mutations (p = 0.01). All cases were positive for the RET51 isoform expression while only 72/83 (86.7%) were positive for RET9 isoform expression. A statistically significant higher expression of the RET51 isoform was found in cases positive for RET somatic mutation than in cases either positive for RAS mutation (p = 0.0006) or negative for both mutations (p = 0.001). According to our data, RET gene over-expression does not play a role in MTC tumorigenesis, neither as an entire gene or as an isoform. At variance, the RET gene, and in particular the RET51 isoform, is expressed higher in RET mutated cases. On the basis of these results we can hypothesize that the overexpression of RET, and in particular of RET51, could potentiate the transforming activity of mutated RET, making these cases more aggressive.

Expression of RET is associated with Oestrogen receptor expression but lacks prognostic significance in breast cancer

BackgroundThe Rearranged during Transfection (RET) protein is overexpressed in a subset of Estrogen Receptor (ER) positive breast cancer, with both signalling pathways functionally interacting. This cross-talk plays a pivotal role in the resistance of breast cancer cells to anti-endocrine therapies, and RET expression is assumed to correlate with poor prognosis based on findings in small patient cohorts. The aim of our study was to investigate the impact of RET expression on patient outcome in human breast cancer.MethodsWe performed an immunohistochemical analysis of RET protein expression on a tissue microarray encompassing 990 breast cancer patients and correlated its expression with clinicopathological parameters and survival data.ResultsExpression of RET was detected in 409 out of 990 cases (41.3%). RET and ER expression significantly correlated (p < 0.0001). The Luminal B HER2-positive subtype showed the highest expression rate (48.9%). In univariate and multivariate survival analyses, RET expression had no impact on overall survival.ConclusionWe confirmed the co-expression of RET and ER, but we did not find RET expression to be an independent prognostic factor in human breast cancer. Clinical trials with newly developed RET inhibitors are needed to evaluate if RET inhibition has a beneficial impact on patient survival in ER positive breast cancer.

Down-regulation of microRNA-216a confers protection against yttrium aluminium garnet laser-induced retinal injury via the GDNF-mediated GDNF/GFRα1/RET signalling pathway

Retinal injury plays a leading role in the onset of visual impairment. Current forms of treatment are not able to ameliorate scarring, cell death and tissue and axon regeneration. Recently, microRNA-216a (miR-216a) has been reported to regulate snx5, a novel notch signalling pathway component during retinal development. This study aims to elucidate the role of miR-216a in yttrium aluminium garnet (YAG) laser-induced retinal injury by targeting glial cell line-derived neurotrophic factor (GDNF) via GDNF/GDNF family neurotrophic factor receptor α1 (GFRα1)/rearranged during transfection (RET) signalling pathway. Wistar male rats were first randomly assigned into control and model groups. Immunohistochemistry was performed to detect the GDNF positive expression rate and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining for apoptotic index (AI) of retinal tissue. Retinal neurons were divided into normal, blank, negative control (NC), miR-216a mimic, miR-216a inhibitor, siRNA-GDNF and miR-216a inhibitor?siRNA-GDNF groups. Dual luciferase reporter assay was conducted in order to identify the targeting relationship between GDNF and miR-216a. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot were used for the analysis of mRNA and protein levels of miR-216a and related genes. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine cell proliferation and flow cytometry was used to observe cell cycle and apoptosis. Results show that the model group had an increased GDNF positive rate, AI of retinal tissue and mRNA and protein levels of cellular oncogene fos (c-fos), vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), GDNF, GFRα1 and bcl-2-associated X protein (bax), declined miR-216a level and mRNA and protein levels of RET and bcl-2 compared with the control group. GDNF was verified as the target gene for miR-216a. Compared with the blank and NC groups, the miR-216a mimic and siRNA-GDNF groups had higher mRNA and protein levels of c-fos, VEGF and bax, cell number in the G1 phase and increased cell apoptosis but reduced BDNF, GDNF, GFRα1, RET and bcl-2 expression, cell proliferation and cell numbers in the S phase, while the opposite trend was observed in the miR-216a inhibitor group. Taken together, our findings demonstrate that elevated GDNF levels can reduce the retinal injury, whereby down-regulated miR-216a aggravates the YAG laser-induced retinal injury by targeting the GDNF level through the GDNF/ GFRα1/RET signalling pathway.

