D1 Cell Research Articles

Plinabulin Rapidly (within 24 Hours) Reverses Myelosuppression Induced By Chemotherapy

▪IntroductionPlinabulin (Plin), a small molecule selective immune-enhancing microtubulin binding agent (SIMBA) has anti-cancer activity, and also prevents chemotherapy (Chemo) induced neutropenia (CIN). Plin has a rapid onset CIN prevention (1 st week) and G-CSF a delayed onset (2 nd week) of the cycle. Single agent Plin has non-inferior protection against CIN compared to Peg. Combining Plin with pegfilgrastim (Peg) has superior protection against CIN throughout the entire Chemo cycle (Blayney ASCO 2021). Plinabulin protected LSK cells in mice receiving myelosuppressive Chemo, which are cells equivalent to CD34+ in humans (Tonra Cancer Chemotherapy and Pharmacology. 2020). We evaluated the mechanism of Plin's rapid onset for CIN prevention in patients (pts).MethodsData from the CIN phase 2/3 Plin trials PROTECTIVE-1 (NCT03102606) with docetaxel 75 mg/m2 in pts with NSCLC, Breast Cancer, Prostate Cancer and PROTECTIVE-2 (NCT0329457) with docetaxel 75 mg/m2, doxorubicin 50 mg/m2, cyclophosphamide 500 mg/m2 (TAC) in early stage Breast Cancer was analyzed for pts receiving either Plin (n=228) or Control (no Plin); n=172). Both studies 105 and 106 had patients randomized to either Plin or Pegfilgrastim, which latter was given on the next day of Chemo, on day (D)2. The safety blood draw, taken on D2 at predose Pegfilgrastim, served as a no-treatment Control on D2 in this blood cell analysis for comparison with Plin, which was given on D1. The Plin dose was 20 mg/m2 or 40 mg, and was given as a single dose per cycle, by 30 min IV infusion, 30 min after Chemo on day (D)1. The 40 mg fixed dose is equivalent to 20 mg/m2 Plin. Absolute neutrophil count (ANC), monocyte (M), eosinophil (E), basophil (B), lymphocyte (L), erythrocyte (RBC), and platelet (P) counts were obtained from peripheral blood draws and analyzed by Central Laboratory (Covance). The primary analysis was to compare cell counts between Plin and Control in blood draws taken 24 hour (hr)post-Chemo (thus D2). Separately, we also established Spearman correlation coefficients (r) of scatter plots between ANC and M,E,B,L,RBC, or P count on D2, in a broader dataset that also included Plin doses of 5,10 and 30 mg/m2 in addition to 0 and 20 mg/m2 (40 mg fixed) doses to allow for dose-response evaluation (total n=451). D2 cell counts were expressed as change from pre-dose D1, in absolute values.ResultsAt predose D1 (baseline), counts of ANC, M,E,B,L,RBC, and P were similar between the Plin and Control groups (p=NS). On D2, at 24 hr post-Chemo, ANC, M,E and B ,had all significantly decreased compared to D1 in the Control group (indicating myelosuppression by Chemo), whereas significantly increased with Plin (suggesting reversal of this myelosuppression), albeit within normal range (Table below). At 24 hr post-Chemo, counts for L and RBC had significantly decreased compared to D1 with both Control and Plin, however this decrease was ~50% less with Plin compared to Control (P<0.001). No difference in P counts were observed between Plin and Control at 24 hr post Chemo (p=0.69). Correlative analyses showed that at 24 hr post-Chemo (D2), ANC was positively correlated with counts of M (r=+0.42; p<0.0001), E (r=+0.16; p=0.0008), B (r=+0.20; p<0.0001), L (r=+0.32; p<0.0001), RBC (r=+0.07; p=0.1187) and P (r=+0.18; p=0.0002). Thus, with larger increase in ANC counts on D2, the larger were the increase in the other blood cell types. The observed results were Plin dose-dependent, and consistent between pts receiving either docetaxel alone or TAC . Shown below are mean (SD) of absolute cell count on D2, expressed as change from predose D1.ConclusionPlin rapidly (within 24 hours) reversed myelosuppression induced by Chemo, by protecting granulocyte-monocyte-progenitor (GMP) stem cells (responsible for producing ANC, M, B and E cells) or progenitor cells further upstream in the hematopoietic lineage tree. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

LncRNA DLGAP1-AS2 regulates miR-503/cyclin D1 to promote cell proliferation in non-small cell lung cancer

