Non-targeting siRNA Research Articles

Extracellular acidification-induced CXCL8 production through a proton-sensing receptor OGR1 in human airway smooth muscle cells: a response inhibited by dexamethasone

BackgroundHuman airway smooth muscle cells (ASMCs) contribute to bronchial contraction and airway hyperresponsiveness in patients with bronchial asthma. They also generate cytokines, chemokines, and matricellular proteins. Ovarian cancer G protein-coupled receptor 1 (OGR1) senses extracellular protons and mediates the production of interleukin-6 (IL-6) and connective tissue growth factor (CTGF) in ASMCs.MethodsASMCs were stimulated for the indicated time by pH 6.3 or pH 7.4-adjusted Dulbecco’s Modified Eagle Medium (DMEM) containing 0.1% bovine serum albumin (BSA) (0.1% BSA-DMEM). As a control stimulant, pH 7.4-adjusted 0.1% BSA-DMEM containing 10 ng/mL tumor necrosis factor-α (TNF-α) was used. Interleukin-8/C-X-C motif chemokine ligand 8 (CXCL8) mRNA expression in ASMCs was quantified by RT-PCR using real-time TaqMan technology. CXCL8 secreted from ASMCs was measured by enzyme-linked immunosorbent assay (ELISA). Phosphorylation at serine 536 of NF-κB p65 and binding of p65 to oligonucleotide containing an NF-κB consensus binding site were analyzed by Western blotting and an ELISA-based kit.ResultsAcidic pH induced a significant increase of CXCL8 mRNA expression and CXCL8 protein secretion in ASMCs. ASMCs transfected with small interfering RNA (siRNA) targeted for OGR1 produced less CXCL8 compared with those transfected with non-targeting siRNA. Protein kinase C (PKC) inhibitor, MEK1/2 inhibitor, and the inhibitor of IκB phosphorylation reduced acidic pH-stimulated CXCL8 production in ASMCs. Dexamethasone also inhibited acidic pH-stimulated CXCL8 production of ASMCs in a dose-dependent manner. Dexamethasone did not affect either phosphorylation or binding to the consensus DNA site of NF-κB p65.ConclusionsCXCL8 released from ASMCs by extracellular acidification may play a pivotal role in airway accumulation of neutrophils. Glucocorticoids inhibit acidic pH-stimulated CXCL8 production independent of serine 536 phosphorylation and the binding to DNA of NF-κB p65, although NF-κB activity is essential for CXCL8 production in ASMCs.

Abstract P5-03-01: LIN9 regulation of NEK2 underlies taxol resistance in triple-negative breast cancer

Abstract Currently, there are no targeted strategies to combat triple negative breast cancer, resulting in poor patient survival. TNBC initially respond well to cytotoxic chemotherapies such as paclitaxel, yet resistance and metastatic recurrence are common. Paclitaxel causes defects in mitotic spindle formation and centrosome function, as well as improper chromosome segregation leading to cell death. While being some of the most effective drugs for this disease, taxanes are associated with high toxicity. Thus discovering new therapeutic targets that are selective for TNBC should yield novel approaches for improving patient outcomes. We discovered that LIN9, a transcriptional regulator of mitosis, is overexpressed in 66% of TNBC and associated with poor survival. We further found that both LIN9 mRNA and protein expression are upregulated in paclitaxel-resistant versus sensitive cells and directly correlates with paclitaxel IC50 values across nine breast cancer cell lines. In MDA-MB-231 and MDA-MB-468 cell lines, enforced overexpression of LIN9 increases multi- and micronucleation, indicators of chromosomal instability. Conversely, LIN9 silencing also results in multi- and micronucleation, and supernumerary centrosomes. Most importantly, LIN9 silencing increases sensitivity to paclitaxel in TNBC cells with intrinsic (BT549) or acquired (MDA-MB-231 and MDA-MB-468) resistance. We have previously reported that treating TNBC cells with Bromodomain and ExtraTerminal protein inhibitors (BETi) reduces LIN9 expression, thus we determined if BETi could reverse paclitaxel resistance. Treatment with the BETi, JQ1, and paclitaxel caused a greater induction of apoptosis compared to either drug alone. Dual treatment also resulted in a potentiation of abnormal centrosomes, multinucleation, and micronucleation compared to that caused by either BETi or JQ1 alone. To identify the mechanism(s) by which genetic or therapeutic suppression of LIN9 reverses paclitaxel resistance, we compared the transcriptomes of TNBC cells transiently transfected with non-targeting or LIN9-targeted siRNAs in MDA-MB-231 and HCC70 cell lines. We further narrowed the list of candidate LIN9 targets by identifying genes that were bound by LIN9 in a published ChIP-Seq dataset from HeLa cells whose expression is also correlated with LIN9 expression and associated with reduced breast cancer patient survival. Using this approach, we identified NIMA-related Kinase 2 (NEK2), a serine/threonine kinase required for centrosome separation during mitosis as a potential mediator of the effects of LIN9 suppression. NEK2 is overexpressed in 47% of basal breast cancers and is associated with poor survival. In addition, NEK2 is upregulated in paclitaxel-resistant cells and LIN9 silencing decreases expression of NEK2. Silencing NEK2 expression also restores sensitivity to paclitaxel in resistant cells. Together, these data indicate that increased LIN9 expression in TNBC promotes paclitaxel resistance by contributing to centrosome dysfunction through upregulation of NEK2. They also indicate that targeting LIN9 expression in addition to paclitaxel treatment may be a viable therapeutic approach for TNBC patients. Citation Format: Shively MS, Gayle SS, Sahni JM, Keri RA. LIN9 regulation of NEK2 underlies taxol resistance in triple-negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-01.