Abstract 2881: RET signaling in pancreatic cancer: A novel target by metformin to suppress cancer progression

Abstract Metformin has recently gained attention as an anti-cancer drug and/or a chemoprevention agent because of its roles in inhibiting mTOR, lowering hyperinsulinemia, modulating inflammatory responses, and selectively killing cancer stem cells. However, the key underlying molecular mechanisms for the inhibitory effects of metformin on pancreatic cancer progression remain largely unknown. RET (REarranged during Transfection), a single-pass transmembrane receptor tyrosine kinase and its ligand, glial cell-derived neurotrophic factor (GDNF), were strongly expressed in pancreatic cancer and correlated to invasion and worse survival after surgical resection. In this study, we investigated the roles of RET in the inhibitory effects of metformin on pancreatic cancer cell growth and migration. Real-time PCR and Western blot were used to determine mRNA and protein levels of molecular markers, respectively. siRNAs were adopted to specifically knockdown target genes. Boyden chamber assay was applied to assess cell migration in vitro. We observed that metformin treatment significantly reduced the mRNA and protein expression of RET in a dose-dependent manner in pancreatic cancer cells PANC-1 and MIA PaCa-2. The inhibitory effects of metformin on RET expression were not diminished when AMPK was knockdown by AMPK siRNA or the specific inhibitor, Compound C, suggesting that metformin may suppress RET in an AMPK-independent manner. Metformin treatment or RET knockdown by RET siRNA significantly decreased the phosphorylation of NF-κB and p70S6K, while modestly reduced the phosphorylation of the ERK, AKT, or STAT3. Furthermore, metformin treatment or RET knockdown significantly inhibited GDNF-induced cell migration. These data indicate that targeting RET with metformin may be an attractive and novel strategy for the prevention and treatment of pancreatic cancer progression and metastasis. Further in vitro and in vivo studies are warranted to investigate how metformin modulates RET signaling to inhibit the progression and metastasis of pancreatic cancer. Citation Format: Huailong Chang, Zhiyong Xiao, Tao Li, Lanjing Zhang, Yong Lin, Darren Carpizo, Xianglin Tan. RET signaling in pancreatic cancer: A novel target by metformin to suppress cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2881.

65 YEARS OF THE DOUBLE HELIX: Exploiting insights on the RET receptor for personalized cancer medicine.

The focus of precision cancer medicine is the use of patient genetic signatures to predict disease occurrence and course and tailor approaches to individualized treatment to improve patient outcomes. The rearranged during transfection (RET) receptor tyrosine kinase represents a paradigm for the power of personalized cancer management to change cancer impact and improve quality of life. Oncogenic activation of RET occurs through several mechanisms including activating mutations and increased or aberrant expression. Activating RET mutations found in the inherited cancer syndrome multiple endocrine neoplasia 2 permit early diagnosis, predict disease course and guide disease management to optimize patient survival. Rearrangements of RET found in thyroid and lung tumors provide insights on potential disease aggressiveness and offer opportunities for RET-targeted therapy. Aberrant RET expression in a subset of cases is associated with tumor dissemination, resistance to therapies and/or poorer prognosis in multiple cancers. The potential of RET targeting through repurposing of small-molecule multikinase inhibitors, selective RET inhibitors or other novel approaches provides exciting opportunities to individualize therapies across multiple pathologies where RET oncogenicity contributes to cancer outcomes.

