Downregulation Of CCND1 Research Articles

Cardiomyocytes derived small extracellular vesicles plays an important role in heart development

Abstract Introduction Neonatal rats have the capacity to regenerate their hearts in response to injury, but this potential is lost after the first week of life. Cardiac maturation lays the foundation for postnatal heart development and disease, yet little is known about the contributions of the microenvironment to cardiomyocyte maturation. Extracellular vesicles (EV) are bilayer-membrane nanoparticles released by all cell types, carrying proteins, lipids, and nucleic acids, which reflect the activation state of parental cells. Secreted small extracellular vesicles (sEV), prominently figure among extracellular signals that regulate cell function. Purpose We aim to determine whether cardiomyocyte (CM) derived sEV carried miRNA that has a role in heart development with specific regards to cardiomyocyte maturation. Methods sEV were isolated from rat cardiomyocyte at day 0 and 7 after birth by serial ultracentrifugation. sEV were characterized by NTA and analyzed by Western blot for the presence of classical EV markers (TSG101, Syntenin-1). The role of sEV in cardiomyocyte proliferation was assessed by analysis of EdU incorporation on neoantal rat CM treated with sEV_p0 or sEV_p7. miRNA content on sEV was assessed using a rat-miRNome MicroRNA Profiling Kit and the identified miRNA's targets confirmed by RealTime-PCR and Western Blot. Results NTA and Western Blot analysis confirmed the presence of sEV in both the extracellular vesicles preparation. sEV_p0 showed to be able to sligtly increase EdU incorporation in treated cardiomyocyte (1.15-Fold) while sEV_p7 significantly inihibit CM proliferation (0.78-Fold) toghether with a change in cardiomyocyte citoscheletal architecture. Data from miRNome analysis showed in sEV_p7 a significan increase in miRNA with cyclines as tagets. Downregulation of Cdk1; Cdk4; Cdk2; CcnB1 and CcnD1 was confirmed on CM trated with sEV_p7 compared to Ctrl. Downregulation of CCND1 was aslo confrmed at the protein level by Western Blot analysis. Conclusions These preliminary resultes showed an important role in heart develompent of cardiomyocyte derived-sEV. A deeper investigation of the pathaway activated by sEV may have a potential interest for the identification of possible regulators for stimulating heart regeneration. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Fondazione Leonardo, Lugano, Switzerland

Abstract P233: TAS0953/HM06 is effective in preclinical models of diverse tumor types driven by RET alterations

Abstract Fusions involving RET receptor tyrosine kinase are a common driver of tumors across different tissue types, such as lung, thyroid, colorectal, soft tissue and others. TAS0953/HM06 (hereby referred to as HM06) is a novel 2ndgeneration RET-specific inhibitor that is effective against RET solvent front (G810) and gatekeeper (V804) mutations. Here, we evaluated the efficacy of HM06 in lung and thyroid carcinomas, and soft-tissue sarcoma cell lines and PDXs derived from RET inhibitor-naive tumor samples or from tumors with acquired resistance to selpercatinib. HM06 was more effective than the RET multi-kinase inhibitors cabozantinib and vandetanib, and as effective as selpercatinib and pralsetinib in inhibiting growth of patient-derived and isogenic lung, thyroid and sarcoma cell lines (IC50=0.02-0.1 µM) harboring different RET fusions (KIF5B-RET, CCDC6-RET, TRIM33-RET, SPECCL1-RET) or activating mutations (RET C634W). Growth of non-tumor cells was up to 80-fold less sensitive to HM06 (IC50= 1.6 µM). Treatment of RET fusion-positive lung cancer cells with HM06 resulted in a dose-dependent inhibition of RET phosphorylation (Y905 and Y1062) and the downstream effectors AKT, ERK1/2, p70S6K and S6. Caspase 3/7 activity and markers of apoptosis (BIM, cleaved PARP) were induced by HM06 to a similar extent as pralsetinib and selpercatinib (dose range: 0.05-1 µM). HM06 induced changes in the core mediators of cell cycle regulation (upregulation of p27, downregulation of CCND1) and suppressed expression of MYC and ETV5. In vivo, HM06 blocked tumor growth and/or induced regression of up to 65% in seven patient-derived xenograft (PDX) models with RET fusions (five NSCLC PDXs, one sarcoma PDX and one NSCLC cell-line xenograft) to a similar extent as pralsetinib and selpercatinib. However, 6 weeks after cessation of treatment of the SPECCL1-RET-driven sarcoma PDX model, growth of tumors treated with HM06 was suppressed completely, whereas 3/5 pralsetinib-treated tumors and 1/5 selpercatinib-treated tumor regrew. Combination of HM06 and the MET inhibitor capmatinib effectively blocked growth of PDX tumors in a model that was derived from a patient sample that expressed RET fusion and METamplification, and was resistant to selpercatinib. These results suggest that HM06 may be an effective therapy for RET-driven tumors in a tissue-type agnostic manner and can effectively address common on-target and off-target resistance mechanisms such as RET G810X and V804X mutations. HM06 is currently in a phase 1 and 2 clinical trial for patients with advanced solid tumors with RET gene abnormalities (margaRET, NCT 04683250). Citation Format: Igor Odintsov, Renate I. Kurth, Kota Ishizawa, Lukas Delasos, Allan J.W. Lui, Inna Khodos, Connor J. Hagen, Qing Chang, Marissa S. Mattar, Morana Vojnic, Siddharth Kunte, Annalisa Bonifacio, Claudio Giuliano, Elisa De Stanchina, Emily Cheng, Emanuela Lovati, Marc Ladanyi, Romel Somwar. TAS0953/HM06 is effective in preclinical models of diverse tumor types driven by RET alterations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P233.