BackgroundLncRNA DLGAP1-AS2 plays an oncogenic role in glioma, while its role in other cancers is unknown. This study aimed to study the role of DLGAP1-AS2 in non-small cell lung cancer (NSCLC).MethodsExpression of DLGAP1-AS2 in NSCLC and paired non-tumor tissues from 64 NSCLC patients and the prognostic value of DLGAP1-AS2 for NSCLC were analyzed by performing a 5-year follow-up study. The interaction between DLGAP1-AS2 and miR-503 was confirmed by dual luciferase reporter assay, and their relationship was explored in NSCLC cells transfected with DLGAP1-AS2 expression vector or miR-503 mimic. The roles of DLGAP1-AS2 and miR-503 in regulating cyclin D1 expression were analyzed by RT-qPCR and Western blot. Cell proliferation was analyzed by CCK-8 assay.ResultsDLGAP1-AS2 was upregulated in NSCLC and predicted poor survival. Interaction between DLGAP1-AS2 and miR-503 was confirmed by dual luciferase activity assay. Overexpression experiments showed that DLGAP1-AS2 and miR-503 overexpression failed to significantly affect the expression of each other. Interestingly, DLGAP1-AS2 overexpression upregulated cyclin D1, a target of miR-503, increased cell proliferation and reduced the effects of miR-503 overexpression on cyclin D1 expression and cell proliferation.ConclusionsDLGAP1-AS2 may regulate miR-503/cyclin D1 to promote cell proliferation in NSCLC.

High-performance SOD mimetic enzyme Au@Ce for arresting cell cycle and proliferation of acute myeloid leukemia

SOD-like activity of CeO2 nanoparticles (Ce NPs) is driven by Ce3+/Ce4+, high oxidative stress can oxidize Ce3+ to reduce the ratio of Ce3+/Ce4+, inactivating the SOD activity of Ce NPs. Herein, we found Au@Ce NPs, assembled by Au NPs and Ce NPs, exhibited high-performance of SOD mimetic enzyme activity even upon the oxidation of H2O2. Ce NPs supported by nano-Au can acquire the electrons from Au NPs through the enhanced localized surface plasmon resonance (LSPR), maintaining the stability of Ce3+/Ce4+ and SOD-like activity. Meanwhile, Au@Ce NPs retained the peroxidase function and catalase function. As a result, Au@Ce NPs effectively scavenged O2•- and the derived ROS in AML cells, which are the important signaling source that drives AML cell proliferation and accelerates cell cycle progression. When HL-60 cells were treated by Au@Ce NPs, the removal of endogenous ROS signal significantly arrested cell cycle at G1 phase and suppressed the cell proliferation by blocking the mitogen-activated protein kinases (MAPKs) signaling and the Akt/Cyclin D1 cell cycle signaling. Importantly, this treatment strategy showed therapeutic effect for subcutaneous transplantation of AML model as well as a satisfactory result in diminishing the leukocyte infiltration of liver and spleen particularly. Thus, assembled Au@Ce NPs show the high-performance SOD-like activity, promising the potential in treating AML and regulating abnormal ROS in other diseases safely and efficiently.

Maduramicin arrests myocardial cells at G0/G1 phase of the cell cycle through inhibiting AKT-Cyclin D1 signaling.

Maduramicin, a polyether ionophore antibiotic used as an anticoccidial feed additive in poultry, is toxic to animals and humans and can cause heart failure. The present study was initiated to explore the underlying mechanism of toxicity in H9c2 myocardial cells. We observed using cell imaging and counting methods that maduramicin inhibited cell growth in a concentration-dependent manner. Furthermore, MTT assays showed that maduramicin inhibited cell proliferation in a concentration- and time-dependent manner, and was also confirmed by the finding that maduramicin time dependently blocked the incorporation of BrdU into DNA in H9c2 myocardial cells. Further studies revealed that maduramicin induced accumulation of the cells at G0/G1 phase of the cell cycle and concurrently, there was down regulation of expression of Cyclin D1. In addition, exposure to maduramicin pruned phosphorylation of AKT at both T308 and S473 sites. Finally, we found that pre-treatment of H9c2 myocardial cells with AKT activator SC79, attenuated the inhibitory effects of maduramicin on Cyclin D1 expression and cell proliferation. Collectively, our results suggest that maduramicin-suppressed AKT-Cyclin D1 signaling which results in G0/G1 phase cell cycle arrest, leading to the inhibition of myocardial cell proliferation.

