Transient Silencing Research Articles

Expression and genotype-dependent catalytic activity of N-acetyltransferase 2 (NAT2) in human peripheral blood mononuclear cells and its modulation by Sirtuin 1

N-acetyltransferase 2 (NAT2) catalyzes the biotransformation of numerous arylamine and hydrazine drugs and carcinogens. Genetic polymorphisms of NAT2 modify drug efficacy and toxicity and susceptibility to diseases such as cancer and type 2 diabetes. Expression of NAT2 has been documented in the liver and gastrointestinal tract but not in other tissues. Deacetylation of cytosolic proteins by sirtuins is a post-translational modification important in regulatory networks of diverse cellular processes. The aim of the present study was to investigate NAT2 expression in peripheral blood mononuclear cells (PBMC) and the effects of NAT2 genotype and Sirtuin 1 (SIRT1). Both NAT2 and SIRT1 proteins were expressed on PBMC. Their expression was more prevalent on CD3+ compared to CD19+ and CD56+ cell populations. N-acetylation capacity of PBMC exhibited a NAT2 gene-dose response toward the N-acetylation of isoniazid. Subjects with rapid NAT2 genotype showed an apparent Vmax of 42.1 ± 2.4; intermediate NAT2 genotypes an apparent Vmax of 22.6 ± 2.2; and slow acetylator NAT2 genotypes an apparent Vmax of 19.9 ± 1.7 nM acetyl-isoniazid/24 h/million cells. The N-acetylation capacity of NAT2 in the presence of SIRT1 enhancer was significantly decreased (p < 0.001), conversely, the transient silencing of SIRT1 resulted in an increase of N-acetylation capacity (p < 0.001). These findings are the first report of NAT2 genotype-dependent expression on PBMC and post-translational modification by SIRT1. These findings constitute a substantial advance in our understanding of human N-acetyltransferase expression and a new much less invasive method for measurement of human NAT2 expression and phenotype.

Evolutionarily conserved partial gene duplication in the Triticeae tribe of grasses confers pathogen resistance

BackgroundThe large and highly repetitive genomes of the cultivated species Hordeum vulgare (barley), Triticum aestivum (wheat), and Secale cereale (rye) belonging to the Triticeae tribe of grasses appear to be particularly rich in gene-like sequences including partial duplicates. Most of them have been classified as putative pseudogenes. In this study we employ transient and stable gene silencing- and over-expression systems in barley to study the function of HvARM1 (for H. vulgare Armadillo 1), a partial gene duplicate of the U-box/armadillo-repeat E3 ligase HvPUB15 (for H. vulgare Plant U-Box 15).ResultsThe partial ARM1 gene is derived from a gene-duplication event in a common ancestor of the Triticeae and contributes to quantitative host as well as nonhost resistance to the biotrophic powdery mildew fungus Blumeria graminis. In barley, allelic variants of HvARM1 but not of HvPUB15 are significantly associated with levels of powdery mildew infection. Both HvPUB15 and HvARM1 proteins interact in yeast and plant cells with the susceptibility-related, plastid-localized barley homologs of THF1 (for Thylakoid formation 1) and of ClpS1 (for Clp-protease adaptor S1) of Arabidopsis thaliana. A genome-wide scan for partial gene duplicates reveals further events in barley resulting in stress-regulated, potentially neo-functionalized, genes.ConclusionThe results suggest neo-functionalization of the partial gene copy HvARM1 increases resistance against powdery mildew infection. It further links plastid function with susceptibility to biotrophic pathogen attack. These findings shed new light on a novel mechanism to employ partial duplication of protein-protein interaction domains to facilitate the expansion of immune signaling networks.

Transient Magnetothermal Neuronal Silencing Using the Chloride Channel Anoctamin 1 (TMEM16A).

Determining the role and necessity of specific neurons in a network calls for precisely timed, reversible removal of these neurons from the circuit via remotely triggered transient silencing. Previously, we have shown that alternating magnetic field mediated heating of magnetic nanoparticles, bound to neurons, expressing temperature-sensitive cation channels TRPV1 remotely activates these neurons, evoking behavioral responses in mice. Here, we demonstrate how to apply magnetic nanoparticle heating to silence target neurons. Rat hippocampal neuronal cultures were transfected to express the temperature gated chloride channel, anoctamin 1 (TMEM16A). Spontaneous firing was suppressed within seconds of alternating magnetic field application to anoctamin 1 (TMEM16A) channel expressing, magnetic nanoparticle decorated neurons. Five seconds of magnetic field application leads to 12 s of silencing, with a latency of 2 s and an average suppression ratio of more than 80%. Immediately following the silencing period spontaneous activity resumed. The method provides a promising avenue for tether free, remote, transient neuronal silencing in vivo for both scientific and therapeutic applications.

P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes.