MiRNA-194 predicts favorable prognosis in gastric cancer and inhibits gastric cancer cell growth by targeting CCND1.

MicroRNAs (MiRNAs) play critical roles in regulating target gene expression and multiple cellular processes in human cancer malignant progression. However, the function of miR‐194 in gastric cancer (GC) remains unclear and controversial. In this study, we identified a series of miRNAs that can serve as prognostic biomarkers for GC by analysis of miRNA expression using The Cancer Genome Atlas data. Among them, miR‐100, miR‐125b, miR‐199a, and miR‐194 were the four most promising prognostic biomarkers in GC due to their significant associations with various clinical characteristics of patients. miR‐100, miR‐125b, and miR‐199a predicted poor prognosis in GC, while miR‐194 predicted favorable prognosis in GC. We also provide the first comprehensive transcriptome analysis of miR‐194 in GC. Our data suggest that miR‐194 tends to regulate target genes by binding to their 3′ UTRs in a 7‐mer‐A1, 7‐mer‐m8, or 8‐mer manner. KEGG pathway analysis showed that the cell cycle was one of the pathways most affected by miR‐194 in GC. Moreover, CCND1 was shown to be a novel target gene of miR‐194 in GC. Additionally, downregulation of CCND1 by miR‐194 in GC further led to cell growth inhibition and cell cycle arrest. In conclusion, miR‐100, miR‐125b, miR‐199a, and miR‐194 may have potential as prognostic and diagnostic biomarkers for GC. miR‐194 suppresses GC cell growth mainly through targeting CCND1 and induction of cell cycle arrest.

Zearalenone exposure affects the Wnt/β-catenin signaling pathway and related genes of porcine endometrial epithelial cells in vitro.

ObjectiveZearalenone (ZEA) has estrogen-like effects. Our previous study has shown that ZEA (0.5 to 1.5 mg/kg) could induce abnormal uterine proliferation through transforming growth factor signaling pathway. To further study the other regulatory networks of uterine hypertrophy caused by ZEA, the potential mechanism of ZEA on porcine endometrial epithelial cells (PECs) was explored by the Illumina Hiseq 2000 sequencing system.MethodsThe PECs were treated with ZEA at 0 (ZEA0), 5 (ZEA5), 20 (ZEA20), and 80 (ZEA80) μmol/L for 24 h. The collected cells were subjected to cell cycle, RNA-seq, real-time quantitative polymerase chain reaction, immunofluorescence, and western blot analysis.ResultsThe proportion of cells in the S and G2 phases decreased (p<0.05), but the proportion of cells in the G1 phase increased (p<0.05) in the ZEA80 treatment. Data analysis revealed that the expression of Wnt pathway-related genes, estrogen-related genes, and mitogen-activated protein kinase pathway-related genes increased (p<0.05), but the expression of genetic stability genes decreased (p<0.05) with increasing ZEA concentrations. The relative mRNA and protein expression of WNT1, β-catenin, glycogen synthase kinase 3β (GSK-3β) were increased (p<0.05) with ZEA increasing, while the relative mRNA and protein expression of cyclin D1 (CCND1) was decreased (p<0.05). Moreover, our immunofluorescence results indicate that β-catenin accumulated around the nucleus from the cell membrane and cytoplasm with increasing ZEA concentrations.ConclusionIn summary, ZEA can activate the Wnt/β-catenin signaling pathway by up-regulating WNT1 and β-catenin expression, to promote the proliferation and development of PECs. At the same time, the up-regulation of GSK-3β and down-regulation of CCND1, as well as the mRNA expression of other pathway related genes indicated that other potential effects of ZEA on the uterine development need further study.

NFE2L3 Inhibition Induces Cell Cycle Arrest at the G0/G1 Phase in Colorectal Cancer Cells through Downregulating CCND1 and pRb1-ser807/811.