Enhancing the sensitivity of ovarian cancer cells to olaparib via microRNA-20b-mediated cyclin D1 targeting.

We previously reported that cyclin D1 silencing interferes with RAD51 accumulation and increases the sensitivity of BRCA1 wild-type ovarian cancer cells to olaparib. However, the mechanisms associated with cyclin D1 overexpression in ovarian cancer are not fully understood. TargetScan predicted the potential binding sites for microRNA-20b (miR-20b) and the 3'-untranslated region of cyclin D1 mRNA; thus, we used luciferase reporter assay to verify those binding sites. The Kaplan-Meier method and log-rank test were used to examine the relationship between miR-20b and progression-free survival of ovarian cancer patients in The Cancer Genome Atlas (n = 367) dataset. In vitro experiments were performed to evaluate the effects of miR-20b on cyclin D1 expression, cell cycle and response to olaparib. A peritoneal cavity metastasis model of ovarian cancer was established to determine the effect of miR-20b on the sensitivity of olaparib. Immunohistochemistry was performed to evaluate molecular mechanisms. In this work, we demonstrated that miR-20b down-regulates cyclin D1, increases the sensitivity of ovarian cancer cells to olaparib, reduces the expression of RAD51, and induces cell cycle arrest in G0/G1 phase. Ovarian cancer patients with higher expression of miR-20b had significantly longer progression-free survival. These results indicate that miR-20b may be a potential clinical indicator for the sensitivity of ovarian cancer to olaparib and the survival of ovarian cancer patients. Our findings suggest that miR-20b may have therapeutic value in combination with olaparib treatment for ovarian cancer.

YWHAE-NUTM2 oncoprotein regulates proliferation and cyclin D1 via RAF/MAPK and Hippo pathways

Endometrial stromal sarcoma (ESS) is the second most common subtype of uterine mesenchymal cancer, after leiomyosarcoma, and oncogenic fusion proteins are found in many ESS. Our previous studies demonstrated transforming properties and diagnostic relevance of the fusion oncoprotein YWHAE–NUTM2 in high-grade endometrial stromal sarcoma (HG-ESS) and showed that cyclin D1 is a diagnostic biomarker in these HG-ESS. However, YWHAE–NUTM2 mechanisms of oncogenesis and roles in cyclin D1 expression have not been characterized. In the current studies, we show YWHAE-NUTM2 complexes with both BRAF/RAF1 and YAP/TAZ in HG-ESS. These interactions are functionally relevant because YWHAE-NUTM2 knockdown in HG-ESS and other models inhibits RAF/MEK/MAPK phosphorylation, cyclin D1 expression, and cell proliferation. Further, cyclin D1 knockdown in HG-ESS dephosphorylates RB1 and inhibits proliferation. In keeping with these findings, we show that MEK and CDK4/6 inhibitors have anti-proliferative effects in HG-ESS, and combinations of these inhibitors have synergistic activity. These findings establish that YWHAE-NUTM2 regulates cyclin D1 expression and cell proliferation by dysregulating RAF/MEK/MAPK and Hippo/YAP-TAZ signaling pathways. Recent studies demonstrate Hippo/YAP-TAZ pathway aberrations in many sarcomas, but this is among the first studies to demonstrate a well-defined oncogenic mechanism as the cause of Hippo pathway dysregulation.

Sirtuin 1 and 2 inhibitors enhance the inhibitory effect of sorafenib in hepatocellular carcinoma cells

Multidrug resistance (MDR) counteracts the efficiency of sorafenib, an important first-line therapy for hepatocellular carcinoma (HCC). Sirtuins (SIRTs) 1 and 2 are associated with tumor progression and MDR. We treated 2D and 3D cultures (which mimic the features of in vivo tumors) from HCC cells with sorafenib alone or in the presence of SIRTs 1 and 2 inhibitors (cambinol or EX-527; combined treatments). Cultures subjected to combined treatments showed a greater fall in cellular viability, proliferation (PCNA, cyclin D1 and Ki-67 expression and cell cycle analysis), migration and invasion when compared with cultures treated only with sorafenib. Similarly, combined treatments produced more apoptosis (annexin V/PI, caspase-3/7 activity) than sorafenib alone. Since cell cycle dysregulation and apoptotic blockage are reported mechanisms of MDR, the modulation found in PCNA, cyclin D1, Ki-67 and caspase-3/7 proteins by cambinol and EX-527 are probably playing a role in enhancing the sensitivity of HCC cell lines to sorafenib. EX-527 reduced MRP3 and BCRP expression in sorafenib-treated HCC cells. Since ABC transporters contribute to MDR, MRP3 and BCRP could be also influencing in the response of HCC cells to sorafenib. Overall, 2D and 3D cultures behave similarly except that 3D cultures were less sensitive to treatments, reinforcing the clinical relevance of the current study. Findings presented in this manuscript support a potential application for SIRTs 1 and 2 inhibitors since we demonstrated that these compounds enhance the inhibitory effect of sorafenib upon treatment of hepatocellular carcinoma cells lines.