Purinergic receptor-7 (P2X7R) signaling controls Th17 and Th1 generation/differentiation, while NOD-like receptor P3 (NLRP3) acts as a Th2 transcriptional factor. Here, we demonstrated the existence of a P2X7R/NLRP3 pathway in T cells that is dysregulated by a P2X7R intracellular region loss-of-function mutation, leading to NLRP3 displacement and to excessive Th17 generation due to abrogation of the NLRP3-mediated Th2 program. This ultimately resulted in poor outcomes in cardiac-transplanted patients carrying the mutant allele, who showed abnormal Th17 generation. Transient NLRP3 silencing in nonmutant T cells or overexpression in mutant T cells normalized the Th profile. Interestingly, IL-17 blockade reduced Th17 skewing of human T cells in vitro and abrogated the severe allograft vasculopathy and abnormal Th17 generation observed in preclinical models in which P2X7R was genetically deleted. This P2X7R intracellular region mutation thus impaired the modulatory effects of P2X7R on NLRP3 expression and function in T cells and led to NLRP3 dysregulation and Th17 skewing, delineating a high-risk group of cardiac-transplanted patients who may benefit from personalized therapy.

PO-500 A novel role for junctional adhesion molecule-a (JAM-A) in HER2-positive gastro-oesophageal cancers

IntroductionJunctional Adhesion Molecule-A (JAM-A) is a cell-cell adhesion protein that regulates physiological adhesion in epithelial and endothelial cells. Our previous work has shown that increased JAM-A expression on breast tumour epithelial tissue associates with poor prognosis in patients with invasive breast cancer, and identified a correlation between high JAM-A expression and levels of the important oncogene human epidermal growth factor receptor-2 (HER2). An important role for HER2 in gastro-oesophageal cancer is emerging, however the specific role of JAM-A in this setting is unclear.Material and methodsIn a pilot study, gastric cancer patient tissue sections (n=11) were stained for JAM-A expression. A novel, weighted ranking system was developed to semi-quantitatively score the extremely heterogeneous JAM-A membranous staining in gastric tissues; ultimately ranking tissues as low, moderate or high JAM-A expression. JAM-A scores were also correlated with HER2 status in each section. In parallel, a panel of gastro-oesophageal cell lines was used to investigate potential molecular relationships between JAM-A and HER2. Finally, HER2 protein expression was examined following transient JAM-A gene silencing in the gastro-oesophageal junction cancer cell line ESO26.Results and discussionsThe pilot study revealed that high JAM-A expression significantly correlated with HER2 positivity, and noted a novel staining pattern for JAM-A in diffuse gastric cancers. Molecular studies showed that JAM-A gene silencing reduced HER2 protein expression in gastric cancer cells. These data suggests that JAM-A may act as a novel upstream regulator of HER2 in gastro-oesophageal cancer cells, and warrant further investigation. Current studies are focussed on elucidating the mechanisms underpinning regulation of HER2 expression by JAM-A.ConclusionPromising preliminary indications that JAM-A plays a role in regulating HER2 expression in non-breast cancers may suggest it as both an unfavourable biomarker, as well as a novel putative pharmacological target for HER2-positive gastro-oesophageal cancers.This abstract has emanated from research conducted with the financial support of Science Foundation Ireland (SFI) under grant number 13/IA/1994 (to AMH).

PO-163 Junctional adhesion molecule-a is a novel upstream regulator of human epidermal growth factor receptor-3 signalling in breast cancer

IntroductionJunctional Adhesion Molecule-A (JAM-A) is a transmembrane protein with important physiological functions in regulating cell-cell adhesion. JAM-A levels have been reported to regulate HER2 expression (Brennan et al, Oncogene 2013;32(22):2799–804), thus we hypothesised that JAM-A also regulates expression of HER3 in breast cancer cells. HER3 is the most potent binding partner of HER2 in activating tumour growth signalling, and accumulating evidence suggests that HER3 plays an important role in resistance to anti-HER2 therapies. Furthermore HER3 is frequently overexpressed in HER2-negative breast cancers, and, along with other HER family members, may drive HER2-independent tumourigenic mechanisms.Material and methodsUsing in vitro assays with a panel of breast cell lines, we are investigating whether JAM-A knockdown or over-expression influences expression or activity of FOXA1 and HER3 at mRNA and protein level, and DNA-protein binding assay to show FOXA1/HER3 promoter gene interaction.Results and discussionsResults from our study showed that stable overexpression of JAM-A in MCF7 breast cancer cells (MCF7-JAM) increased both mRNA and protein expression of HER3. Correspondingly, transient gene silencing of JAM-A reduced the mRNA and protein expression of HER3 in MCF7 and SKBR3 cells. In MCF7 cells, JAM-A gene silencing phenocopied that of HER3 gene silencing by reducing protein expression of the HER downstream effectors phospho-AKT and phospho-ERK, in parallel with significant reductions in cell viability (measured by Alamar Blue assay). To begin exploring the mechanism whereby JAM-A regulates HER3 expression, we focused on the HER3 transcription factor FOXA1 following a bioinformatic search. JAM-A gene silencing reduced FOXA1 expression in all cell lines tested; while JAM-A overexpression had the opposite effect in increasing FOXA1 expression. FOXA1 gene silencing was sufficient to reduce HER3 expression in the same cells. Furthermore FOXA1 in nuclear extracts of breast cancer cells bound specifically to an oligonucleotide sequence corresponding to the HER3 gene promoter, and gene silencing of JAM-A reduced FOXA1 binding activity to this sequence. Taken together, our data provide novel evidence of a direct relationship between levels of JAM-A, FOXA1 and HER3 in breast cancer cells.ConclusionIn conclusion, we suggest that JAM-A merits investigation as a novel target to inhibit HER3-dependent tumourigenic signalling in breast cancer.