The molecular mechanism for colorectal cancer to develop remains unelucidated. To find biomarkers related to colorectal cancer development, we analyzed the gene expression profile of 380 colorectal cancer patients and 51 healthy controls by R software. Finally, 1579 upregulated differential expression genes (DEGs) and 3218 downregulated DEGs were identified. Then, the top 20 upregulated DEGs were compared with 181 upregulated DEGs that we reported previously, and 11 overlapped DEGs were found. NFE2L3 (nuclear factor, erythroid 2-like 3) was among those overlapped DEGs and was rarely reported in colorectal cancer. Real-time polymerase chain reaction (PCR) results showed that higher NFE2L3 expression levels were identified in paired tumor samples than in paratumor samples (48 paired samples). Flow cytometry analysis revealed that the cell cycle was arrested at the G0/G1 phase after inhibition of NFE2L3 in both HCT116 and SW480 cell lines. Western blot detection showed that CCND1 and phosphorylated Rb transcriptional corepressor 1 at ser-807/811 (pRb1-ser807/811) expression levels were downregulated when NFE2L3 was inhibited in those two cell lines. A significant positive correlation was observed between NFE2L3 and CCND1 expression levels in colorectal tissue samples. These evidences indicate that downregulation of NFE2L3 induces cell cycle arrest at the G0/G1 phase through downregulation of CCND1 and pRb1-ser807/811.

Inhibition of GPR158 by microRNA-449a suppresses neural lineage of glioma stem/progenitor cells and correlates with higher glioma grades

To identify biomarkers for glioma growth, invasion and progression, we used a candidate gene approach in mouse models with two complementary brain tumour phenotypes, developing either slow-growing, diffusely infiltrating gliomas or highly proliferative, non-invasive primitive neural tumours. In a microRNA screen we first identified microRNA-449a as most significantly differentially expressed between these two tumour types. miR-449a has a target dependent effect, inhibiting cell growth and migration by downregulation of CCND1 and suppressing neural phenotypes by inhibition of G protein coupled-receptor (GPR) 158. GPR158 promotes glioma stem cell differentiation and induces apoptosis and is highest expressed in the cerebral cortex and in oligodendrogliomas, lower in IDH mutant astrocytomas and lowest in the most malignant form of glioma, IDH wild-type glioblastoma. The correlation of GPR158 expression with molecular subtypes, patient survival and therapy response suggests a possible role of GPR158 as prognostic biomarker in human gliomas.

Abstract GS6-05: Gain-of-function kinase library screen identifies FGFR1 amplification as a mechanism of resistance to antiestrogens and CDK4/6 inhibitors in ER+ breast cancer

Abstract Background: CDK4/6 inhibitors have been approved in combination with endocrine therapy for treatment of ER+ metastatic breast cancer. The goal of this study was to discover mechanisms of resistance to ER antagonists alone and in combination with CDK4/6 inhibitors. Results: To achieve this goal, we used lentiviral vectors to individually express 559 human kinase open reading frames (ORFs) in ER+ MCF7 human breast cancer cells treated with fulvestrant ± the CDK4/6 inhibitor ribociclib. In MCF7 cells treated with fulvestrant alone or with ribociclib, we identified 15 and 17 kinases associated with resistance, respectively. Ten of these kinases overlapped in both groups. In a secondary screen, MCF7 cells were stably transduced with V5-tagged lentiviruses expressing the positive 'hits' for treatment with fulvestrant/ribociclib. Five of 17 kinases (FGFR1, FRK, HCK, FGR, CRKL) were confirmed to induce resistance to fulvestrant/palbociclib and fulvestrant/ribociblib. Survey of TCGA for copy number alterations and/or expression of these 5 genes showed only FGFR1 to be amplified/overexpressed in ˜15% of ER+ breast cancers. Experiments in vitro showed that ER+/FGFR1-amplified (amp) MDA-134, CAMA-1 and HCC1500 human breast cancer cells and MCF7 cells stably transduced with FGFR1 were relatively resistant to estrogen deprivation, fulvestrant and fulvestrant/palbociclib compared to non-FGFR1 amp MCF7 cells. This resistance was abrogated by treatment with the FGFR tyrosine kinase inhibitor (TKI) lucitanib. Treatment with fulvestrant or palbociclib alone modestly delayed growth of ER+/FGFR1-amp breast cancer patient-derived xenografts (PDX) established in nude mice. However, addition of the FGFR TKI erdafitinib to fulvestrant/palbociclib resulted in marked PDX regression in all mice without associated toxicity and a complete cell cycle arrest measured by Ki67. Treatment of FGFR-amp cells with FGF-2 strongly induced CCND1 (cyclin D1) expression. Downregulation of CCND1 with CCND1 RNAi oligonucleotides restored sensitivity of FGFR1-amp cells to fulvestrant/palbociclib, thus phenocopying the effect of FGFR TKIs. Conversely, overexpression of CCND1 in MCF7 cells induced resistance to estrogen deprivation and to fulvestrant ± palbociclib. Finally, we examined next gen sequencing of cell free tumor DNA by Guardant360 in 34 patients before and after progression on CDK4/6 inhibitor. In 10/34 (29%) post-progression specimens, we detected alterations in the FGFR pathway: FGFR1 amplification (n=7), FGFR1 N546K (n=1), FGFR2 N549K (n=1), and FGFR2 V395D (n=1) activating mutations. Conclusions: These data suggest aberrant FGFR signaling is a mechanism of resistance to anti-ER therapies ± CDK4/6 inhibitors. We posit overexpression of cyclin D1 induced by both FGFR signaling and ER transcription plays a role in drug resistance. Based on these findings we propose ER+/FGFR1 amplified breast cancers are endocrine resistant and should be candidates for treatment with combinations of ER and FGFR antagonists. Accordingly, we have initiated a phase Ib trial of fulvestrant, palbociclib and erdafitinib in patients with antiestrogen resistant ER+/HER2-negative breast cancer with FGFR1-4 amplification. Citation Format: Formisano L, Lu Y, Jansen VM, Bauer JA, Hanker A, Gonzalez Ericsson P, Lee K-M, Nixon MJ, Guerrero-Zotano AL, Schwarz LJ, Sanders M, Sudhan D, Dugger TC, Cruz MR, Behdad A, Cristofanilli M, Bardia A, O'Shaughnessy J, Mayer IA, Arteaga CL. Gain-of-function kinase library screen identifies FGFR1 amplification as a mechanism of resistance to antiestrogens and CDK4/6 inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS6-05.