Associative Learning is Supported by State Dependent Changes to Inhibition in Striatal D1 and D2 Cells

Auditory stimulus response associations are in part mediated by projections from the auditory cortex to the auditory striatum. Using chemogenetic techniques in freely moving Mongolian gerbils, we show that suppression of glutamatergic input from auditory cortex layer 5 to the auditory striatum impacts both learning speed and performance in a sound discrimination task. Early cortical suppression during learning caused significant delays in task acquisition, while suppression after task acquisition affected discrimination performance. Whole-cell recordings sampled throughout learning revealed a transient reduction in postsynaptic inhibition in both striatal D1 and D2 cells. In contrast, when baseline striatal inhibitory strengths were reduced by a transient sensory deprivation, learning was associated with briefly augmented inhibition and evoked firing rates. The latter was supported by phenotype-dependent compensatory mechanisms in striatal D1 and D2 cell intrinsic membrane properties. Together, these results reveal a flexible corticostriatal inhibitory synaptic plasticity mechanism that accompanies associative auditory learning.

EXTH-70. CYP450 AND METABOLIC PROFILING OF ANTI-CANCER IMIPRIDONE ONC201

Abstract Imipridone ONC201 is an investigational agent in phase II clinical trials for high grade gliomas as a single agent once weekly. Systemic and tumor exposure to ONC201 exceeds therapeutic thresholds consistently; however, exposure is variable despite fasted conditions and does not strongly covary with body weight or body surface area. Clinical trials with ONC201 have restricted the use of concomitant medication that induce or inhibit cytochrome P450 (CYP) enzymes; however, several supportive medications for glioma patients such as corticosteroids and anti-seizure medications can affect these enzymes. Studies in pooled human liver microsomes revealed that ONC201 is not an inducer of seven major drug-metabolizing human CYP enzymes: CYP 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4. However, ONC201 inhibits multiple CYPs IC50 values ranging from 34.9 to 428.6 μM and is substrate of CYP3A4 and, to lesser extent, other CYPs. Given potential metabolism of ONC201 by these enzymes, we conducted metabolic profiling in human hepatocytes that revealed at least 13 metabolites; however, only one was &amp;gt;10% abundance. The major metabolite, termed ONC207, that lacks a benzyl moiety of ONC201 was assayed by LC-MS-MS in plasma samples obtained from 16 ONC201-treated H3 K27M-mutant glioma patients. Peak plasma concentrations of ONC207 relative to ONC201 was 22% (range: 6%-48%) and its abundance was not correlated with ONC201, suggesting that variable exposure to ONC201 may not be associated with variable metabolism. ONC207 does not inhibit dopamine receptor D2 or cancer cell viability in contrast to the parent compound. In summary, ONC201 is an inhibitor and substrate, but not an inducer, of multiple CYP enzymes. The major metabolite of ONC207 appears to be biologically inactive and is unlikely to explain variable ONC201 exposure or its prolonged biological activity that outlives its systemic presence.

Rieske iron-sulfur protein induces FKBP12.6/RyR2 complex remodeling and subsequent pulmonary hypertension through NF-\u03baB/cyclin D1 pathway

Ca2+ signaling in pulmonary arterial smooth muscle cells (PASMCs) plays an important role in pulmonary hypertension (PH). However, the underlying specific ion channel mechanisms remain largely unknown. Here, we report ryanodine receptor (RyR) channel activity and Ca2+ release both are increased, and association of RyR2 by FK506 binding protein 12.6 (FKBP12.6) is decreased in PASMCs from mice with chronic hypoxia (CH)-induced PH. Smooth muscle cell (SMC)-specific RyR2 knockout (KO) or Rieske iron-sulfur protein (RISP) knockdown inhibits the altered Ca2+ signaling, increased nuclear factor (NF)-κB/cyclin D1 activation and cell proliferation, and CH-induced PH in mice. FKBP12.6 KO or FK506 treatment enhances CH-induced PH, while S107 (a specific stabilizer of RyR2/FKBP12.6 complex) produces an opposite effect. In conclusion, CH causes RISP-dependent ROS generation and FKBP12.6/RyR2 dissociation, leading to PH. RISP inhibition, RyR2/FKBP12.6 complex stabilization and Ca2+ release blockade may be potentially beneficial for the treatment of PH.