Abstract 3109: Investigating the role of wild-type TFE3 in renal cell carcinoma cells harboring TFE3 fusions with spliceosome machinery associated genes

Abstract Background: Translocation Renal Cell Carcinoma (tRCC) represents an aggressive subtype of kidney cancer associated with various gene fusions involving translocation of one of two members of micropthalmia transcription factor (MiT) family, TFE3 or TFEB. Despite the identification of multiple TFE3 gene fusions in tRCC, heterogeneous phenotype and various dysregulated signaling pathways resulting from variety of gene fusion partners pose a challenge to establish effective treatments for these patients. In this work, we assessed the role of wild type TFE3 (wt-TFE3) in RCC cell bearing TFE3 fusion with genes associated with pre-mRNA splicing factor machinery (NONO, SFPQ and PRCC).Methods: Endogenous SFPQ-TFE3 expressing cells, RP-RO7, were generated from patient derived xenograft established in NSG mice. UOK-109 and UOK-146 (kindly provided by Dr. Marston Lenehan, NCI) were used as endogenous NONO-TFE3 and PRCC-TFE3 expressing cells, respectively. RNA interference (RNAi) mediated knockdown with differential exon targeting strategy was employed to study wt-TFE3 loss of function in RP-R07, UOK-109 and UOK-146, respectively. Real time monitoring of cell viability assay with multiplex readout were developed to investigate the effect of wt-TFE3 loss of function in 2D and 3D culture system. Two different 3D models were used;1) Tumor growth model, in which cancer cell interaction with extracellular matrix and stromal cells were represented in a multiculture-spheroid system; 2) Invasion model, in which cell ability to invade basement membrane barrier was modeled with matrix restricted spheroid, and the invasion trajectories were observed and quantified. Exogenous expression of wt-TFE3-EGFP was utilized to study the protein subcellular localization in RP-R07, UOK-109 and UOK-146 2D cultures and 3D culture system.Results: Consistent with the expression of chimeric TFE3, wt-TFE3-EGFP ectopic expression demonstrated strong nuclear localization in RP-R07, UOK-109 and UOK-146. Multiplex readout of cells viability assay showed that transient loss of wt-TFE3 in RP-R07, UOK-109 and UOK-146 curtails the proliferation rate of these cells in 2D and 3D models in fusion partner dependent manner (P&amp;lt;0.05). 3D invasion assay coupled with RNAi strategy in RP-R07, UOK-109 and UOK-146 demonstrated the role wt-TFE3 in invasion potential of these cell in fusion partner dependent fashion (P&amp;lt;0.05). Downstream analysis suggested that the wt-TFE3 transient silencing affected cell proliferation and invasion ability via inhibition of the IRS-PI3K-mTOR axis.Conclusion: Our results suggest the potential role of wt-TFE3 in tRCC oncogenesis irrespective of its different fusion partner. Citation Format: Nur P. Damayanti, Khunsha Ahmed, May Elbanna, Chinghai Kao, Roberto Pili. Investigating the role of wild-type TFE3 in renal cell carcinoma cells harboring TFE3 fusions with spliceosome machinery associated genes [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 3109.

Transient silencing of heat shock proteins showed remarkable roles for HSP70 during adaptation to stress in plants

Heat shock proteins (HSPs) have vital roles during plant adaptation to biotic and abiotic stresses, as well as stress-free conditions. In the present study, we used a heterologous strategy of virus induced gene silencing to investigate the role of different classes of HSPs in Nicotiana benthamiana. Different growth and physiological parameters in silenced plants were evaluated under both control and salt stress conditions. Among the treatments, silencing of HSP70, especially under salinity regime, was found to have stronger impacts on growth, protein concentration, the accumulation of photosynthetic pigments, proline and total soluble carbohydrates content, malondialdehyde, the activity of antioxidant enzymes, performance index, relative water content and the ratio of K+/Na+, suggesting a more prominent role for HSP70 in both stressful and stress-free life cycle of the plants. Principal component analysis and hierarchical clustering indicated that HSP70 gene was silenced, the plants might effectively respond to stress by a higher accumulation of compatible solutes, like proline to protect the cell membranes and proteins from damage.