Sulforaphane Inhibits Liver Cancer Cell Growth and Angiogenesis

Sulforaphane (SFN) exhibits inhibitory effects in different types of cancers. However, its inhibitory effect on liver cancer remains unknown. This study aimed to determine the therapeutic potential of SFN for the treatment of liver cancer and explore the functional mechanisms underlying the inhibitory effects of SFN. Water-Soluble Tetrazolium salt (WST-1) assay was performed to assess the in vitro effect of SFN on cell proliferation in the human liver cancer cell lines, HepG2 and Huh-7. The mRNA levels of Nrf2 target genes and cell cycle-related genes were determined using quantitative RT-PCR. For assessing the inhibitory effect of SFN in vivo, we injected immortalized liver cancer cells into BALB/c nude mice as a xenograft model. SFN was orally administrated daily after tumor inoculation and continued for thirty-five days until their sacrifices. Nrf2 activation, induced by SFN, was confirmed by mRNA upregulation of HO-1, MRP2, and NQO1 in both the cell lines. Significant inhibition of liver cancer cell proliferation by SFN was shown in vitro in a dose-dependent manner by the downregulation of CCND1, CCNB1, CDK1 and CDK2. In in vivo studies, the administration of SFN significantly reduced the subcutaneous tumor burdens at the end of experiments by suppressing tumor cell proliferation, confirmed by Ki67 immunohistochemical analysis. The mRNA levels of CCND1, CCNB1, CDK1 and CDK2 were also decreased in these SFNtreated xenograft tumors. Moreover, CD34 immunostaining elucidated that the intratumoral neovascularization was markedly attenuated in the SFN-treated xenograft tumors. SFN exerts inhibitory effect on human liver cancer cells with antiangiogenic activity. The earlier version of this study was presented at the meeting of AASLD Liver Learning on Oct 2017.

Abstract 1008: Gain-of-function kinase library screen identifies FGFR1 amplification as a mechanism of resistance to antiestrogens and CDK4/6 inhibitors in ER+ breast cancer

Abstract The CDK4/6 inhibitor palbociclib was recently approved in combination with endocrine therapy for treatment of ER+ metastatic breast cancer. The goal of this study was to discover mechanisms of resistance to ER antagonists alone and in combination with CDK4/6 inhibitors. To achieve this goal, we used lentiviral vectors to individually express 559 human kinase open reading frames (ORFs) in ER+ MCF7 human breast cancer cells treated with fulvestrant ± the CDK4/6 inhibitor ribociclib (Novartis). In MCF7 cells treated with fulvestrant alone or with ribociclib, we identified 21 and 17 kinases, respectively, which induced a &amp;gt;30% increase in cell viability compared to control cells; 11 of these kinases overlapped in both treatment groups. In a secondary screen, MCF7 cells were stably transduced with V5-tagged lentiviruses expressing the positive ‘hits’ for treatment with fulvestrant/ribociclib. Five of 11 kinases (FGFR1, FRK, HCK, FGR, CRKL) were confirmed to induce resistance to fulvestrant/palbociclib and fulvestrant/ribociblib. Survey of TCGA for copy number alterations and/or expression of these five genes showed only FGFR1 to be amplified/overexpressed in 17% of ER+ breast cancers. Experiments in vitro showed that ER+/FGFR1-amplified (amp) MDA-134, CAMA-1 and HCC1500 human breast cancer cells and MCF7 cells stably transduced with FGFR1 were relatively resistant to estrogen deprivation, fulvestrant and fulvestrant/palbociclib compared to non-FGFR1 amp MCF7 cells. This resistance was abrogated by treatment with the FGFR tyrosine kinase inhibitor (TKI) lucitanib. Treatment with fulvestrant or palbociclib, each alone, modestly delayed growth of ER+/FGFR1-amp breast cancer patient-derived xenografts (PDX) established in nude mice. However, addition of the FGFR TKI erdafitinib to fulvestrant/palbociclib resulted in marked PDX regressions in all mice without associated toxicity. Treatment of FGFR-amp cells with FGF-2 strongly induced CCND1 (cyclin D1) expression. Downregulation of CCND1 with CCND1 RNAi oligonucleotides or kinase inhibition with erdafitinib restored sensitivity of FGFR1-amp cells to fulvestrant/palbociclib. Conversely, overexpression of CCND1 in MCF7 cells induced resistance to estrogen deprivation and fulvestrant ± palbociclib. At this time, we are examining whether FGFR1 amplification measured by FISH correlates with maintenance of proliferation (Ki67) in 110 patients with ER+/HER2- breast cancer treated with palbociclib for 14 days immediately before surgery (Arnedos et al. ASCO 2016). In summary, using a gain-of-function ORF kinome screen, we identified FGFR1 amplification as a mechanism of resistance to anti-ER therapies ± CDK4/6 inhibitors. Based on these data we propose FGFR inhibitors should be tested in combination with ER antagonists and CDK4/6 inhibitors in patients with ER+/FGFR amplified breast cancer. Citation Format: Luigi Formisano, Yao Lu, Valerie M. Jansen, Joshua A. Bauer, Ariella B. Hanker, Melinda E. Sanders, Paula González-Ericsson, Sunkyu Kim, Monica Arnedos, Fabrice André, Carlos L. Arteaga. Gain-of-function kinase library screen identifies FGFR1 amplification as a mechanism of resistance to antiestrogens and CDK4/6 inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1008. doi:10.1158/1538-7445.AM2017-1008