A negative reciprocal regulatory axis between cyclin D1 and HNF4α modulates cell cycle progression and metabolism in the liver

Hepatocyte nuclear factor 4α (HNF4α) is a master regulator of liver function and a tumor suppressor in hepatocellular carcinoma (HCC). In this study, we explore the reciprocal negative regulation of HNF4α and cyclin D1, a key cell cycle protein in the liver. Transcriptomic analysis of cultured hepatocyte and HCC cells found that cyclin D1 knockdown induced the expression of a large network of HNF4α-regulated genes. Chromatin immunoprecipitation-sequencing (ChIP-seq) demonstrated that cyclin D1 inhibits the binding of HNF4α to thousands of targets in the liver, thereby diminishing the expression of associated genes that regulate diverse metabolic activities. Conversely, acute HNF4α deletion in the liver induces cyclin D1 and hepatocyte cell cycle progression; concurrent cyclin D1 ablation blocked this proliferation, suggesting that HNF4α maintains proliferative quiescence in the liver, at least, in part, via repression of cyclin D1. Acute cyclin D1 deletion in the regenerating liver markedly inhibited hepatocyte proliferation after partial hepatectomy, confirming its pivotal role in cell cycle progression in this in vivo model, and enhanced the expression of HNF4α target proteins. Hepatocyte cyclin D1 gene ablation caused markedly increased postprandial liver glycogen levels (in a HNF4α-dependent fashion), indicating that the cyclin D1-HNF4α axis regulates glucose metabolism in response to feeding. In AML12 hepatocytes, cyclin D1 depletion led to increased glucose uptake, which was negated if HNF4α was depleted simultaneously, and markedly elevated glycogen synthesis. To summarize, mutual repression by cyclin D1 and HNF4α coordinately controls the cell cycle machinery and metabolism in the liver.

STAT3 is a key molecule in the oncogenic behavior of diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is one of the most lethal childhood brain tumors. This tumor is unique because it is detected exclusively in the ventral pons of patients aged between 6 and 7 years, which suggests a developmental nature of its formation. Signal transducer and activator of transcription 3 (STAT3) is a critical molecule for the differentiation of neural stem cells into astrocytes during neurodevelopment. Additionally, STAT3 is associated with oncogenesis and the epithelial-mesenchymal transition (EMT) in various types of tumor. In recent years, several studies have demonstrated the oncogenic role of STAT3 in high-grade gliomas. However, the role of STAT3 in DIPG at the cellular level remains unknown. To assess the possible association between gliogenesis and DIPG, the expression levels of various molecules participating in the differentiation of neural stem cells were compared between normal brain control tissues and DIPG tissues using open public data. All of the screened genes exhibited significantly increased expression in DIPG tissues compared with normal tissues. As STAT3 expression was the most increased, the effect of STAT3 inhibition in a DIPG cell line was assessed via STAT3 short hairpin (sh)RNA transfection and treatment with AG490, a STAT3 inhibitor. Changes in viability, apoptosis, EMT and radiation therapy efficiency were also evaluated. Downregulation of STAT3 resulted in decreased cyclin D1 expression and cell viability, migration and invasion. Additionally, treatment with STAT3 shRNA or AG490 suppressed the EMT phenotype. Finally, when radiation was administered in combination with STAT3 inhibition, the therapeutic efficiency, assessed by cell viability and DNA damage repair, was increased. The present results suggest that STAT3 is a potential therapeutic target in DIPG, especially when combined with radiation therapy.