Transplantation of P53-Silenced Endothelial Progenitor Cells (EPCs) Under Renal Capsule—A Potential Treatment for Diabetic Kidney Disease (DKD)

Diabetic kidney disease (DKD) is one of the major vascular complications of diabetes, which is associated with glomerulosclerosis and poor perfusion. Therefore, improving the renal perfusion may help to halt or reverse the kidney injury. Here, we investigated whether transplanting mouse EPC (that has been modified by transiently silencing p53 gene using Adenovirus transduction ex-vivo), under renal capsule could improve angiogenesis and renal perfusion, by preventing EPC apoptosis in a hyperglycemic milieu. Methods: First, we confirmed hyperglycemia and progressive proteinuria in STZ-induced type 1 diabetic C57BL/6J mouse. Next, we transplanted 0.3 million p53-silenced EPCs,or Ad-Null-EPCs (control), bilaterally, under each kidney capsule. Another comparator was non-STZ normal mouse. Urine was collected weekly for volume and protein estimation. Renal blood flow was measured by laser Doppler. Kidneys were harvested post sacrifice and qRT-PCR were performed for targeted (towards angiogenic genes) gene expression assays. Results: Excess urine volume as noted in the hyperglycemic mouse models was reduced 4-fold post transplantation of p53sh-EPCs compared to null. There was no proteinuria after week 2 in p53sh-EPC transplanted mouse unlike control. Enhanced blood flow by laser doppler (3.2 fold) was also noted with delivery of p53sh-EPCs compared to null EPC. Absence of proteinuria (after 2 weeks) and renal blood flow measurements for p53sh group were similar to the non-STZ mouse. Interestingly, markers for neovascularization, such as eNOS (4.5 fold, p=0.002) and VEGF-A (1.5 fold, p=0.03) upregulated significantly post p53 silenced EPC transplantation compared to null EPC. CD31 staining is pending. Conclusion: Transient silencing of p53 gene in mouse EPCs help to improve proteinuria, diabetic polyuria and renal blood flow, most likely by increasing angiogenesis and perfusion and may have a prominent therapeutic role in DKD. Disclosure N. Kundu: None. L.D. Asico: None. C.C. Domingues: None. N. Ahmadi: None. S. Sen: None.

GDNF family receptor α-1 in catfish: Possible implication to brain dopaminergic activity

Glial cell line-derived neurotrophic factor (GDNF) is a potent trophic factor that preferentially binds to GDNF family receptor α-1 (GFRα-1) by regulating dopaminergic (DA-ergic) neurons in brain. Present study aimed to evaluate the significance of GFRα-1 expression during early brain development in catfish. Initially, the full-length cDNA of GFRα-1 was cloned from adult brain which showed high homology with other vertebrate counterparts. Quantitative PCR analysis of tissue distribution revealed ubiquitous expression of GFRα-1 in the tissues analyzed with high levels in female brain and ovary. Significant high expression was evident in brain at 75 and 100 days post hatch females than the respective age-match males. Expression of GFRα-1 was high in brain during the spawning phase when compared to other reproductive phases. Localization of GFRα-1 revealed its presence in preoptic area-hypothalamus which correlated well with the expression profile in discrete areas of brain in adult catfish. Transient silencing of GFRα-1 through siRNA lowered expression levels of GFRα-1, which further down regulated the expression of certain brain-specific genes. Expression of GFRα-1 in brain declined significantly upon treatment with the 1-methyl-1,2,3,6-tetrahydropyridine causing neurodegeneration which further correlated with catecholamines (CA), L-3,4-dihydroxyphenylalanine, DA and norepinephrine levels. Taken together, GFRα-1 plausibly entrains gonadotropin-releasing hormone and gonadotropin axis either directly or indirectly, at least by partially targeting CA-ergic activity.

Heterogeneous nuclear ribonucleoprotein C1 may control miR-30d levels in endometrial exosomes affecting early embryo implantation.

