Anti-tumor activity and acquired resistance mechanism of dovitinib (TKI258) in RET rearranged lung adenocarcinoma

의과대학/석사RET rearrangement is a newly identified oncogenic mutation in lung adenocarcinoma (LADC). Activity of dovitinib (TKI258), a potent inhibitor of FGFR, VEGFR, and PDGFR, on RET-rearranged LADC has not been known. The aims of the study are to explore antitumor effects and mechanism of acquired resistance of dovitinib in RET-rearranged LADC. In silico analysis results indicated that dovitinib strongly interacts with the surrounding residues in RET kinase domain, dovitinib potently induced cell cycle arrest in G1 phase and does-dependent suppression of phosphorylation of RET and ERK in LC-2/ad LADC cells harboring CCDC6-RET rearrangement. The therapeutic effects of dovitinib were photocopied by siRNA knockdown of RET. Selective inhibition of dovitinib on autophosphorylation of RET kinases was confirmed in HEK293 cells expressing KIF5B-RET or CCDC6-RET. A phospho-RTK array reveals that LC-2/ad cells maintain phosphorylation of FGFRs and VEGFRs in the presence of dovitinib. Dovitinib inhibited ERK phosphorylation more efficiently than vandetanib, sunitinib and sorafenib. The effects on ERK phosphorylation were correlated with the results of cell viability assays. Antitumor effect of dovitinib was observed in tumor xenograft model. To identify the acquired resistance mechanism, dovitinib-resistant cells (LC-2/ad DR) were established by exposure of LC-2/ad to stepwise increasing concentration of dovitinib. Knockdown of RET or dovitinib treatment did not inhibit ERK phosphorylation in LC-2/ad DR cells. Saracatinib, a src kinase inhibitor, suppressed ERK phosphorylation and growth of LC-2/ad DR cells. Taken together, dovitinib can be a potential therapeutic option for RET-rearranged LADC, and acquired resistance to dovitinib can be overcome by targeting SRC.ope

Acetate-Mediated Odorant Receptor OR51E2 Activation Results in Calcitonin Secretion in Parafollicular C-Cells: A Novel Diagnostic Target of Human Medullary Thyroid Cancer

Medullary thyroid cancer originates from parafollicular C-cells in the thyroid. Despite successful thyroidectomy, localizing remnant cancer cells in patients with elevated calcitonin and carcinoembryonic antigen levels remains a challenge. Extranasal odorant receptors are expressed in cells from non-olfactory tissues, including C-cells. This study evaluates the odorant receptor signals from parafollicular C-cells, specifically, the presence of olfactory marker protein, and further assesses the ability of the protein in localizing and treating medullary thyroid cancer. We used immunohistochemistry, immunofluorescent staining, Western blot, RNA sequencing, and real time-PCR to analyze the expression of odorant receptors in mice thyroids, thyroid cancer cell lines, and patient specimens. We used in vivo assays to analyze acetate binding, calcitonin secretion, and cAMP pathway. We also used positron emission tomography (PET) to assess C11-acetate uptake in medullary thyroid cancer patients. We investigated olfactory marker protein expression in C-cells in patients and found that it co-localizes with calcitonin in C-cells from both normal and cancer cell lines. Specifically, we found that OR51E2 and OR51E1 were expressed in thyroid cancer cell lines and human medullary thyroid cancer cells. Furthermore, we found that in the C-cells, the binding of acetate to OR51E2 activates its migration into the nucleus, subsequently resulting in calcitonin secretion via the cAMP pathway. Finally, we found that C11-acetate, a positron emission tomography radiotracer analog for acetate, binds competitively to OR51E2. We confirmed C11-acetate uptake in cancer cells and in human patients using PET. We demonstrated that acetate binds to OR51E2 in C-cells. Using C11-acetate PET, we identified recurrence sites in post-operative medullary thyroid cancer patients. Therefore, OR51E2 may be a novel diagnostic and therapeutic target for medullary thyroid cancer.ope

Antitumor Activity and Acquired Resistance Mechanism of Dovitinib (TKI258) in RET-Rearranged Lung Adenocarcinoma