2066-P: Dyrk1a Antisense Oligonucleotides Conjugated to GLP-1R Agonist Promote Pancreatic Beta-Cell Proliferation

Loss of functional beta cell mass is a major contributor to the pathogenesis of both type 1 and type 2 diabetes. Enhancing proliferation of existing beta cells in the pancreatic islet is one of the promising approaches for restoring functional beta cell mass. Increasing evidence indicates inhibition of dual specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1a) stimulates beta cell proliferation. However, to date DYRK1a small molecule inhibitors have not been therapeutically viable due to their ability to increase proliferation in peripheral tissues. Previously we demonstrated that generation 2.5 antisense oligonucleotides (ASO) conjugated to a glucagon like peptide 1 receptor (GLP-1R) agonist can specifically target the pancreatic beta cell. When compared to control ASO treated isolated islets, islets administered a GLP-1/Dyrk1a ASO conjugate reduces DYRK1a mRNA and protein expression and significantly increases Ki67 mRNA, a cell proliferation marker, in a dose-response manner in vitro. Furthermore, combination treatment of a GLP-1/Dyrk1a ASO conjugate and the small molecule TGF-β inhibitor Ly364947, can synergistically increase Ki67 mRNA expression, cell cycle progression markers cyclin D1, cyclin E2, Cdk1, Cdk4 mRNAs and beta cell markers Pdx1 and Glut2 mRNAs when compared to a DYRK1a conjugated ASO or Ly364947 alone. Administration of a Dyrk1a conjugated ASO to wild type and db/db mice reduced Dyrk1a mRNA and increased Ki67 mRNA expression in islets when compared to a control ASO. Cell cycle markers cyclin D2, cyclin E2 and Cdk1 mRNAs are also significantly upregulated. In conclusion, a GLP-1/Dyrk1a ASO conjugate can specifically inhibit beta cell Dyrk1a mRNA expression and promote beta cell proliferation, suggesting that this may be an important approach for the treatment of type 1 and type 2 diabetes. Disclosure W. Fu: Employee; Self; Ionis Pharmaceuticals, Inc. R. Lee: Employee; Self; Ionis Pharmaceuticals, Inc. E.C. Pirie: Employee; Self; Ionis Pharmaceuticals, Inc. T. Prakash: None. S. Murray: Employee; Self; Ionis Pharmaceuticals. A. Powers: Employee; Self; Ionis Pharmaceuticals, Inc. Stock/Shareholder; Self; Ionis Pharmaceuticals, Inc. A.E. Mullick: None. R.M. Crooke: Employee; Self; Ionis Pharmaceuticals, Inc. Employee; Spouse/Partner; Ionis Pharmaceuticals, Inc.

Macrophage migration inhibitory factor regulates integrin-β1 and cyclin D1 expression via ERK pathway in podocytes

AimsMacrophage migration inhibitory factor (MIF) is found to increase in proliferative glomerulonephritis. MIF binds to the MIF receptor (CD74) that activates MAP kinase (ERK and p38). Integrins and cyclinD1 regulate cell proliferation, differentiation and adhesion. This study evaluates whether MIF can regulate integrin-β1/cyclin D1 expression and cell adhesion of podocytes. Main methodsExpression of integrin-β1 mRNA/protein and cyclin D1 mRNA under stimulation of MIF was evaluated by real-time PCR and Western blotting. MIF receptor (CD74) and MAP kinase under MIF treatment were examined to determine which pathway regulated integrin-β1 and cyclin D1 expression. Cell adhesion was evaluated under MIF treatment and/or anti-integrin-β1 antibody by cell adhesion assay. Key findingsProtein levels of integrin-β1 were up-regulated under MIF treatment in a dosage-dependent manner. CD74 protein levels were not changed after MIF treatment. Integrin-β1 and cyclin D1 mRNA levels were up-regulated after MIF 100 ng/ml treatment. ERK inhibitor U0126 reduced MIF-induced the increase in integrin-β1 mRNA and protein expression following MIF stimulation. However, p38 inhibitor SB 203580 did not inhibit MIF-induced increase in integrin-β1 mRNA and protein expression following MIF stimulation. MIF-induced increase in cyclin D1 mRNA level also was inhibited only by U0126 following MIF stimulation. Podocyte adhesion was increased after MIF treatment, but, anti-integrin-β1 antibody decreased MIF-enhanced podocyte adhesion. SignificanceMIF increases integrin-β1 and cyclin D1 expression through the ERK pathway in podocytes, and the up-regulated expression of integrin-β1 increases podocyte adhesion. These results provide further understanding for the role of MIF in developing proliferative glomerulonephritis.

C-MET-dependent signal transduction mediates retinoblastoma growth by regulating PKM2 nuclear translocation.