Hsa-miR-134 suppresses non-small cell lung cancer (NSCLC) development through down-regulation of CCND1.

Hsa-miRNA-134 (miR-134) has recently been discovered to have anticancer efficacy in different organs. However, the role of miR-134 on non-small cell lung cancer (NSCLC) is still ambiguous. In this study, we investigated the role of miR-134 on the development of NSCLC. The results indicated that miR-134 was significantly down-regulated in primary tumor tissues and very low levels were found in NSCLC cell lines. Ectopic expression of miR-134 in NSCLC cell lines significantly suppressed cell growth as evidenced by cell viability assay, colony formation assay and BrdU staining, through inhibition of cyclin D1, cyclin D2, CDK4 and up-regulation of p57(Kip2) and p21(Waf1/Cip1). In addition, miR-134 induced apoptosis, as indicated by concomitantly with up-regulation of key apoptosis protein cleaved caspase-3, and down-regulation of anti-apoptosis protein Bcl2. Moreover, miR-134 inhibited cellular migration and invasiveness through inhibition of matrix metalloproteinases (MMP)-7 and MMP-9. Further, oncogene CCND1 was revealed to be a putative target of miR-134, which was inversely correlated with miR-134 expression in NSCLC. Taken together, our results demonstrated that miR-134 played a pivotal role on NSCLC through inhibiting cell proliferation, migration, invasion, and promoting apoptosis by targeting oncogenic CCND1.

Abstract B16: NR5A2 as a potential therapeutic target for hepatoblastoma

Abstract Hepatoblastoma (HB) is the most common malignant liver tumor in very young children. Because of multidrug resistance and severe side effects of anti-HB drugs during HB chemotherapy, it is urgent and important to develop new effective therapeutic agents with less toxicity for the treatment of pediatric patients with liver cancer. Nuclear Receptor Subfamily 5 Group A Member 2 (NR5A2; LRH-1) has been linked to various types of malignancies, and it is an emerging therapeutic target for many cancers. However the role of NR5A2 in liver cancer remains elusive. In this study, we found that both mRNA and protein expression levels of NR5A2 were significantly higher in HepG2 and HuH6 human HB cell lines and mouse orthotopic HB liver than the normal liver cell and normal liver tissue. Lentiviral-Mediated Short Hairpin RNA (shRNA) knockdown of NR5A2 inhibited HepG2 and HuH6 cell proliferation and colony formation compared with control cells. Furthermore, we found that silencing of NR5A2 induced downregulation of cell cycle gene CCND1 and the regulator gene c-myc. Similar to the effects of shRNA, we found the NR5A2 antagonist treatment also inhibited the proliferation of human HB cell lines (HepG2, HuH6 and HepT1 cells), reduced colony formation and CCND1 and c-Myc expression. Finally, flow cytometry results showed that the NR5A2 antagonist treatment induced a decreased percentage of cells in S phase which resulted in a significant decrease in the rate of cell proliferation and tumorigenicity, and induced an increased number of cells in the G2/M phase, leading to HB cell cycle arrest at G2/M phase. Collectively, our results suggest that NR5A2 play an important role in HB cancer cell proliferation. Interference of NR5A2 by shRNA or the NR5A2 antagonist inhibits HB cell proliferation and colony formation by inhibiting the cell cycle process through downregulation of CCND1 and c-Myc expression. By targeting NR5A2 signaling pathway, NR5A2 antagonist has promising therapeutic potential in the treatment of HB cancer. Note: This abstract was not presented at the conference. Citation Format: Jingling Jin, Roma Patel, Nan Ge Jin, Yan Shi, Wenjing Sun, Jianhua Yang, Jed G. Nuchtern, Sanjeev A. Vasudevan. NR5A2 as a potential therapeutic target for hepatoblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B16.