Detection of RET (rearranged during transfection) variants and their downstream signal molecules in RET rearranged lung adenocarcinoma patients

BackgroundWe screened resected tumor tissues from patients with lung cancer for EGFR mutations, ALK rearrangements, and rearranged during transfection (RET) gene variants (including RET rearrangements and the Kinesin Family Member 5B (KIF5B)-RET fusion gene) using various methods including reverse transcription polymerase chain reaction (RT-PCR), transcript assays, fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). We also examined the protein expression of associated downstream signaling molecules to assess the effect of these variants on patient outcome. MethodWe constructed a tissue microarray (TMA) comprising 581 resected tumor tissues from patients with lung adenocarcinoma and analyzed the microarray by both FISH (using RET break-apart and KIF5B-RET SY translocation probes) and a commercial RET transcript assay. We evaluated the expression of RET and RET-related signaling molecules, including p-AKT and p-ERK, by TMA -based IHC staining. ResultsAmong the 581 specimens, 51 (8.8%) specimens harbored RET rearrangements, including 12 cases (2.1%) carrying a KIF5B-RET fusion gene. Surprisingly, RET expression was lower in KIF5B-RET fusion gene-positive than in RET wild-type specimens. We detected activating EGFR mutations in 11 (21.6%) of the 51 RET variant-positive specimens. Among the KIF5B-RET fusion gene-positive specimens, p-ERK expression was significantly lower in the EGFR mutation subgroup showing RET expression than in the EGFR mutation subgroup that did not express RET. Similarly, the RET rearrangement group showed significant variation in the expression level of p-AKT (P = 0.028) and p-ERK, whose expression remarkably increased in specimens not expressing RET. The expression of p-ERK markedly increased in the RET rearrangement group regardless of RET expression. ConclusionThis result suggests that a combination of RET and ERK inhibitors may be an effective treatment strategy for lung adenocarcinoma patients harboring RET variants.

Elevated RET expression enhances EGFR activation and mediates EGFR inhibitor resistance in head and neck squamous cell carcinoma

Background and aimCo-activation of EGFR by alternative receptor tyrosine kinases (RTKs) might mediate resistance to EGFR inhibition in head and neck squamous cell carcinoma (HNSCC). Here we found a novel mechanism to improve the efficacy of EGFR inhibitor erlotinib on HNSCC. MethodImmunohistochemistry, western blot, cell migration and invasion assays, cell proliferation, cell cycle analysis and in vivo serial transplantation assays were used to evaluate the role of RET on HNSCC cells. ResultsThe elevated levels of a rearranged during transfection (RET) are observed in HNSCC and that high levels of RET correlate with increased tumor size, advanced tumor stage and decreased overall survival rate. The HNSCC cell proliferation and invasion were inhibited by RET knockdown in vitro and in vivo. The inhibition of RET expression markedly reduced EGFR phosphorylation and downstream EGFR signaling. The inhibition of RET signaling significantly increased the sensitivity of HNSCC cells to the EGFR inhibitor erlotinib in both in vitro and in vivo models. ConclusionOur results offer a preclinical proof-of-concept supporting a role for RET signaling inhibition in a targeted therapeutic approach to improve the efficacy of EGFR inhibition in HNSCC.

Targeting RET-interleukin-6 crosstalk to impair metastatic dissemination in breast cancer.

RET (rearranged during transfection) is a receptor tyrosine kinase overexpressed in a subset of oestrogen receptor (ER)-positive breast cancers whose expression is regulated by ER signalling. The article from the Hynes group has reported for the first time that RET expression can also be regulated by the inflammatory cytokine IL-6. Importantly, RET and IL-6 interact at a functional level to control migration and the metastatic potential of ER-positive breast cancer cells, in a process that is mediated by FAK activation. Further, targeting RET with receptor tyrosine kinase inhibitors was reported to be more effective than endocrine therapies in impairing metastatic dissemination in vivo, thereby indicating a level of RET regulation that is independent of ER.