RET rearrangement is a newly identified oncogenic mutation in lung adenocarcinoma (LADC). Activity of dovitinib (TKI258), a potent inhibitor of FGFR, VEGFR, and PDGFR, in RET-rearranged LADC has not been reported. The aims of the study are to explore antitumor effects and mechanisms of acquired resistance of dovitinib in RET-rearranged LADC. Using structural modeling and in vitro analysis, we demonstrated that dovitinib induced cell-cycle arrest at G0-G1 phase and apoptosis by selective inhibition of RET kinase activity and ERK1/2 signaling in RET-rearranged LC-2/ad cells. Strong antitumor effect of dovitinib was observed in an LC-2/ad tumor xenograft model. To identify the acquired resistance mechanisms to dovitinib, LC-2/ad cells were exposed to increasing concentrations of dovitinib to generate LC-2/ad DR cells. Gene-set enrichment analysis of gene expression and phosphor-kinase revealed that Src, a central gene in focal adhesion, was activated in LC-2/ad DR cells. Saracatinib, an src kinase inhibitor, suppressed ERK1/2 phosphorylation and growth of LC-2/ad DR cells. Taken together, these findings suggest that dovitinib can be a potential therapeutic option for RET-rearranged LADC, in which acquired resistance to dovitinib can be overcome by targeting Src.ope

후각표지단백질에 의한 뇌하수체 프로락틴분비세포의 프로락틴 분비 조절 기전 연구

Olfactory marker protein (OMP) is a marker of olfactory receptor-mediated chemoreception, even outside the olfactory system. Here, we report that OMP expression in the pituitary gland plays a role in basal and thyrotropin-releasing hormone (TRH)-induced prolactin (PRL) production and secretion. We found that OMP was expressed in human and rodent pituitary glands, especially in PRL-secreting lactotrophs. OMP knockdown in GH4 rat pituitary cells increased PRL production and secretion via extracellular signal-regulated kinase (ERK)1/2 signaling. Real-time PCR analysis and the Ca2+ influx assay revealed that OMP was critical for TRH-induced PRL secretion. OMP-knockout mice showed lower fertility than control mice, which was associated with increased basal PRL production via activation of ERK1/2 signaling and reduced TRH-induced PRL secretion. However, both in vitro and in vivo results indicated that OMP was only required for hormone production and secretion because ERK1/2 activation failed to stimulate cell proliferation. Additionally, patients with prolactinoma lacked OMP expression in tumor tissues with hyperactivated ERK1/2 signaling. These findings indicate that OMP plays a role in PRL production and secretion in lactotrophs through the modulation of Ca2+ and TRH signaling. 후각표지단백질 (OMP)은 후각 상피 내의 후각 수용세포에서 특징적으로 발현되는 세포질 단백질이다. 최근 시퀀싱 기술이 발달하면서 OMP가 내분비계와 같은 후각 기관계가 아닌 조직에서도 발현되는 것으로 알려졌다. 본 연구에서는 OMP가 인간과 마우스 뇌하수체 전엽의 프로락틴 분비세포에서 발현하는 것을 확인하였고, OMP가 후각수용세포의 세포내 칼슘을 조절하여 신호전달의 종결을 조절하는 것처럼 프로락틴 호르몬 분비에 미치는 영향 및 기전을 연구하였다. 프로락틴을 분비하는 GH4 세포에서 OMP발현을 억제하자 ERK1/2의 인산화가 증가되었고 프로락틴 생성 및 분비를 증가시켰다. 또한, Real-time PCR 분석과 칼슘 유입분석 결과를 통해 OMP가 다른 신경호르몬 도파민과 에스트로젠이 아닌 갑상샘자극 호르몬-분비호르몬에 의한 프로락틴 생성 및 분비에 관여하는 것을 확인하였다. OMP가 결핍된 마우스에서는 대조군 마우스에 비해 ERK1/2 활성 증가 및 기저 프로락틴 분비가 증가되었고, 생식능력이 떨어지는 표현형을 보였다. OMP에 의해 증가된 ERK1/2 인산화는 세포증식에는 관여하지 않고 프로락틴의 생산과 분비에만 관여하였다. 프로락틴 분비 뇌하수체선종 환자에서는 ERK1/2 인산화 및 프로락틴 발현이 정상인의 조직에 비교했을 때 확연히 증가되어 있는 반면, OMP의 발현은 정상인에 비해 확연하게 감소되어 있었다. 결론적으로, OMP는 칼슘과 갑상샘자극호르몬-분비호르몬 신호 기전을 조절하여 프로락틴 분비세포의 프로락틴 생산과 분비에 중요한 역할을 담당한다고 할 수 있다.open박

Downregulation of miR-216a-5p and miR-652-3p is associated with growth and invasion by targeting JAK2 and PRRX1 in GH-producing pituitary tumours