Mesenchymal epithelial transition (C-MET) factor overexpression has been found in many types of cancer and has served as an important molecular target for therapeutic intervention. However, the role of C-MET in retinoblastoma remains largely unclear. The present study aimed to investigate the potential role and mechanism of C-MET in Y79 retinoblastoma cells. We found that C-MET was highly expressed in Y79 retinoblastoma cells, and, in addition, the levels of C-MET were positively correlated with cell proliferation and retinoblastoma growth. Inhibition of C-MET suppressed Y79 retinoblastoma cell proliferation and tumour growth. Mechanistically, we showed that HGF-induced C-MET-dependent signal transduction resulted in ERK 1/2 phosphorylation, which subsequently promoted the nuclear translocation of PKM2. Nuclear PKM2 further interacted with histone H3 and contributed to C-MET-dependent cyclin D1 and c-Myc expression and cell proliferation. These findings highlight the role of C-MET in Y79 retinoblastoma cells and reveal a C-MET-dependent signal transduction mechanism. C-MET may be a potential therapeutic target for retinoblastoma. SIGNIFICANCE OF THE STUDY: We demonstrated a new target of retinoblastoma, C-MET. C-MET-dependent signal transduction promotes Y79 retinoblastoma cell proliferation and tumour growth through ERK 1/2/PKM2/histone H3 signalling pathway. C-MET may be a potential target for retinoblastoma therapy.

Differentiation-inducing factor-1 suppresses cyclin D1-induced cell proliferation of MCF-7 breast cancer cells by inhibiting S6K-mediated signal transducer and activator of transcription 3 synthesis.

Differentiation‐inducing factor‐1 (DIF‐1) has been reported to inhibit the proliferation of various mammalian cells by unknown means, although some possible mechanisms of its action have been proposed, including the activation of glycogen synthase kinase‐3 (GSK‐3). Here, we report an alternative mechanism underlying the action of DIF‐1 in human breast cancer cell line MCF‐7, on which the effects of DIF‐1 have not been examined previously. Intragastric administration of DIF‐1 reduced the tumor growth from MCF‐7 cells injected into a mammary fat pad of nude mice, without causing adverse effects. In cultured MCF‐7, DIF‐1 arrested the cell cycle in G0/G1 phase and suppressed cyclin D1 expression, consistent with our previous results obtained in other cell species. However, DIF‐1 did not inhibit the phosphorylation of GSK‐3. Investigating an alternative mechanism for the reduction of cyclin D1, we found that DIF‐1 reduced the protein levels of signal transducer and activator of transcription 3 (STAT3). The STAT3 inhibitor S3I‐201 suppressed cyclin D1 expression and cell proliferation and the overexpression of STAT3 enhanced cyclin D1 expression and accelerated proliferation. Differentiation‐inducing factor‐1 did not reduce STAT3 mRNA or reduce STAT3 protein in the presence of cycloheximide, suggesting that DIF‐1 inhibited STAT3 protein synthesis. Seeking its mechanism, we revealed that DIF‐1 inhibited the activation of 70 kDa and/or 85 kDa ribosomal protein S6 kinase (p70S6K/p85S6K). Inhibition of p70S6K/p85S6K by rapamycin also reduced the expressions of STAT3 and cyclin D1. Therefore, DIF‐1 suppresses MCF‐7 proliferation by inhibiting p70S6K/p85S6K activity and STAT3 protein synthesis followed by reduction of cyclin D1 expression.

Omethoate induces pharyngeal cancer cell proliferation and G1/S cell cycle progression by activation of Akt/GSK-3β/cyclin D1 signaling pathway

Omethoate is a broad category of organophosphorous pesticides (OPs) and has toxic effects on human health under long-term, low-dose exposure. However, the role of omethoate in cancer development remains elusive. The incidence of global head and neck squamous cell carcinomas (HNSCC) has markedly increased in recent years. Thus, we examined whether omethoate induced the proliferation of FaDu cells (a cell line of HNSCC) and if so, what the underlying mechanism was. The study revealed that omethoate induced FaDu cell growth in a dose- and time-dependent manner. Omethoate stimulated FaDu cell proliferation was mainly due to enhancing the G1 to S phase transition by flow cytometry analysis. We also found that omethoate up-regulated cyclin D1, a key gene controlling the G1-S transition. Furthermore, we showed that omethoate was capable of activating the Akt/GSK-3β signaling pathway. Blockage of Akt by siRNA or small molecule inhibitor significantly suppressed omethoate-induced cyclin D1 expression and cell proliferation. Collectively, these findings demonstrated for the first time that omethoate could induce the pharyngeal cancer cell proliferation by activation of the Akt/GSK-3β/cyclin D1 signaling pathway.