SA62 - TRANSCRIPTIONAL CONSEQUENCES OF REDUCED EXPRESSION OF THE SCHIZOPHRENIA SUSCEPTIBILITY GENE SETD1A

Background Loss of function alleles in SETD1A are a genetic risk factor for developing schizophrenia. SETD1A encodes a protein possessing histone methyltransferase activity. Specifically, it catalyses the methylation of histone H3 at Lys4, a modification indicative of active regulatory DNA function. Reduced SETD1A expression is therefore likely to have a major impact on global gene expression via alterations to the histone code. To understand the downstream genes and pathways altered by SETD1A expression we performed gene expression profiling of a neural cell line in which SETD1A expression had been reduced. Methods SETD1A expression was reduced in the SH-SY5Y neuroblastoma cell line using two non-overlapping siRNAs. A non-targeting siRNA was used as a control. Four days after transfection cells were harvested for RNA extraction. Transcriptional profiling was performed using Illumina microarrays. Differential expressed probes were identified using t tests and gene ontology enrichment analysis performed using DAVID. Quantitative PCR was used to verify the microarray findings for a subset of gene transcripts. We tested for enrichment of association with schizophrenia amongst the differential expressed probes using MAGMA. Results Both targeting siRNAs reduced SETD1A RNA expression ~50% compared to control. Gene expression profiling identified 1131 differentially expressed probes (p Discussion Reduced SETD1A levels lead to widespread changes in the expression of genes involved in diverse process, consistent with its role in general transcription. However, we observed altered expression of known regulators of neuronal development and function. Our results also highlight mitochondrial function as important downstream consequences of reduced SETD1A expression. Importantly, we show that gene expression changes after knock-down of a rare variant schizophrenia risk gene are enriched for a common variant association signal, indicating a point of molecular convergence in risk mechanisms.

Investigating the role of COQ8B in aortic smooth muscle cells.

Background and Hypothesis: 
 Thoracic aortic aneurysm (TAA) is an aortopathy characterized by aortic enlargement and life-threatening complications such as aortic dissection and sudden cardiac death. Previous studies identified COQ8B as a candidate genetic modifier of TAA severity. COQ8B is important for mitochondrial biosynthesis of coenzyme Q, but its precise functions are not defined. We hypothesize that alteration of COQ8B influences TAA pathogenesis via energy and oxidant metabolism pathways. 
 Experimental Design: 
 Smooth muscle cells (SMCs) were cultured directly from leftover healthy aortic tissues acquired during cardiac transplant operations. At confluence of 50-70%, cells were transfected with siRNA targeting COQ8B or a non-targeting negative control siRNA. Gene expression was measured using real-time quantitative polymerase chain reaction (RT-qPCR). Production of the reactive oxygen species hydrogen peroxide (H2O2) was measured using the fluorescence-based Amplex Red Hydrogen Peroxide Assay (Invitrogen) in basal growth medium. 
 Results: 
 Expression of COQ8B decreased by approximately 75% to 85% at 48 hours following siRNA transfection compared with negative control. This was associated with approximately 1.5 fold upregulation of the SMC contractile gene CNN1 (p<0.05). Knockdown of COQ8B did not appear to alter H2O2 production measured at timepoints of 48 or 72 hours. 
 Conclusion and Impact: 
 Based on these preliminary data, decreased COQ8B expression appears to alter the contractile phenotype of SMCs but may not significantly influence extracellular levels of H2O2 under basal conditions. Exogenous activation of pathways important for TAA pathogenesis may be required to elucidate the role of COQ8B. Ultimately, this work may lead to improved clinical approaches.