Is there a specific mechanism to load the microRNA (miRNA), hsa-miR-30d, into exosomes to facilitate maternal communication with preimplantation embryos? The heterogeneous nuclear ribonucleoprotein C1 (hnRNPC1) is involved in the internalization of endometrial miR-30d into exosomes to prepare for its subsequent incorporation into trophectoderm cells. Our group previously described a novel cell-to-cell communication mechanism involving the delivery of endometrial miRNAs from the maternal endometrium to the trophectoderm cells of preimplantation embryos. Specifically, human endometrial miR-30d is taken up by murine blastocysts causing the overexpression of certain genes involved in embryonic adhesion (Itb3, Itga7 and Cdh5) increasing embryo adhesion rates. Transfer of maternal miR-30d to preimplantation embryos was confirmed by co-culture of wild-type (WT) and miR-30d knockout (KO) murine embryos with primary cultures of human endometrial epithelial cells (hEECs) in which mir-30d was labeled with specific Molecular Beacon (MB) or SmartFlare probes. Potential molecules responsible for the miR-30d loading into exosomes were purified by pull-down analysis with a biotinylated form of miR-30d on protein lysates from human endometrial exosomes, identified using mass spectrometry and assessed by flow cytometry, western blotting and co-localization studies. The role of hnRNPC1 in the miR-30d loading and transportation was interrogated by quantification of this miRNA in exosomes isolated from endometrial cells in which hnRNPC1 was transiently silenced using small interference RNA. Finally, the transfer of miR-30d to WT and KO embryos was assessed upon co-culture with sihnRNPC1 transfected cells. Murine embryos from miR-30d WT and KO mice, (strain MirC26tm1Mtm/Mmjax), were obtained by oviduct flushing of superovulated females. Endometrial Exosomes were purified by ultracentrifugation of supernatants from primary cultures of hEECs or Ishikawa cells. MB and Smartflare miR-30d probes were detected by confocal and/or transmission electron microscopy (TEM). hEECs and exosomes derived from them were subjected to pull-down with a biotinylated form of miR-30d. Captured proteins were identified by mass spectrometry (MS/MS). Western blotting was performed to detect hnRNPC1 and CYR61 in whole lysates, subcellular fractions and secreted vesicles from hEECs. Co-localization studies of the selected proteins with the exosomal marker CD63 were performed. FACS analysis was carried out to determine the presence of hnRNPC1 inside exosomes. Silencing of hnRNPC1 was conducted in the Ishikawa Cell Line with the Smart Pool Accell HNRNPC siRNA at a final concentration of 50 nM. RT-qPCRs were done to determine the messenger levels of miR-30d in cells and exosomes. Co-cultures of WT and KO embryos were established with Ishikawa cells double-transfected with sihnRPNC1 and MB probes. MS/MS analysis allowed us to identify hnRNPC1 as a possible protein to influence miR-30d loading into exosomes. Co-localization studies of hnRNPC1 with CD63 and FACS analyses suggested the presence of hnRNPC1 inside exosomes. Silencing of hnRNPC1 in Ishikawa cells resulted in a sharp decrease of the levels of miR-30d in both epithelial-like cells (P = 0.0001) and exosomes (P = 0.0152), suggesting its potential role in miR-30d biogenesis and transfer. Co-culture assays of miR-30d KO embryos with sihnRNPC1 hEECs revealed a decrease in embryo-miR-30d acquisition during the adhesion and invasion stages. In turn, transient silencing of hnRNPC1 results in a significant decrease of blastocyst adhesion compared to mock transfection conditions using Block-it, in both WT [Mean ± SD; 67 ± 10.0% vs. 38 ± 8.5%(P = 0.0006)] and miR-30d KO embryos [Mean ± SD; 50 ± 11.5% vs. 26 ± 8.8% (P = 0.0029) (n = 2); 14 embryos transferred per condition tested]. MS/MS data are available via ProteomeXchange with identifier PXD008773. The Ishikawa Cell Line was used as a model of hEECs in silencing experiments due to the low survival rates of primary hEECs after transfection. The data show that hnRNPC1 may be involved in the internalization of miR-30d inside exosomes. The decreased rates of embryo adhesion in endometrial epithelial-like cells transiently silenced with sihnRNPC1evidence that hnRNPC1 could be an important player in the maternal-embryo communication established in the early stages of implantation. This work was supported by the Miguel Servet Program Type I of Instituto de Salud Carlos III [CP13/00038]; FIS project [PI14/00545] to F.V.; the 'Atracció de Talent' Program from VLC-CAMPUS [UV-INV-PREDOC14-178329 to NB]; a Torres-Quevedo grant (PTQ-13-06133) by the Spanish Ministry of Economy and Competitiveness to IM and MINECO/FEDER Grant [SAF2015-67154-R] to C.S. The authors declare there is no conflict of interest.

Curcumin inhibits autocrine growth hormone-mediated invasion and metastasis by targeting NF-κB signaling and polyamine metabolism in breast cancer cells.

Curcumin is assumed to be a plant-derived therapeutic drug that triggers apoptotic cell death in vitro and in vivo by affecting different molecular targets such as NF-κB. Phase I/II trial of curcumin alone or with chemotherapeutic drugs has been accomplished in pancreatic, colon, prostate and breast cancer cases. Recently, autocrine growth hormone (GH) signaling-induced cell growth, metastasis and drug resistance have been demonstrated in breast cancer. In this study, our aim was to investigate the potential therapeutic effect of curcumin by evaluating the molecular machinery of curcumin-triggered apoptotic cell death via focusing on NF-κB signaling and polyamine (PA) metabolism in autocrine GH-expressing MCF-7, MDA-MB-453 and MDA-MB-231 breast cancer cells. For this purpose, a pcDNA3.1 (+) vector with a GH gene insert was transfected by a liposomal agent in all breast cancer cells and then selection was conducted in neomycin (G418) included media. Autocrine GH-induced curcumin resistance was overcome in a dose-dependent manner and curcumin inhibited cell proliferation, invasion-metastasis and phosphorylation of p65 (Ser536), and thereby partly prevented its DNA binding activity in breast cancer cells. Moreover, curcumin induced caspase-mediated apoptotic cell death by activating the PA catabolic enzyme expressions, which led to generation of toxic by-products such as H2O2 in MCF-7, MDA-MB-453 and MDA-MB-231 GH+ breast cancer cells. In addition, transient silencing of SSAT prevented curcumin-induced cell viability loss and apoptotic cell death in each breast cancer cells. In conclusion, curcumin could overcome the GH-mediated resistant phenotype via modulating cell survival, death-related signaling routes and activating PA catabolic pathway.