CD44 and OTP Are Strong Prognostic Markers for Pulmonary Carcinoids

Pulmonary carcinoids are well-differentiated neuroendocrine tumors showing usually a favorable prognosis. However, there is a risk for late recurrence and/or distant metastasis. Because histologic classification in typical and atypical carcinoids is difficult and its reliability to predict disease outcome varies, we evaluated three genes as potential prognostic markers, that is, orthopedia homeobox (OTP), CD44, and rearranged during transfection (RET). These genes were analyzed in 56 frozen carcinoids by quantitative real-time PCR (qRT-PCR). RET was further studied by methylation and mutation analysis. Immunohistochemistry for CD44 and OTP protein expression was conducted on 292 carcinoids. Low mRNA expression levels of CD44 (P = 1.8e(-5)) and OTP (P = 0.00054), and high levels of RET (P = 0.025), were strongly associated with a low 20-year survival of carcinoid patients. High RET expression was not related to promoter hypomethylation or gene mutations. A direct link between gene expression and protein levels was confirmed for CD44 and OTP but not for RET. Within all carcinoids as well as atypical carcinoids, absence of CD44 protein was significantly associated with low 20-year survival (P = 0.00014 and 0.00013, respectively). The absence of nuclear OTP followed by complete loss of expression was also significantly associated with unfavorable disease outcome in all carcinoids (P = 5.2(-6)). Multivariate analyses revealed that age at diagnosis, histopathology, stage, and cytoplasmic OTP immunoreactivity were independent predictors of prognosis. Our study indicates that CD44 and OTP are strong indicators of poor outcome. We therefore argue for implementation of these markers in routine diagnostics in addition to histopathology to improve subclassification of pulmonary carcinoids into prognostically relevant categories.

Invasive properties of papillary thyroid cancer with concurrent BRAF(V600E) mutation and rearranged during transfection proto-oncogene protein expression

To investigate the aggressive properties of papillary thyroid cancer (PTC) with concurrent BRAF(V600E) mutation and rearranged during transfection (RET) proto-oncogene protein expression. Fifty pathologically confirmed PTC patients who had received thyroidectomy were enrolled in this study. BRAF(V600E) mutation was detected by real time polymerase chain reaction (RT-PCR), while RET protein expression was measured by immunohistochemical SP method. Clinical and pathological features were compared between the concurrent BRAF(V600E) mutation and RET protein expression group (n=24) and BRAF(V600E) mutation or RET protein expression alone group (n=19). Seven patients were ruled out from the final analysis due to the absence of either BRAF(V600E) mutation or RET protein expression. Of these 50 patients, BRAF(V600E) mutation and RET protein expression were detected in 38 patients (76%) and 28 patients (56%), respectively. Concurrent BRAF(V600E) mutation and RET expression was detected in 24 patients (48%). Compared with the concurrent BRAF(V600E) mutation and RET protein expression group, the BRAF(V600E) mutation or RET protein expression alone group had relatively poorer tissue differentiation and higher prognostic score (P=0.011, P=0.022). PTC patients with concurrent BRAF(V600E) mutation and RET expression present poorer differentiation, more highly aggressive variant in carcinoma tissues, and higher cancer-related mortality risk.

Abstract 4547: Treatment of human medullary thyroid carcinoma (MTC) with either proteasome (Pr) or histone deacetylase (HDAC) inhibitors leads to a fall in RET mRNA levels and, in turn, a decrease in RET protein expression providing alternate strategies to reduce RET expression in a tyrosine-kinase driven disease