CLIC1 Cooperates with Integrins to Promote Thrombus Formation and Angiogenesis

Chloride intracellular channel 1 (CLIC1) is a member of a family of six highly homologous membrane proteins (CLIC1-6), which have been shown to be co-regulated with integrins suggesting their involvement in cell adhesion, migration and proliferation. CLIC1 is a metamorphic protein that functions as an oxidoreductase in the cytoplasm as well as an ion channel in the cell membrane. CLIC1 is upregulated in angiogenic endothelial and metastatic tumor cells. In addition, studies in CLIC1 knockout mice have shown that CLIC1 promotes platelet function. Here, we hypothesize that CLIC1 supports cell adhesive functions in platelets as well as endothelial cells and in doing so mediates critical vascular biological processes such as thrombus formation, vascular repair and angiogenesis.To assess the role of CLIC1 in endothelial cells, we monitored human umbilical venous endothelial cells (HUVEC) after transfection with CLIC1 Smartpool® or non-targeted siRNA by phase contrast microscopy. This experiment revealed a significant anti-proliferative effect in CLIC1 knockdown cells in conventional 2-dimensional culture that coincided with reduced cell spreading. The deficit in spreading was reiterated after embedding the CLIC1-depleted HUVEC in a 3-dimensional matrix of fibrin clot and this loss in anchorage led to a dramatic increase in cell death.Immunocytochemistry and fluorescence microscopy of HUVEC 10 minutes after initial cell attachment showed strong CLIC1 expression in nascent lamellipodia, where it colocalized with F-actin. Recruitment of CLIC1 to the cell membrane was associated with lamellipodia formation and was lost at later stages of adhesion when HUVEC began to generate stress fibers. CLIC1 cell membrane expression was inhibited after knocking down integrin β3 or treating HUVEC with the small molecule CLIC1 inhibitor IAA94. Notably, inhibiting CLIC1 or integrin β3 caused a significant reduction of F-actin assembly in lamellipodia suggesting that both molecules cooperate during the initial phase of cell spreading.Paralleling our experiments with HUVEC, we observed that CLIC1 co-localizes with F-actin in the leading edges as well as in the cortical ring of newly attached platelets. Moreover, using flow cytometry, we detected robust CLIC1 cell surface expression in platelets after treatment with ADP, which was enhanced after addition of the β3 integrin ligand fibrinogen and reduced when we blocked integrins with peptides representing the integrin recognition motif RGD. These data suggest a close coordination of CLIC1 membrane recruitment with the adhesive machinery in activated platelets. Moreover, inhibition of CLIC1 relocation to the platelet membrane with IAA94 hindered activation of integrin αIIbβ3, which resulted in impaired platelet aggregation.Lastly, we tested whether CLIC1 inhibition has antithrombotic effects in vivo. To this end, we assessed vasoocclusion in a murine dorsal skinfold chamber model where blood flow was monitored by intravital fluorescence microscopy. Intraperitoneal injections of mice with the CLIC1 inhibitor IAA94 (20mg/kg) 19 hours and 1 hour before the photochemical induction of thrombus formation caused a significant delay in vaso-occlusion in response to CLIC1 inhibition compared to vehicle-treated controls indicating that CLIC1 inhibition has a significant anti-thrombotic effects in vivo. Together, our data show that CLIC1 cooperates with integrins during cell adhesion and as such mediates functions related to platelet homeostasis, thrombus formation, and angiogenesis. DisclosuresNo relevant conflicts of interest to declare.

SiRNA-mediated protein knockdown in precision-cut lung slices

Small interfering RNA (siRNA) can induce RNA interference, which leads to the knockdown of messenger RNA (mRNA) and protein. As a result, siRNA is often used in vitro and in vivo to unravel the function of genes and as a therapeutic agent to disrupt excessive expression of disease-related genes. However, there is a large gap between in vitro and in vivo models in terms of simplicity, flexibility, throughput, and translatability. This gap could be bridged by using precision-cut tissue slices, which represent viable explants prepared from animal or human tissue that can be cultured ex vivo. Previously, we demonstrated that self-deliverable siRNA (Accell siRNA) induced significant mRNA knockdown in lung slices. The goal of this study, however, was to investigate whether Accell siRNA also induced protein knockdown in murine lung slices. Slices were incubated for up to 96 h with no siRNA (untransfected), non-targeting siRNA (control), or gene-targeting siRNA (Gapdh, Ppib, Serpinh1, and Bcl2l1). Overall, untransfected and transfected slices remained viable during an incubation of 96 h. In addition, gene-targeting siRNAs induced not only significant and specific mRNA knockdown but also protein knockdown. Finally, protein knockdown of fibrogenesis-related targets (Ppib, Serpinh1, and Bcl2l1) was shown to influence fibrogenesis on mRNA level, thereby demonstrating this model its utility in functional genomics and translational research.

Baicalein stimulates fibroblast growth factor 21 expression by up-regulating retinoic acid receptor-related orphan receptor α in C2C12 myotubes

Retinoic acid receptor-related orphan receptor (ROR) α has been implicated in various physiological functions, including the immune system, inflammation, and circadian rhythms. In the present study, the synthetic RORα/γ agonist SR1078 stimulated the production and gene expression of fibroblast growth factor 21 (FGF21) in C2C12 myotubes. FGF21, a member of the FGF family, plays important roles in the regulation of peripheral glucose tolerance and lipid metabolism and improves metabolic health. The mRNA expression and secretion of FGF21 was significantly weaker in Rora-silenced cells than in cells transfected with non-targeting control siRNA. SR1078 significantly up-regulated C/EBP homologous protein (CHOP), an established marker of ER stress, in a dose-dependent manner in C2C12 myotubes, while CHOP expression was decreased in Rora-silenced C2C12 cells, suggesting that RORα is involved in the regulation of FGF21 expression and stimulates ER stress in C2C12 myotubes. The naturally occurring compound baicalein up-regulated FGF21 expression and secretion in C2C12 myotubes. Additionally, the up-regulation of CHOP mRNA and protein expression was observed in C2C12 myotubes after the baicalein treatment. Furthermore, the knockdown of RORα prevented the augmentation of FGF21 and up-regulation of CHOP in response to baicalein in C2C12 cells. Collectively, these results suggest that baicalein stimulates the ER stress response and FGF21 expression through an RORα-dependent mechanism in C2C12 myotubes, and indicate the potential of baicalein as an effective anti-obesity therapy via its ability to enhance FGF21 production.