Reduced BCL2 and CCND1 mRNA expression in human cervical cancer HeLa cells treated with a combination of everolimus and paclitaxel

Aim of the studyCervical cancer is the second most common malignancy in women worldwide. Everolimus displays direct effects on growth and proliferation of cancer cells via inhibition of mammalian target of rapamycin (mTOR) protein, which is known to be associated with drug resistance. In this study, we aimed to investigate the effects of everolimus, gemcitabine, and paclitaxel in terms of cell viability and mRNA expression levels of GRP78, CCND1, CASP2, and BCL2 genes.Material and methodsHeLa cells were treated with different doses of everolimus, gemcitabine, and paclitaxel. Cell viability was assessed using MTT assay, and obtained dose response curves were used for the calculations of inhibitory concentration (IC) values. At the end of the treatment times with selected doses, RNA isolation and cDNA synthesis were performed. Finally, GRP78, CCND1, CASP2, and BCL2 genes mRNA expression levels were analysed using quantitative PCR.ResultsThe IC50 value of everolimus was 0.9 µM for 24-hour treatment. Moreover, the IC50 value of gemcitabine and paclitaxel was found to be around 18.1 µM and 7.08 µM, respectively. Everolimus, gemcitabine, and paclitaxel treatments alone did not change the GRP78, CCND1, BCL2 and CASP2 mRNA expression levels significantly. However, combined treatment of everolimus and paclitaxel significantly reduced BCL2 and CCND1 mRNA expression (p < 0.05). In contrast, this combination did not change GRP78 and CASP2 mRNA expression levels (p > 0.05).ConclusionsDown-regulation of CCND1 and BCL2 expression may be an important mechanism by which everolimus increases the therapeutic window of paclitaxel in cervical cancers.

The Wnt Pathway Target Gene &amp;lt;i&amp;gt;CCND1&amp;lt;/i&amp;gt; Changes Mitochondrial Localization and Decreases Mitochondrial Activity in Colorectal Cancer Cell Line SW480

Mutations leading to constitutive activation of the Wnt pathway and its target genes are frequently observed in cancer. The Wnt pathway promotes cell proliferation and increasing evidence supports its role also in cancer cell metabolism. This study aims to elucidate the role of the Wnt/β-catenin target gene CCND1 in these processes in colorectal cancer. We analyzed whether knock-down of CCND1 affects cell cycle progression and energy metabolism in a colorectal cancer cell line. Down-regulation of CCND1 led to retardation of the cell cycle. The proportion of cells in the G0 phase increased, while the amount of cells in the S- and G2/M phase decreased. Interestingly, knock-down of CCND1 changed the perinuclear localization of mitochondria into a homogeneous distribution within the cytosol. In addition CCND1 knock-down led to an increase of the intracellular ATP level indicating that cyclin D1 reduced mitochondrial activity. Our findings suggest that in addition to its role in cell cycle regulation, the Wnt target gene CCND1 regulates mitochondrial localization and inhibits mitochondrial activity in colorectal cancer cells.

Promoting roles of the secreted frizzled-related protein 2 as a Wnt agonist in lung cancer cells.

The secreted frizzled-related protein 2 (SFRP2) plays a pivotal role in the Wnt pathway, however, it functions as an agonist or an antagonist is still controversial. We profiled SFRP2 expression in several lung cancer cell lines, and found that A549 and 95-D exhibited the lowest and the highest level of SFRP2, respectively. Then we employed the SFRP2-overexpressing plasmid and siRNA to transfect A549 and 95-D cells, respectively. Through MTT assays, we found that SFRP2 knockdown inhibited cell proliferation, and halted the 95-D cells in G1 phase of the cell cycle by downregulation of CCND1 and CDK4, indicating that SFRP2 has the ability of promoting lung cancer cell proliferation. We further checked the cell properties of migration and invasion, using wound scratch assay and Transwell assays. The data showed decreased migrated and invasive 95-D cells after SFRP2 knockdown, and the observations were the opposite in the overexpressing model, implying that SFRP2 promoted lung cancer cell invasion. Moreover, the canonical Wnt pathway was also studied through detection of β-catenin by western blotting. In the SFRP2 overexpressing model, A549 cells presented stronger expression of β-catenin compared with controls, while it was the opposite in 95-D cells. These results suggested that SFRP2 serves as a Wnt agonist in lung cancer cells. Together, the findings of this study implied that SFRP2 is not only an agonist of Wnt pathway, but also a cancer promoting protein for lung cancer, indicating SFRP2 as a promising therapeutic target for lung cancer treatment.