Expression of aberrant microRNA (miRNA) is associated with tumour formation, migration, and invasion. However, there is limited information about the epigenetics of pituitary tumorigenesis. This study investigated the role of miRNA expression during the tumorigenesis of growth hormone (GH)-secreting pituitary tumours. miRNA profiling and real-time PCR were used to analyse the mRNA expression profile in sequential pituitary tissues of a unique animal model with a GH-producing pituitary tumour. Selected miRNAs were further validated in GH-producing cell lines and human pituitary tumour samples. The expression of significantly altered miRNAs and their predicted targets, as detected by microarray, was evaluated by real-time PCR, Western blotting, and immunohistochemistry using samples from mouse models and human pituitary tumours. The effect of miRNAs on tumour proliferation and invasion was examined in GH3 cells using the MTS and Matrigel invasion assays. Among the 14 miRNAs whose expression was significantly changed, miR-216a-5p (fold change = -5.638, P -value = 0.014) and miR-652-3p (fold change = -3.482, P -value = 0.010) were constantly and significantly downregulated. Transfection with mimics of miR-216a-5p and miR-652-3p inhibited GH3 proliferation and invasion, whereas inhibitors promoted them. The direct target genes of miR-216a-5p and miR-652-3p were Jak2 and Prrx1, respectively, which were downregulated in GH3 cells transfected with mimics and in serial pituitary gland tissues, including hyperplasic tissues and tumours of acromegalic animal models and pituitary tumour tissues of acromegalic patients. Downregulated miR-216a-5p and miR-652-3p expression may contribute to tumour progression by targeting JAK2 and PRRX1 on GH-producing pituitary tumours.restrictio

An open label, multicenter, phase II study of dovitinib in advanced thyroid cancer

BACKGROUND: This phase 2 study investigated the efficacy and safety of dovitinib (TKI258), a receptor tyrosine kinase inhibitor with potent activity against fibroblast growth factor receptor (FGFR) and vascular endothelial growth factor receptor (VEGFR), in locally advanced or metastatic thyroid cancer patients. PATIENTS AND METHODS: Patients with advanced thyroid cancer that was refractory or not appropriate for (131)I received dovitinib orally, 500mg once daily for five consecutive days, followed by a 2-day rest every week. The primary end-point was objective response rate. Secondary end-points were progression-free survival (PFS), overall survival (OS), duration of response, changes in tumour markers and safety. RESULTS: Between January 2013 and October 2014, a total of 40 patients were enrolled. There were 23 (57.5%) papillary thyroid cancer, 12 (30%) medullary thyroid cancer and 5 (12.5%) follicular thyroid cancer patients. One patient had withdrawn consent before the administration of dovitinib. The overall response rate was 20.5% (8/39) and disease control rate was 69.1% (26/39). Median PFS was 5.4 months (95% confidence interval (CI), 2.0-8.8) and median OS was not reached with 8.4 months follow-up duration. Common treatment-related adverse events were diarrhoea (53.8%), anorexia (35.8%), vomiting (25.6%), fatigue (23%) and nausea (20.5%), most of which were grade 1 or 2. There were no grade 4 events or treatment-related deaths. Dose interruption occurred in 12 (30.7%) patients, and 19 (48.7%) patients experienced dose reduction due to adverse events. CONCLUSIONS: Dovitinib has a modest activity with manageable toxicity in locally advanced or metastatic thyroid cancer.ope

Serum glucose excretion after Roux-en-Y gastric bypass: a potential target for diabetes treatment

Objective: The mechanisms underlying type 2 diabetes resolution after Roux-en-Y gastric bypass (RYGB) are unclear. We suspected that glucose excretion may occur in the small bowel based on observations in humans. The aim of this study was to evaluate the mechanisms underlying serum glucose excretion in the small intestine and its contribution to glucose homeostasis after bariatric surgery. Design: 2-Deoxy-2-[18F]-fluoro-D-glucose (FDG) was measured in RYGB-operated or sham-operated obese diabetic rats. Altered glucose metabolism was targeted and RNA sequencing was performed in areas of high or low FDG uptake in the ileum or common limb. Intestinal glucose metabolism and excretion were confirmed using 14C-glucose and FDG. Increased glucose metabolism was evaluated in IEC-18 cells and mouse intestinal organoids. Obese or ob/ob mice were treated with amphiregulin (AREG) to correlate intestinal glycolysis changes with changes in serum glucose homeostasis. Results: The AREG/EGFR/mTOR/AKT/GLUT1 signal transduction pathway was activated in areas of increased glycolysis and intestinal glucose excretion in RYGB-operated rats. Intraluminal GLUT1 inhibitor administration offset improved glucose homeostasis in RYGB-operated rats. AREG-induced signal transduction pathway was confirmed using IEC-18 cells and mouse organoids, resulting in a greater capacity for glucose uptake via GLUT1 overexpression and sequestration in apical and basolateral membranes. Systemic and local AREG administration increased GLUT1 expression and small intestinal membrane translocation and prevented hyperglycaemic exacerbation. Conclusion: Bariatric surgery or AREG administration induces apical and basolateral membrane GLUT1 expression in the small intestinal enterocytes, resulting in increased serum glucose excretion in the gut lumen. Our findings suggest a novel, potentially targetable glucose homeostatic mechanism in the small intestine.restrictio