Fermitin family homolog-2 (FERMT2) is highly expressed in human placental villi and modulates trophoblast invasion

BackgroundIntegrins are transmembrane receptors that mediate cell–extracellular matrix (ECM) and cell-cell adhesion and trophoblast cells undergo changes in integrin expression as they differentiate. However, the mechanism(s) of integrin activation leading to integrin-mediated signaling in trophoblast cell differentiation is unknown. The Fermitin family proteins are integrin activators that help mediate integrin-mediated signaling, but have never been studied in detail within the human placenta. Thus, we examined the spatiotemporal pattern of expression of Fermitin family homolog-2 (FERMT2) in human chorionic villi throughout gestation and its role in trophoblast-substrate adhesion and invasion.MethodsPlacental villous tissue was obtained from patients undergoing elective terminations by dilatation and curettage at weeks 8–12 (n = 10), weeks 13–14 (n = 8), as well as from term deliveries at weeks 37–40 (n = 6). Tissues were fixed, processed and sections utilized for immunofluorescence analysis of FERMT2 expression during gestation. Additionally, HTR8-SVneo human trophoblast cells were transfected by electroporation with FERMT2-specific siRNAs or non-targeting siRNAs (control) and used in cell-substrate adhesion as well as invasion assays.ResultsFERMT2 was more commonly expressed in the basal domain of villous cytotrophoblast cells and prominently localized around the periphery of individual extravillous trophoblast cells. siRNA-mediated knockdown of FERMT2 in HTR8-SVneo cells resulted in significantly decreased trophoblast-substrate attachment (p < 0.05) as well as significantly decreased trophoblast invasion (p < 0.05) relative to control cells.ConclusionsThe detection of FERMT2 throughout extravillous trophoblast columns and the results of invasion assays demonstrated that this protein is likely an important regulator of integrin activation in extravillous cells to modulate migration and invasion.

Targeting the Warburg effect via LDHA inhibition engages ATF4 signaling for cancer cell survival.

Nutrient restriction reprograms cellular signaling and metabolic network to shape cancer phenotype. Lactate dehydrogenase A (LDHA) has a key role in aerobic glycolysis (the Warburg effect) through regeneration of the electron acceptor NAD+ and is widely regarded as a desirable target for cancer therapeutics. However, the mechanisms of cellular response and adaptation to LDHA inhibition remain largely unknown. Here, we show that LDHA activity supports serine and aspartate biosynthesis. Surprisingly, however, LDHA inhibition fails to impact human melanoma cell proliferation, survival, or tumor growth. Reduced intracellular serine and aspartate following LDHA inhibition engage GCN2-ATF4 signaling to initiate an expansive pro-survival response. This includes the upregulation of glutamine transporter SLC1A5 and glutamine uptake, with concomitant build-up of essential amino acids, and mTORC1 activation, to ameliorate the effects of LDHA inhibition. Tumors with low LDHA expression and melanoma patients acquiring resistance to MAPK signaling inhibitors, which target the Warburg effect, exhibit altered metabolic gene expression reminiscent of the ATF4-mediated survival signaling. ATF4-controlled survival mechanisms conferring synthetic vulnerability to the approaches targeting the Warburg effect offer efficacious therapeutic strategies.

Spinal protein phosphatase 1 constrains respiratory plasticity after sustained hypoxia

Plasticity is an important aspect of the neural control of breathing. One well-studied form of respiratory plasticity is phrenic long-term facilitation (pLTF) induced by acute intermittent but not sustained hypoxia. Okadaic acid-sensitive protein phosphatases (PPs) differentially regulate phrenic nerve activity with intermittent vs. sustained hypoxia, at least partially accounting for pLTF pattern sensitivity. However, okadaic acid inhibits multiple serine/threonine phosphatases, and the relevant phosphatase (PP1, PP2A, PP5) for pLTF pattern sensitivity has not been identified. Here, we demonstrate that sustained hypoxia (25 min, 9-10.5% O2) elicits phrenic motor facilitation in rats pretreated with bilateral intrapleural injections of small interfering RNAs (siRNAs; Accell-modified to preferentially transfect neurons, 3.33 μM, 3 days) targeting PP1 mRNA (48 ± 14% change from baseline, n = 6) but not PP2A (14 ± 9% baseline, n = 6) or nontargeting siRNAs (4 ± 10% baseline, n = 7). In time control rats (no hypoxia) treated with siRNAs ( n = 6), no facilitation was evident (-9 ± 9% baseline). siRNAs had no effect on the hypoxic phrenic response. Immunohistochemistry revealed PP1 and PP2A protein in identified phrenic motoneurons. Although PP1 and PP2A siRNAs significantly decreased PP1 and PP2A mRNA in PC12 cell cultures, we were not able to verify "knockdown" in vivo after siRNA treatment. On the other hand, PP1 and PP2A siRNAs significantly decreased PP1 and PP2A mRNA in PC12 cell cultures, verifying the intended siRNA effects. In conclusion, PP1 (not PP2A) is the relevant okadaic acid-sensitive phosphatase constraining phrenic motor facilitation after sustained hypoxia and likely contributing to pLTF pattern sensitivity. NEW & NOTEWORTHY This study demonstrates that the relevant okadaic acid-sensitive Ser/Thr protein phosphatase (PP) constraining facilitation after sustained hypoxia is PP1 and not PP2A. It suggests that PP1 may be critical in the pattern sensitivity of hypoxia-induced phrenic motor plasticity.