Function of the ERFL1a Transcription Factor in Wheat Responses to Water Deficiency.

The APETALA2/ethylene response factor (AP2/ERF) superfamily is involved in the responses of plants to biotic and abiotic stresses; however, the functions and mechanisms of some members of this family in plants are unclear. In our previous study, expression of TaERFL1a, a member of the AP2/ERF family, was remarkably induced in wheat seedlings suffering freezing stress. In this study, we show that its expression was rapidly upregulated in response to salt, cold, and water deficiency, suggesting roles in the responses to abiotic stresses. Further, transient barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) resulted in significantly reduced tolerance to 20% PEG6000-stimulated water deficiency. Subcellular localization and transcriptional activation assays separately showed that TaERFL1a was targeted to the nucleus and possessed transcriptional activation activity. Yeast two-hybrid library screening identified six interacting proteins, and of these, the interactions between TaERFL1a and TaSGT1, and TaERFL1a and TaDAD2 proteins were further confirmed by yeast co-transformation and bimolecular fluorescent complementation (BiFC). Collectively, our results suggest that TaERFL1a is a stress-responsive transcription factor, which could be functionally related to proteins involved in the abiotic stress responses of plants.

Abstract 252: Innovative Cell-therapy for Diabetic Kidney Disease (DKD)

Diabetic kidney disease (DKD) is one of the major vascular complications of diabetes, which is associated with glomerulosclerosis and poor perfusion. Therefore, improving the renal perfusion may help to halt or reverse the kidney injury. Here, we investigated whether transplanting mouse EPC (that has been modified by transiently silencing p53 gene using Adenovirus transduction ex-vivo), under renal capsule could improve angiogenesis and renal perfusion, by preventing EPC apoptosis in a hyperglycemic milieu. Methods: First, we confirmed hyperglycemia and progressive proteinuria in STZ-induced type 1 diabetic C57BL/6J mouse. Next, we transplanted 0.3 million p53-silenced EPCs,or Ad-Null-EPCs (control), bilaterally, under each kidney capsule. Another comparator was non-STZ normal mouse. Urine was collected weekly for volume and protein estimation. Renal blood flow was measured by laser Doppler. Kidneys were harvested post sacrifice and qRT-PCR were performed for targeted (towards angiogenic genes) gene expression assays. Results: Excess urine volume as noted in the hyperglycemic mouse models was reduced 4-fold post transplantation of p53sh-EPCs compared to null. There was no proteinuria after week 2 in p53sh-EPC transplanted mouse unlike control. Enhanced blood flow by laser doppler (3.2 fold) was also noted with delivery of p53sh-EPCs compared to null EPC. Absence of proteinuria (after 2wks) and renal blood flow measurements for p53sh group were similar to the non-STZ mouse. Interestingly, markers for neovascularization, such as eNOS (4.5 fold, p=0.002) and VEGF-A (1.5 fold, p=0.03) upregulated significantly post p53 silenced EPC transplantation compared to null EPC. CD31 staining is pending. Conclusion: Transient silencing of p53 gene in mouse EPCs help to improve proteinuria, diabetic polyuria and renal blood flow, most likely by increasing angiogenesis and perfusion and may have a prominent therapeutic role in DKD.

Enhancing Understanding of the Visual Cycle by Applying CRISPR/Cas9 Gene Editing in Zebrafish.

During the vertebrate visual cycle, all-trans-retinal is exported from photoreceptors to the adjacent RPE or Müller glia wherein 11-cis-retinal is regenerated. The 11-cis chromophore is returned to photoreceptors, forming light-sensitive visual pigments with opsin GPCRs. Dysfunction of this process perturbs phototransduction because functional visual pigment cannot be generated. Mutations in visual cycle genes can result in monogenic inherited forms of blindness. Though key enzymatic processes are well characterized, questions remain as to the physiological role of visual cycle proteins in different retinal cell types, functional domains of these proteins in retinoid biochemistry and in vivo pathogenesis of disease mutations. Significant progress is needed to develop effective and accessible treatments for inherited blindness arising from mutations in visual cycle genes. Here, we review opportunities to apply gene editing technology to two crucial visual cycle components, RPE65 and CRALBP. Expressed exclusively in the human RPE, RPE65 enzymatically converts retinyl esters into 11-cis retinal. CRALBP is an 11-cis-retinal binding protein expressed in human RPE and Muller glia. Loss-of-function mutations in either protein results in autosomal recessive forms of blindness. Modeling these human conditions using RPE65 or CRALBP murine knockout models have enhanced our understanding of their biochemical function, associated disease pathogenesis and development of therapeutics. However, rod-dominated murine retinae provide a challenge to assess cone function. The cone-rich zebrafish model is amenable to cost-effective maintenance of a variety of strains. Interestingly, gene duplication in zebrafish resulted in three Rpe65 and two Cralbp isoforms with differential temporal and spatial expression patterns. Functional investigations of zebrafish Rpe65 and Cralbp were restricted to gene knockdown with morpholino oligonucleotides. However, transient silencing, off-target effects and discrepancies between knockdown and knockout models, highlight a need for more comprehensive alternatives for functional genomics. CRISPR/Cas9 in zebrafish has emerged as a formidable technology enabling targeted gene knockout, knock-in, activation, or silencing to single base-pair resolution. Effective, targeted gene editing by CRISPR/Cas9 in zebrafish enables unprecedented opportunities to create genetic research models. This review will discuss existing knowledge gaps regarding RPE65 and CRALBP. We explore the benefits of CRISPR/Cas9 to establish innovative zebrafish models to enhance knowledge of the visual cycle.