Abstract AS25: An adeno-associated virus (AAV) 9 vector delivering modified human mullerian inhibiting substance (MIS) as a gene therapy for ovarian cancer

Abstract Purpose: Ovarian cancer is the most lethal malignancy of the female reproductive tract; as such, effective treatments are needed to improve patient survival. MIS has previously been shown to inhibit growth of a stem-like subset of the total cell population in ovarian cancer cell lines (Wei et al, 2010; Meirelles et al, 2012). We have recently engineered novel peptide modifications to human MIS (LR-MIS), which increase production and potency in vitro and in vivo (Pepin et al, 2013), and inserted it into an AAV vector. AAV delivered gene therapy has undergone a clinical resurgence with a good safety profile and sustained gene expression (Zhang et al, 2011). Therefore, we assessed the efficacy of a single intraperitoneal (ip) dose of AAV9-LR-MIS to inhibit tumor growth of an established cell line for subsequent evaluation in patient-derived ovarian cancer xenografts (PDX). Methods: 6 week old female nude mice were treated with a single ip injection of 3e11 or 1e12 virions of AAV9 LR-MIS or GFP. Serum levels of human MIS were monitored weekly by ELISA. After serum steady state was achieved, the established ovarian cancer cell line, OVCAR5 was xenografted subcutaneously at 1e6 cells per animal. Tumors treated with AAV were measured twice weekly. Additionally, Patient ascites were processed to isolate the cancer cell population, which was specifically amplified in vitro to create low passage primary cell lines. Results: AAV9-LR-MIS treatment resulted in elevated and sustained blood concentrations of MIS (Fig1A) in nude mice without any signs of toxicity, and significantly inhibited OVCAR5 tumor growth (p=0.0003) (Fig1B) in two separate experiments. AAV9-GFP revealed that the pancreas, omentum, gut mesentery, and body wall muscles were the primary sites of infection. Treatment with MIS induced downregulation of CCND1 in tumors. A panel of 15 primary ovarian cancer cell lines, all expressing the MIS type II receptor were generated from patient ascites and cells are being tested for their ability to grow as PDX in Nude, NOD/SCID, and NSG mice. Figure 1.AAV-LR-MIS treatment results in stable expression of MIS and inhibition of tumor growth of OVCAR5 xenografts. A) Weekly MIS ELISA of serum from mice after single doses of 1e10 to 1e12 virions. B) Tumor growth of 1e6 OVCAR5 cells xenografted into nude mice (N=5) pretreated with 3e11/mouse (1.5e13/kg) LR-MIS or GFP virus (p=0.0003 by two-way ANOVA).Figure 1. AAV-LR-MIS treatment results in stable expression of MIS and inhibition of tumor growth of OVCAR5 xenografts. A) Weekly MIS ELISA of serum from mice after single doses of 1e10 to 1e12 virions. B) Tumor growth of 1e6 OVCAR5 cells xenografted into nude mice (N=5) pretreated with 3e11/mouse (1.5e13/kg) LR-MIS or GFP virus (p=0.0003 by two-way ANOVA). Conclusions: AAV9 virally delivered human LR-MIS can produce adequate sustained levels of MIS that inhibited the growth of the human ovarian cancer cell line, OVCAR 5. AAV9 LR MIS provides a well-tolerated, low toxicity treatment of ovarian cancer, which can now be evaluated in a phase 0 trial using PDX models being prepared from human ovarian cancer ascites. Citation Format: David Pepin, Amanda Sosulski, Katherine Hendren, Li Hua Zhang, Fotini Nicolaou, Dan Wang, Guangping Gao, Patricia K Donahoe. An adeno-associated virus (AAV) 9 vector delivering modified human mullerian inhibiting substance (MIS) as a gene therapy for ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS25.

Cornelia de Lange Syndrome: NIPBL haploinsufficiency downregulates canonical Wnt pathway in zebrafish embryos and patients fibroblasts

Cornelia de Lange Syndrome is a severe genetic disorder characterized by malformations affecting multiple systems, with a common feature of severe mental retardation. Genetic variants within four genes (NIPBL (Nipped-B-like), SMC1A, SMC3, and HDAC8) are believed to be responsible for the majority of cases; all these genes encode proteins that are part of the ‘cohesin complex'. Cohesins exhibit two temporally separated major roles in cells: one controlling the cell cycle and the other involved in regulating the gene expression. The present study focuses on the role of the zebrafish nipblb paralog during neural development, examining its expression in the central nervous system, and analyzing the consequences of nipblb loss of function. Neural development was impaired by the knockdown of nipblb in zebrafish. nipblb-loss-of-function embryos presented with increased apoptosis in the developing neural tissues, downregulation of canonical Wnt pathway genes, and subsequent decreased Cyclin D1 (Ccnd1) levels. Importantly, the same pattern of canonical WNT pathway and CCND1 downregulation was observed in NIPBL-mutated patient-specific fibroblasts. Finally, chemical activation of the pathway in nipblb-loss-of-function embryos rescued the adverse phenotype and restored the physiological levels of cell death.

MiR-449b inhibits the proliferation of SW1116 colon cancer stem cells through downregulation of CCND1 and E2F3 expression

Colorectal cancer is one of the leading causes of cancer-related mortality worldwide. Cancer stem cells are cell populations with stem cell nature presenting in tumor tissues and are the root of tumor formation and metastasis. CCND1 and E2F3 play important roles in cell cycle regulation. The 3'UTRs of CCND1 and E2F3 contain miR-449 binding sites. By transfecting pre-miR-449b and inhibiting miR-449b, we found that cell cycle, cell proliferation ability and cell cycle regulatory protein expression levels of colon cancer stem cells were altered. The correlation between CCND1, E2F3 and miR-449b showed that miR-449b could downregulate CCND1 and E2F3 expression. This, in turn, reduced the proliferative ability of colon cancer stem cells. These data suggest that miR-449b plays a tumor-suppressive role in colon cancer stem cells.