The effect of caveolin-1 knockdown on interleukin-1β-induced chemokine (C-C motif) ligand 2 expression in synovial fluid-derived fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease leading to destructive changes in peripheral joints and their irreversible deformity. The influx of chemoattractant-mediated inflammatory cells to the joints is one of the main features of RA. The aim of this study was to investigate the effect of a knockdown of caveolin-1 (CAV1), a known regulator of multiple cell signaling pathways, on chemokine (C-C motif) ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1) expression in synovial fluid-derived fibroblast-like synoviocytes (sfd-FLSs) obtained from patients with RA. Primary cell cultures of sfd-FLSs were established from RA synovial fluids. Cells were transiently transfected with small interfering RNA (siRNA) specific for CAV1, and then incubated with interleukin (IL)-1β to induce CCL2 expression. The expression levels of CAV1 and CCL2 were assessed at transcript level, using quantitative polymerase chain reaction (qPCR) and at protein level by enzyme-linked immunosorbent assay (ELISA) and western blotting analysis. A transient CAV1 knockdown in sfd-FLSs resulted in a decrease in the IL-1β-induced CCL2 mRNA expression level vs non-transfected cells and cells transfected with non-targeting siRNA. The concentration of secreted CCL2 was not affected significantly. Our study demonstrates that CCL2 expression in sfd-FLSs is CAV1-dependent, but only at transcript level. As the function of CAV1 has not been unequivocally determined, more studies are needed to confirm the role of CAV1 in inflammatory processes related to RA.

Abstract A17: NUAK1 acts as a growth suppressor in epithelial ovarian cancer

Abstract Epithelial ovarian cancer (EOC) cells form multicellular aggregates, or spheroids, and enter a dormant state during intraperitoneal metastasis. Dormant spheroids reduce anabolic metabolism and cell proliferation, which are linked to chemo-resistance. Liver kinase B1 (LKB1), encoded by the STK11 gene, is a critical regulator of stress metabolic signaling; LKB1 phosphorylates the downstream substrates AMP-activated protein kinase (AMPK) and AMPK-related kinases (ARKs) to mediate stress signaling. We have demonstrated that LKB1 expression and activity is required for EOC spheroid cell survival, yet knockdown of AMPKα1/α2 has no effect on viability. In addition, we have preliminary data demonstrating that phosphorylated AMPK is still maintained in EOC spheroids generated from CRISPR-mediated STK11-deleted cells, which completely lack LKB1 expression. Taken together, these results implicate the importance of other AMPK-independent effectors of LKB1 signaling in spheroid cell viability. Therefore, to identify critical downstream targets of LKB1-mediated signaling in EOC spheroids, we employed multiplex inhibitor beads/mass spectrometry (MIB/MS) using OVCAR8 STK11-knockout cells and OVCAR8 control cells grown in adherent and spheroid culture conditions. Using this proteomic approach, we identified NUAK1 as the sole ARK out of 12 different family members that is negatively affected by LKB1 loss. In fact, both phosphorylated and total NUAK1 protein expression are decreased in STK11-knockout cells and spheroids. NUAK1 can negatively control cell growth and proliferation by direct regulation of cell cycle checkpoint proteins in several cell systems; however, it has been relatively understudied in EOC. In analysis of the serous ovarian carcinoma data from TCGA, NUAK1 is infrequently altered (&amp;lt;2% of tumors) and it has the highest mean mRNA expression level as compared with the other 11 ARK genes. However, using immunoblot analysis, we show that NUAK1 protein is largely underexpressed in many established (n=15) and new ascites-derived EOC cell lines (n=22). In fact, further NUAK1 knockdown increased spheroid cell viability, size, and reattachment capability, whereas knockdown of its closely-related family member NUAK2 had no effect compared with the non-targeting siRNA control. Likewise, treatment with the pharmacologic NUAK1/2 inhibitor WZ4003 increased EOC cell growth and clonogenicity. Conversely, ectopic overexpression of NUAK1 reduced EOC cell growth and clonogenicity. Collectively, our data indicate that NUAK1 acts as a newly identified growth suppressor downstream of LKB1 metabolic stress signaling in EOC. We are currently investigating mechanisms regulating NUAK1 protein stability and activity in EOC cells, and whether activated LKB1-NUAK1 signaling promotes stress metabolic signaling during tumor dormancy in the context of EOC metastasis. Citation Format: Parima Saxena, Olga Collins, Yudith Ramos Valdes, Adrian Buensuceso, Kyle Francis, Kevin Brown, Karen Colwill, Anne-Claude Gingras, Robert Rottapel, Gabriel E. DiMattia, Trevor G. Shepherd. NUAK1 acts as a growth suppressor in epithelial ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A17.