Abstract Medullary thyroid carcinoma (MTC), a neuro-endocrine cancer of parafollicular C-cells accounts for 4-10% of all thyroid cancers and has low response rates to “cytotoxic chemotherapy”. The RET proto-oncogene encodes the RET (Rearranged during Transfection) transmembrane tyrosine kinase (TK) receptor, a pivotal player and a logical target for treatment of MTC. While trials with vandetanib (V) a RET TK inhibitor (TKI) in patients with MTC have been promising, we recognized a TKI is unlikely to have maximum benefit administered alone. We sought to combine V + a drug with a broad activity profile and no overlapping toxicities. Recognizing the failure of traditional “cytotoxic” agents we investigated the proteasome (Pr) inhibitor, bortezomib (B) not yet explored clinically in MTC. Initial experiments using TT MTC cells with a mutant C634W RET, found V + B non-antagonistic in cytotoxicity assays, and V showed decreased phospho-RET expression in TT cells with 0.5 or 1 µM in 1.5h. But while V did not change RET protein levels, B decreased RET protein 7 fold and secondarily phospho-RET levels. That this was secondary to Pr inhibition and not unique to B was confirmed by treating TT cells 6h or 24h with three other Pr inhibitors – epoxomycin, lactacystin or MG132. We observed 5-9 fold decreases in RET protein, which followed a time course and a dose response, with greater reduction at 24h than at 6h. To assess if the decrease in RET protein was due to decreased RET mRNA levels, we assessed RET mRNA expression and were surprised to find a 4-9 fold decrease with Pr inhibitors. Since Pr inhibitors reduced RET by reducing mRNA levels, and reasoning this would provide an alternate strategy to impact RET function, we studied the effect of three HDAC inhibitors on RET mRNA and protein expression. Treatment of TT cells with vorinostat, belinostat, or romidepsin (R) decreased RET mRNA 3-4 fold at 24h and protein levels 4 and 7-14 fold, at 24 and 48h respectively. Furthermore B + R had greater impact than either drug separately, depressing RET protein in TT cells an additional 2-3 fold to levels 4-12 fold less than that in untreated cells, suggesting mechanisms whereby Pr and HDAC inhibitors reduce mRNA levels might differ. We have pursued the latter by examining mRNA and protein levels of 3 candidate transcription factors identified in a literature search. To date we have shown TTF1 and E2F1 mRNA levels are decreased 4-58 fold by both Pr and HDAC inhibitors whereas EGR1 levels only fall with Pr inhibitors. siRNA studies that modulate transcription factor levels will clarify their possible roles in the Pr and HDAC inhibitor drug effects and help elucidate whether these inhibitors, either alone or in combination, can prove useful in MTC therapy by reducing the crucial RET TK. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4547. doi:10.1158/1538-7445.AM2011-4547

Combination of RET siRNA and irinotecan inhibited the growth of medullary thyroid carcinoma TT cells and xenografts via apoptosis

Medullary thyroid carcinoma (MTC) is a rare endocrine tumor that frequently metastasizes, and treatment with irinotecan (CPT-11) is limited because of side effects. Mutations in the Rearranged during transfection (RET) proto-oncogene are considered the causative event of MTC. The objective of this study was to examine whether small interfering RNA (siRNA) and its combined treatment with CPT-11 could inhibit MTC cell growth in vitro and in vivo. The transfection of RET siRNA suppressed RET expression, reduced proliferation, and increased caspase-3/7 activity via the down-regulation of Bcl-2 expression. Combined treatments with CPT-11 or SN-38 significantly increased caspase 3/7 activity compared with RET siRNA, CPT-11 or SN-38 treatment alone. Importantly, intratumoral injection of RET siRNA along with intravenous injection of CPT-11 significantly inhibited the tumor growth of MTC xenografts via an increased apoptotic effect. These findings that RET siRNA enhanced sensitivity for CPT-11 will provide a novel strategy for the treatment of MTC with RET mutation.

RET/PTC activation in hyalinizing trabecular tumors of the thyroid.

Hyalinizing trabecular tumor (HTT) of the thyroid is a neoplasm of follicular derivation with a histogenesis that is still the subject of debate. Morphologic affinities between HTT and papillary carcinoma, including nuclear pseudoinclusions and grooves, suggest that these tumors may be of similar origin. The authors investigated the relationship between these two types of tumors by assessing HTT for the presence of rearrangements of the proto-oncogene rearranged during transfection (RET) that, in thyroid tumors, are specific for papillary carcinoma. A series of 14 HTTs, including two cases associated with classic papillary carcinoma, was studied by means of immunohistochemistry and reverse transcription-polymerase chain reaction. Seven follicular adenomas with focal hyalinized trabecular areas served as control cases. Three of the 14 HTT cases under consideration displayed rearrangements of RET generating the RET/papillary thyroid carcinoma type 1 (PTC1) oncogene. In another case, RET expression was detected focally by immunohistochemistry alone. Finally, in one mixed HTT-papillary carcinoma sample, RET/PTC1 expression was detected, but only in the papillary component. None of the control follicular adenomas contained rearrangements of RET/PTC. These findings demonstrate that a comparable percentage (28.6%) of HTTs and papillary carcinomas exhibit the same RET proto-oncogene alterations. Thus, HTT may represent the "hyalinizing trabecular" variant of papillary carcinoma rather than a separate entity.