Inverse Correlation of miRNA and Cell Cycle-Associated Genes Suggests Influence of miRNA on Benign Thyroid Nodule Tumorigenesis

The molecular etiology of cold and benign thyroid nodules (CBTNs) is largely unknown. Increased thyroid epithelial cell proliferation is a hallmark of CBTNs. MicroRNAs (miRNAs) are prominent regulators of cell proliferation. Our objective was to assess the influence of miRNAs on the increased proliferation and thus the molecular etiology of CBTNs. By using microarrays, we defined the molecular pattern of increased proliferation of CBTNs as a differential expression of cell-cycle-associated genes and miRNAs. In silico integration of differentially expressed miRNAs and mRNAs showed an inverse correlation between the expression of 59 miRNAs and 133 mRNAs. Inverse correlations between cell-cycle-associated genes such as CDKN1C and miR-221, CCND1 and miR-31, GADD45A and miR-130b, or CDKN1A and let-7f suggest a modulation of proliferation in CBTNs by miRNAs. Their expression was validated using quantitative RT-PCR and functionally characterized in cell line models. Comparative quantitative RT-PCR of 20 samples of CBTNs and their surrounding tissue revealed an 11-fold down-regulation of miR-31 with a 2.6-fold up-regulation of CCND1, and a 2.6-fold up-regulation of miR-130b with a 2.3-fold down-regulation of its target GADD45A. Using HTori and FTC-133 cell lines, we analyzed proliferation, cell cycle, and apoptosis after transfection of miRNA-31 and miRNA-130b mimic and inhibitors. Overexpression of miR-31 and the resultant down-regulation of CCND1 led to an arrest in the cell cycle phase G1. Overexpression of miR-130b led to an increase of apoptosis and necrosis within 72 h. miR-31 and miR-130b may have an effect on tumorigenesis of CBTNs by regulating proliferation and apoptosis and the cell cycle through cyclin D1.

Inhibition of Fatty-acid Synthase Suppresses P-AKT and Induces Apoptosis in Bladder Cancer

To investigate the role of fatty acid synthase (FASN) in bladder transitional cell carcinoma (BTCC). FASN expression was investigated in non-muscle-invasive BTCC tissue specimens by immunohistochemistry and BTCC cell lines by Western blot. After treatment with FASN-siRNA or FASN inhibitor cerulenin (Cer), the proliferation and apoptosis of BTCC cell lines 5637 and 253 J were determined by cell counting Kit-8 (CCK8) assay and flow cytometry respectively. The expression of p-AKT, cyclin D1 (CCND1), and apoptosis-related proteins were detected by Western blot. High levels of FASN expression were observed in 59% (32/54) of non-muscle-invasive BTCC tissue specimens, and FASN expression was associated with histologic grade (P < .05) and recurrence (P < .05). FASN expression was high in 6 BTCC cell lines. FASN inhibitor Cer and FASN-siRNA produced the increased apoptosis and decreased proliferation of bladder cancer cells, and caused inactivity of AKT and downregulation of CCND1. Furthermore, treatment of BTCC cell lines with Cer resulted in apoptosis via the caspase-dependent pathway involving inactivation of antiapoptotic bcl-2 protein. Our data suggest that FASN plays an important role in BTCC development. Targeting FASN may be a new therapeutic strategy for BTCC.

Differential roles of cyclin D1 and D3 in pancreatic ductal adenocarcinoma

BackgroundThe cyclin D1 (CCND1) and cyclin D3 (CCND3) are frequently co-overexpressed in pancreatic ductal adenocarcinoma (PDAC). Here we examine their differential roles in PDAC.ResultsCCND1 and CCND3 expression were selectively suppressed by shRNA in PDAC cell lines with expression levels of equal CCND1 and CCND3 (BxPC3), enhanced CCND1 (HPAC) or enhanced CCND3 (PANC1). Suppression of cell proliferation was greater with CCND3 than CCND1 downregulation. CCND3 suppression led to a reduced level of phosphorylated retinoblastoma protein (Ser795p-Rb/p110) and resulted in decreased levels of cyclin A mRNA and protein. A global gene expression analysis identified deregulated genes in D1- or D3-cyclin siRNA-treated PANC1 cells. The downregulated gene targets in CCND3 suppressed cells were significantly enriched in cell cycle associated processes (p < 0.005). In contrast, focal adhesion/actin cytoskeleton, MAPK and NF B signaling appeared to characterize the target genes and their interacting proteins in CCND1 suppressed PANC1 cells.ConclusionsOur results suggest that CCND3 is the primary driver of the cell cycle, in cooperation with CCND1 that integrates extracellular mitogenic signaling. We also present evidence that CCND1 plays a role in tumor cell migration. The results provide novel insights for common and differential targets of CCND1 and CCND3 overexpression during pancreatic duct cell carcinogenesis.