Expression profiling of c-kit and its impact after esiRNA silencing during gonadal development in catfish

Receptor, c-Kit is a member of a family of growth factor receptors that have tyrosine kinase activity, and are involved in the transduction of growth regulatory signals across plasma membrane by activation of its ligand, kitl/scf. The present study analyzed mRNA and protein expression profiles of c-kit in the gonads of catfish, Clarias gariepinus, using real time PCR, in situ hybridization and immunohistochemistry. Tissue distribution analysis revealed higher expression mainly in the catfish gonads. Ontogeny studies showed minimal expression during early developmental stages and highest during 50–75 days post hatch, and the dimorphic expression in gonads decreased gradually till adulthood, which might suggest an important role for this gene around later stages of sex differentiation and gonadal development. Expression of c-kit was analyzed at various phases of gonadal cycle in both male and female, which showed minimal expression during the resting phase, and higher expression during the pre-spawning phase in male compared to females. In vitro and in vivo induction using human chorionic gonadotropin elevated the expression of c-kit indicating the regulatory influence of hypothalamo-hypophyseal axis. In vivo transient gene silencing using c-kit-esiRNA in adult catfish during gonadal recrudescence showed a decrease in c-kit expression, which affected the expression levels of germ cell meiotic marker sycp3, as well as several factors and steroidogenic enzyme genes that are involved in germ cell development. Decrease in the levels of 11-ketotestosterone and testosterone in serum were also observed after esiRNA silencing. The findings suggests that c-kit has an important role in the process of germ cell proliferation, development and maturation during gonadal development and recrudescence in catfish.

Coralyne, a protoberberine alkaloid, causes robust photosenstization of cancer cells through ATR-p38 MAPK-BAX and JAK2-STAT1-BAX pathways

Photodynamic therapy (PDT) provides an effective cancer treatment option but it requires sufficient cellular oxygen concentration to exert its photosensitizing effects. Due to hypoxic nature of most tumors, widespread clinical application of PDT is restricted and warrants development of photosensitizers which can kill cancer cells in ROS independent manner. Previously, we reported significant enhancement of the anti-cancer property of coralyne in presence of ultraviolet-A (UVA) light exposure against several human carcinoma cell lines. This study aimed at unravelling molecular cascades of events in CUVA treatment (coralyne and UVA light)-mediated photosensitization of human skin cancer. The CUVA-treatment caused robust apoptosis of A431 cancer cells, primarily through mitochondrial and lysosomal dysfunctions. Silencing of BAX conferred a significant protection against CUVA-induced apoptosis. Both lysosomal proteases and caspase-8 activation contributed to BID cleavage. Further, our results revealed that a dual signaling axis e.g., ATR-p38 MAPK and JAK2-STAT1 pathways functioned upstream of BAX activation in apoptosis response. Moreover, transient silencing of ATR and pharmacological inhibition of p38-MAPK or JAK2 significantly abolished the effect of CUVA treatment induced BAX expression and cell death, linking the extrinsic and intrinsic pathways with the observed cell death. Our data suggest that coralyne, which is known topoisomerase-I inhibitor, may be an attractive agent for photo-chemotherapeutic treatment of human skin cancers.

The Use of RNA Sequencing and Correlation Network Analysis to Study Potential Regulators of Crabapple Leaf Color Transformation.

Anthocyanins are plant pigments that contribute to the color of leaves, flowers and fruits, and that are beneficial to human health in the form of dietary antioxidants. The study of a transformable crabapple cultivar, 'India magic', which has red buds and green mature leaves, using mRNA profiling of four leaf developmental stages, allowed us to characterize molecular mechanisms regulating red color formation in early leaf development and the subsequent rapid down-regulation of anthocyanin biosynthesis. This analysis of differential gene expression during leaf development revealed that ethylene signaling-responsive genes are up-regulated during leaf pigmentation. Genes in the ethylene response factor (ERF), SPL, NAC, WRKY and MADS-box transcription factor (TF) families were identified in two weighted gene co-expression network analysis (WGCNA) modules as having a close relationship to anthocyanin accumulation. Analyses of network hub genes indicated that SPL TFs are located in central positions within anthocyanin-related modules. Furthermore, cis-motif and yeast one-hybrid assays suggested that several anthocyanin biosynthetic or regulatory genes are potential targets of SPL8 and SPL13B. Transient silencing of these two genes confirmed that they play a role in co-ordinating anthocyanin biosynthesis and crabapple leaf development. We present a high-resolution method for identifying regulatory modules associated with leaf pigmentation, which provides a platform for functional genomic studies of anthocyanin biosynthesis.