Characterization of molecular signatures of supratentorial ependymomas.

Ependymomas show poor correlation between World Health Organization grade and clinical outcome. A subgroup of supratentorial ependymomas are characterized by C11orf95-RELA fusions, presumed to be secondary to chromothripsis of chromosome 11, resulting in constitutive activation of the NF-κB signaling pathway and overexpression of cyclin D1, p65, and L1 cell adhesion molecule (L1CAM). These RELA-fused ependymomas are recognized as a separate, molecularly defined World Health Organization entity and might be associated with poor clinical outcome. In this study, we show that immunohistochemistry for NF-κB signaling components, such as L1CAM, p65, and cyclin D1, can help distinguish RELA-fused from non-RELA-fused supratentorial ependymomas. Furthermore, these three markers can reliably differentiate RELA-fused ependymomas from a variety of histologic mimics. Lastly, we report that RELA-fused ependymomas may be associated with different chromosomal copy number changes and molecular alterations compared to their non-RELA-fused counterparts, providing additional insight into the genetic pathogenesis of these tumors and potential targets for directed therapies.

2,3,7,8-Tetrachlorodibenzo-p-dioxin Promotes Proliferation of Astrocyte Cells via the Akt/STAT3/Cyclin D1 Pathway

ObjectiveThe compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a persistent organic pollutant, is harmful to the nervous system, but its effects on the brain are still unclear. This study aimed to investigate the effects of TCDD on astrocytes proliferation and underlying molecular mechanism. MethodsThe cell proliferation was measured by EdU-based proliferation assay and PI staining by flow cytometry. Protein expression levels were detected by Western blotting. Immunofluorescence, cytoplasmic and nuclear fractions separation were used to assess the distribution of signal transducer and activator of transcription 3 (STAT3). ResultsC6 cells treated with 10 and 50 nmol/L TCDD for 24 h showed significant promotion of the proliferation of. The exposure to TCDD resulted in the upregulation in the expression levels of phosphorylated protein kinase B (p-Akt), phosphorylated STAT3, and cyclin D1 in a dose- and time-dependent manner. The inhibition of Akt expression with LY294002 or STAT3 expression with AG490 abolished the TCDD-induced cyclin D1 upregulation and cell proliferation. Furthermore, LY294002 suppressed the activation of STAT3. Finally, TCDD promoted the translocation of STAT3 from the cytoplasm to the nucleus, and LY294002 treatment blocked this effect. ConclusionTCDD exposure promotes the proliferation of astrocyte cells via the Akt/STAT3/cyclin D1 pathway, leading to astrogliosis.

Indole-3-Carbinol Inhibits Laryngeal Cancer Growth Through Cell Cycle Arrest

The growth of a variety of tumors are inhibited by indole-3-carbinol (I3C) obviously. But, its role in laryngeal cancer is not clear. The goal of this study was to research the probable roles that laryngeal cancer cell apoptosis and proliferation Hep-2 was influenced by I3C. I3C dose-dependently therapy obviously inhibited Hep-2cell proliferation, and, I3C promoted apoptosis and induced cell morphological changes at 100, 200, 300, 400 μM doses. We discovered that I3C shows anticancer effect through various signal pathways after Hep-2 cells I3C therapy. In Hep-2laryngeal cancer cell line, through decreasing cell cycle-related proteins that include cyclin D1, CDK6, CDK4, and pRb, G1 arrest was induced by I3C. Apart from this, BALB/c nude mice constructed tumor-bearing mouse models. BALB/c nude mice were divided into three groups: treated with I3C, untreated control group and pretreated with I3C. After 8 weeks treatment, the untreated control group developed bigger tumors compared to mice treated or pretreated with I3C, and in the tumors such as cyclin D1, CDK6, CDK4 and pRb cell cycle-related proteins were obviously decreased. Further, the study result showed there was no harmful side effect in the heart, liver and kidney of the I3C-treated nude mice. In conclusion, both in vivo and in vitro I3C inhibited proliferation and induced the Hep-2 cells apoptosis, and showed low toxicity to normal cells. By suppressing the expression of cyclin families and CDK, we deduce that I3C can inhibit the Hep-2 cells growth in vitro. On normal organs and tissues, the I3C had no toxic effects and was safe.