Abstract 32: BCL11A is necessary for the expression of extracellular matrix genes and metastatic progression of triple-negative breast cancer

Abstract Cancer stem cells (CSC) contribute to the high incidence of metastatic recurrence in triple-negative breast cancer (TNBC) and elucidating the mechanisms controlling CSC properties should provide novel targets for therapeutic development that improves patient outcomes. The transcription factor BCL11A is highly differentially expressed in TNBC versus all other subtypes and controls breast CSC phenotypes such as marker expression and tumor initiation. However, the genes that BCL11A targets to control CSC biology and whether BCL11A promotes metastatic progression are unknown. To address these questions, we assessed the impact of transiently silencing the expression of BCL11A in TNBC cells using siRNA transfections. Reducing BCL11A expression did not impact TNBC cell viability or in vitro migratory capacity. However, silencing BCL11A significantly decreased invasion of TNBC cell lines, in vitro. Furthermore, stable shRNA-mediated silencing of BCL11A in the highly metastatic MDA-MB-231 cell line reduced metastatic outgrowth when xenografted into immunocompromised mice, suggesting that BCL11A is critical for metastatic progression of TNBC. To identify the BCL11A-regulated transcriptome in TNBC, we performed RNA-seq analysis of cells transiently transfected with non-targeting or BCL11A-targeted siRNAs in the MDA-MB-231 cell line. Consistent with the impact of BCL11A on invasion, numerous genes involved in adhesion and extracellular matrix were reduced with BCL11A silencing. The gene encoding the matrix metalloproteinase, MMP1, was the most significantly differentially expressed gene, with a 10-fold decrease in RNA expression following BCL11A suppression. We confirmed this down-regulation by both qRT-PCR and western blots of independent samples, as well as in the HCC1143 TNBC cell line. Current studies are assessing the functional impact of MMP1 regulation by BCL11A on invasion and metastatic progression of TNBC. Citation Format: Darcie D. Seachrist, Natasha N. Ingles, Molly M. Hannigan, Donny D. Licatalosi, Ruth A. Keri. BCL11A is necessary for the expression of extracellular matrix genes and metastatic progression of triple-negative breast cancer [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 32.

Abstract 5481: Targeting the kynurenine pathway as a novel metabolic treatment for head and neck cancer

Abstract Introduction: Head and neck squamous cell carcinoma (HNSCC) still claims 330,000 lives globally each year, indicating a critical need for novel therapies. Recently, we identified high levels of kynurenine (K) in HNSCC patients through global metabolic profiling of HNSCC tumor tissues via mass spectrometry (MS). Indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3- dioxygenase (TDO) are enzymes that degrade tryptophan to K. Elevated K allows tumor cells to evade immune response more easily, promoting cancer progression, while increased IDO levels are associated with poorer prognosis. We hypothesize that restoring immune response to tumors by blocking IDO1 will be an effective therapeutic strategy for sensitizing HNSCC. Methods: Validated human HNSCC cell lines were grown in 2D culture. Kynurenine levels were measured by MS. IDO1, IDO2 and TDO levels in tissue samples were measured by Western blot (WB) analysis. siRNAs for IDO1, IDO2, and TDO were used to knock down tryptophan pathway enzymes (non-targeting siRNA controls). Knockdown efficiency was evaluated by immunocytochemistry. After knockdown of IDO1, RNA was isolated, reverse transcribed and cancer associated pathways were analyzed using RT2 cancer pathway profiler (data analyzed using GeneGlobe; Qiagen). Results: Evaluation of the expression levels of enzymes that play a critical role in kynurenine pathway by WB analysis indicated upregulation of IDO1, IDO2 and TOD in both primary and metastatic tumor samples. siRNA-mediated knockdown of these enzymes indicated that only IDO1 knockdown was able to significantly reduce the levels of kynurenine by more than 80% (p&amp;lt;0.001), whereas knockdown of IDO2 and TDO reduced the kynurenine levels only by 10-25% (p&amp;gt;0.05%). Regulation of IDO1 function evaluated by the RT2 cancer pathway finder indicated downregulation of several genes involved in angiogenesis (ANGPT1, MCP-1, FGF2, TEK, VEGF, SERPINF1), pro-survival (c-IAP2 and XIAP), EMT transition (CDH2, OCLN and SNAI1), and DNA repair (LIG4, POLB, ERCC3 and DDB2), whereas genes involved in glycolysis (ACLY, G6PD,COX5A,LPL and PFKL), DNA damage (GADD34, GADD45G etc) and apoptosis (CASP7, CASP9, BCL2L11) were upregulated. Conclusion: Kynurenine is highly upregulated in HNSCC with checkpoint inhibition of IDO1 leading to increased apoptosis in vitro as well as increases in several glycolytic enzymes. Further translational analysis of glycolytic influences on the tryptophan pathway and immune checkpoint inhibition of IDO1 will provide mechanistic support for development of novel metabolic therapies for HNSCC. Citation Format: Chitra Subramanian, Thekkelnaycke M. Rajendiran, Tanu Soni, Mark S. Cohen. Targeting the kynurenine pathway as a novel metabolic treatment for head and neck cancer [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 5481.

Integrated Human Evaluation of the Lysophosphatidic Acid Pathway as a Novel Therapeutic Target in Atherosclerosis

Variants in the PLPP3 gene encoding for lipid phosphate phosphohydrolase 3 have been associated with susceptibility to atherosclerosis independently of classical risk factors. PLPP3 inactivates lysophosphatidic acid (LPA), a pro-inflammatory, pro-thrombotic product of phospholipase activity. Here we performed the first exploratory analysis of PLPP3, LPA, and LPA receptors (LPARs 1–6) in human atherosclerosis. PLPP3 transcript and protein were repressed when comparing plaques versus normal arteries and plaques from symptomatic versus asymptomatic patients, and they were negatively associated with risk of adverse cardiovascular events. PLPP3 localized to macrophages, smooth muscle, and endothelial cells (ECs) in plaques. LPAR 2, 5, and especially 6 showed increased expression in plaques, with LPAR6 localized in ECs and positively correlated to PLPP3. Utilizing in situ mass spectrometry imaging, LPA and its precursors were found in the plaque fibrous cap, co-localizing with PLPP3 and LPAR6. In vitro, PLPP3 silencing in ECs under LPA stimulation resulted in increased expression of adhesion molecules and cytokines. LPAR6 silencing inhibited LPA-induced cell activation, but not when PLPP3 was silenced simultaneously. Our results show that repression of PLPP3 plays a key role in atherosclerosis by promoting EC activation. Altogether, the PLPP3 pathway represents a suitable target for investigations into novel therapeutic approaches to ameliorate atherosclerosis.