Abstract P1-03-07: Junctional adhesion molecule-A is a novel upstream regulator of human epidermal growth factor receptor-3 signaling in breast cancer

Abstract Junctional Adhesion Molecule-A (JAM-A) is a transmembrane protein with important physiological functions in regulating cell-cell adhesion. Pathophysiologically, its high expression in breast tumour tissue has been shown to correlate with that of Human Epidermal Growth Factor Receptor-2 (HER2), whilst JAM-A knockdown in breast cancer cells has been shown to reduce HER2 expression and signaling. Although HER2 has been successfully targeted in the oncology setting for several years, the problem of clinical drug resistance to HER2-targeted therapies (among other factors) has recently put the spotlight on other HER family members and their upstream regulators as potential drug targets. HER3 is the most potent binding partner of HER2 in activating tumor growth signaling, and accumulating evidence suggests that HER3 plays an important role in resistance to anti-HER2 therapies. Furthermore HER3 is frequently overexpressed in HER2-negative breast cancers, and, along with other HER family members, may drive HER2-independent tumorigenic mechanisms. Since JAM-A levels have been reported to regulate HER2 expression (Brennan et al, Oncogene 2013 32(22):2799-804), we hypothesised that JAM-A also regulates expression of HER3 in breast cancer cells. Results from our study showed that stable overexpression of JAM-A in MCF7 breast cancer cells (MCF7-JAM) increased both mRNA and protein expression of HER3. Correspondingly, transient gene silencing of JAM-A reduced the mRNA and protein expression of HER3 in MCF7 and MCF7-JAM cells. JAM-A silencing also reduced HER3 expression in HER2-positive BT474 breast cancer cells. As the cell lines tested were all Estrogen Receptor-α(ERα)-positive, we examined whether ERα was required to permit JAM-dependent regulation of HER3. However concomitant silencing of ERα in MCF7 cells did not alter the capacity of JAM-A silencing to reduce HER3 protein levels. In MCF7 and MCF7-JAM cells, JAM-A gene silencing phenocopied that of HER3 gene silencing by reducing protein expression of the HER downstream effectors phospho-AKT and phospho-ERK, in parallel with significant reductions in cell viability (measured by Alamar Blue assay). To begin exploring the mechanism whereby JAM-A regulates HER3 expression, we focused on the HER3 transcription factor FOXA1. JAM-A knockdown reduced expression of FOXA1 in MCF7 and MCF7-JAM cells, and knockdown of FOXA1 was sufficient to reduce HER3 expression in the same cells. Taken together, our data provide novel evidence of a direct relationship between levels of JAM-A, FOXA1 and HER3 in breast cancer cells. The relationship appears to be uni-directional, since silencing of HER3 did not alter JAM-A expression in any cell line tested. In conclusion, we suggest that JAM-A merits investigation as a novel target to inhibit HER3-dependent tumorigenic signaling in breast cancer. Our ongoing investigations will determine the pharmacological value of inhibiting JAM-A signaling in breast cancer models, and its potential significance in the setting of resistance to HER2-targeted therapies. Citation Format: Cruz RGB, Hopkins AM. Junctional adhesion molecule-A is a novel upstream regulator of human epidermal growth factor receptor-3 signaling in 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 P1-03-07.

(110) - Optogenetic silencing of CGRPα-expressing sensory neurons alleviates neuropathic pain

Calcitonin gene-related peptide (CGRP) α-expressing sensory neurons are essential for thermal sensation in both the naïve and injured state, but for mechanical sensation only after injury. Previously, it was demonstrated that chemical ablation of these neurons decreased the heat hypersensitivity that develops following both neuropathic and inflammatory injuries. To determine if the silencing of intact neurons results in similar or different behavioral outcomes, and to further investigate the involvement of this cell population in spared nerve injury (SNI), postoperative pain and Complete Freund's Adjuvant (CFA) inflammation, we used mice that selectively express Archaerhodopsin (Arch) in CGRPα-positive neurons. We report that the presence of intact CGRPα neurons after injury results in different behavioral phenotypes than when these neurons are ablated before injury. Further, our results reveal that intact peripheral CGRPα neurons are required for SNI-induced mechanical and thermal hyperalgesia, but not inflammatory-induced mechanical and heat hyperalgesia. In addition, we find that CGRPα peptide signaling does not play a role in SNI-mediated mechanical or thermal hyperalgesias, but instead is responsible for inflammation-induced mechanical and heat hyperalgesias. Furthermore, our data show that isolated sensory neurons are sensitized to mechanical stimuli after SNI and that this sensitization can be reversed by Arch activation. These results reveal previously unknown functions for CGRPα-expressing neurons in the establishment of hyperalgesia following neuropathic injury. Furthermore, transient peripheral silencing of these neurons evades the compensatory mechanisms or circuitry changes that might occur from permanent deletion of CGRPα-expressing neurons, thus revealing critical targets for the treatment of neuropathic pain.