Peripheral serotonin receptor 2B and transient receptor potential channel 4 mediate pruritus to serotonergic antidepressants in mice

Peripheral serotonin receptor 2B and transient receptor potential channel 4 mediate pruritus to serotonergic antidepressants in mice

Analysis of NFKB2‑mediated regulation of mechanisms underlying the development of Hodgkin's lymphoma.

Nuclear factor-κB (NF-κB) is widely involved in various lymphoid malignancies. However, its exact functional role and potential regulatory mechanisms in Hodgkin's lymphoma (HL) remains unclear. The present study aimed to investigate the regulatory mechanism of NF-κB in HL by analysis of a gene expression profile that was obtained from HL cells with or without NF-κB subunit 2 (NFKB2) knockdown. The GSE64234 dataset containing 6 HL cell line specimens transfected with small interfering (si)RNA against NFKB2 and 6 control specimens transfected with non-targeting siRNA sequences was downloaded from the Gene Expression Omnibus database. Based on these data, differentially expressed genes (DEGs) were screened for following data preprocessing. Functional enrichment analysis was subsequently conducted among the identified upregulated and downregulated DEGs. Additionally, a protein-protein interaction (PPI) network was constructed and module analyses were performed. Finally, microRNAs (miRNAs/miRs) targeting the identified DEGs were predicted for the construction of a miRNA-target regulatory network. A total of 253 DEGs were identified, consisting of 109 upregulated and 144 downregulated DEGs. Pathway enrichment analysis revealed that B-cell lymphoma 2-like 1 (BCL2L1) was significantly enriched in the NF-κB signaling pathway, and colony-stimulating factor 2 (CSF2) and BCL2L1 were enriched in the Jak-signal transducer and activator of transcription (STAT) signaling pathway. BCL2L1 and CSF2 were determined to be hub genes in the PPI network. A total of 6 miRNAs, including let-7a-5p, miR-9-5p, miR-155-5p, miR-135a-5p, miR-17-5p and miR-375, were identified in the miRNA-target regulatory network. The results of the present study indicated that NFKB2 may be involved in HL development through regulation of BCL2L1, CSF2, miR-135a-5p, miR-155-5p and miR-9-5p expression, as well as the modulation of Jak-STAT and NF-κB signaling pathways.

Piezo2 Mechanosensitive Ion Channel Role in Primary Enterochromaffin (EC) Cell Mechanosensitivity

INTRODUCTIONIntestinal enterochromaffin (EC) cells are the specialized epithelial mechanosensors of the gastrointestinal (GI) epithelium and are functionally and developmentally like Merkel cells in the skin. EC cells release serotonin (5‐HT) in response to mechanical stimuli. Recently, we discovered that like Merkel cells, human and mouse EC cells express the mechanosensitive (MS) ion channel Piezo2. However, little is known about the functional properties of this channel in primary EC cells.AIMTo determine the role of Piezo2 in primary EC cells.METHODSThe enteroendocrine cell transcription factor NeuroD1 was used to mark the late stage of EC cell development and generate NeuroD1‐cre::GCaMP5/tdTomato mouse to identify EC cells and examine their Ca2+ dynamics. We examined colocalization of tdTomato with 5‐HT and Piezo2 by immunohistochemistry. We established and used primary colon epithelium cultures for whole‐cell patch clamp and Ca2+ imaging experiments and mechanically stimulated by membrane displacement with a piezoelectrically driven glass probe or shear flow.RESULTSImmunostaining showed no tdTomato labeling outside of the GI epithelium, and epithelial tdTomato cells were 5‐HT+ and Piezo2+. Mechanical stimulation of primary EC cells by membrane displacement produced inward MS currents (20.8±4.9 pA/pF, Cm=3.5 pF, n=6) with fast activation and inactivation kinetics (τinact 11.1±2.8 ms), steep mechanosensitivity (Boltzmann function fit with a mid‐point of 3.46±0.80 μm and slope 0.79±0.27 μm) and a linear current‐voltage relationship (slope 0.61±0.17 pA/mV, x‐intercept −4.26±1.34 mV, n=5). Shear flow (20 s) transiently increased intracellular calcium ((Ca2+)i) (2.15±0.5 ΔF/F0, upstroke 3–5 s, return to baseline in 60 s, n=12). MS currents were inhibited by the MS ion channel blocker Gd3+ (20.8±4.9 pA/pF versus 5.3±1.9 pA/pF), Piezo channel blocker GsMTx‐4 (20.8±4.9 pA/pF versus 2.5±0.9 pA/pF) and Piezo2 siRNA (20.8±4.9 pA/pF versus 1.1±0.3 pA/pF), but not non‐targeted (NT) siRNA (20.8±4.9 pA/pF versus 16.0±5.2 pA/pF and) (n=4–6 for each condition, p&lt;0.05 for all except NT siRNA). Similarly, the (Ca2+)i responses to force were inhibited by Gd3+ (2.28±0.7 ΔF/F0 to 0.09±0.05 ΔF/F0), GsMTx‐4 (2.0±0.6 ΔF/F0 to 0.3±0.1 ΔF/F0), Piezo2 siRNA (2.15±0.5 ΔF/F0 to 0.08±0.05 ΔF/F0), but not NT siRNA (2.15±0.5 ΔF/F0 to 2.7±0.3 ΔF/F0) (n=4–6, p&lt;0.05 for all except NT siRNA).CONCLUSIONSIn the mouse epithelium, Piezo2+ EC cells were specifically marked by NeuroD1. Primary colonic NeuroD1+ cells had inward and non‐rectifying MS currents that could be blocked by Gd3+, GsMTx‐4 and Piezo2 siRNA. Similarly, shear flow evoked changes in (Ca2+)i that were also blocked by Piezo2 inhibitors and dependent on extracellular Ca2+. These results suggest that the MS ion channel Piezo2 acts as the primary EC cell mechanosensor, whose activation by force leads to MS ionic current and (Ca2+)i increase that may mediate mechanically‐induced 5‐HT release.Support or Funding InformationThis work was supported by NIH K08 DK106456, AGA Research Scholar Award, Mayo Clinic Center for Biomedical Discovery, Mayo Clinic Center for Cell Signaling in Gastroenterology (P30DK084567) and DK52766.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibin βE (INHBE) is a possible insulin resistance-associated hepatokine identified by comprehensive gene expression analysis in human liver biopsy samples.

The liver plays a major role in whole-body energy homeostasis by releasing secretory factors, termed hepatokines. To identify novel target genes associated with insulin resistance, we performed a comprehensive analysis of gene expression profiles using a DNA chip method in liver biopsy samples from humans with varying degrees of insulin resistance. Inhibin βE (INHBE) was identified as a novel putative hepatokine with hepatic gene expression that positively correlated with insulin resistance and body mass index in humans. Quantitative real time-PCR analysis also showed an increase in INHBE gene expression in independent liver samples from insulin-resistant human subjects. Additionally, Inhbe gene expression increased in the livers of db/db mice, a rodent model of type 2 diabetes. To preliminarily screen the role of Inhbe in vivo in whole-body energy metabolic status, hepatic mRNA was knocked down with siRNA for Inhbe (siINHBE) in db/db mice. Treatment with siINHBE suppressed body weight gain during the two-week experimental period, which was attributable to diminished fat rather than lean mass. Additionally, treatment with siINHBE decreased the respiratory quotient and increased plasma total ketone bodies compared with treatment with non-targeting siRNA, both of which suggest enhanced whole-body fat utilization. Our study suggests that INHBE functions as a possible hepatokine to alter the whole-body metabolic status under obese insulin-resistant conditions.

Phrenic motor neuron adenosine 2A receptors elicit phrenic motor facilitation.

Although adenosine 2A (A2A ) receptor activation triggers specific cell signalling cascades, the ensuing physiological outcomes depend on the specific cell type expressing these receptors. Cervical spinal adenosine 2A (A2A ) receptor activation elicits a prolonged facilitation in phrenic nerve activity, which was nearly abolished following intrapleural A2A receptor siRNA injections. A2A receptor siRNA injections selectively knocked down A2A receptors in cholera toxin B-subunit-identified phrenic motor neurons, sparing cervical non-phrenic motor neurons. Collectively, our results support the hypothesis that phrenic motor neurons express the A2A receptors relevant to A2A receptor-induced phrenic motor facilitation. Upregulation of A2A receptor expression in the phrenic motor neurons per se may potentially be a useful approach to increase phrenic motor neuron excitability in conditions such as spinal cord injury. Cervical spinal adenosine 2A (A2A ) receptor activation elicits a prolonged increase in phrenic nerve activity, an effect known as phrenic motor facilitation (pMF). The specific cervical spinal cells expressing the relevant A2A receptors for pMF are unknown. This is an important question since the physiological outcome of A2A receptor activation is highly cell type specific. Thus, we tested the hypothesis that the relevant A2A receptors for pMF are expressed in phrenic motor neurons per se versus non-phrenic neurons of the cervical spinal cord. A2A receptor immunostaining significantly colocalized with NeuN-positive neurons (89±2%). Intrapleural siRNA injections were used to selectively knock down A2A receptors in cholera toxin B-subunit-labelled phrenic motor neurons. A2A receptor knock-down was verified by a ∼45% decrease in A2A receptor immunoreactivity within phrenic motor neurons versus non-targeting siRNAs (siNT; P<0.05). There was no evidence for knock-down in cervical non-phrenic motor neurons. In rats that were anaesthetized, subjected to neuromuscular blockade and ventilated, pMF induced by cervical (C3-4) intrathecal injections of the A2A receptor agonist CGS21680 was greatly attenuated in siA2A (21%) versus siNT treated rats (147%; P<0.01). There were no significant effects of siA2A on phrenic burst frequency. Collectively, our results support the hypothesis that phrenic motor neurons express the A2A receptors relevant to A2A receptor-induced pMF.