Downstream Gene Expression Changes Research Articles

Cohesin prevents cross-domain gene coactivation.

The contrast between the disruption of genome topology after cohesin loss and the lack of downstream gene expression changes instigates intense debates regarding the structure-function relationship between genome and gene regulation. Here, by analyzing transcriptome and chromatin accessibility at the single-cell level, we discover that, instead of dictating population-wide gene expression levels, cohesin supplies a general function to neutralize stochastic coexpression tendencies of cis-linked genes in single cells. Notably, cohesin loss induces widespread gene coactivation and chromatin co-opening tens of million bases apart in cis. Spatial genome and protein imaging reveals that cohesin prevents gene co-bursting along the chromosome and blocks spatial mixing of transcriptional hubs. Single-molecule imaging shows that cohesin confines the exploration of diverse enhancer and core promoter binding transcriptional regulators. Together, these results support that cohesin arranges nuclear topology to control gene coexpression in single cells.

Methylone is a rapid-acting neuroplastogen with less off-target activity than MDMA.

Post-traumatic stress disorder (PTSD) is a highly prevalent psychiatric disorder that can become chronic and debilitating when left untreated. Available pharmacotherapies are limited, take weeks to show modest benefit and remain ineffective for up to 40% of patients. Methylone is currently in clinical development for the treatment of PTSD. Preclinical studies show rapid, robust and long-lasting antidepressant-like and anxiolytic effects. The mechanism of action underlying these effects is not yet fully understood. This study investigated the downstream gene expression changes and signaling pathways affected by methylone in key brain areas linked to PTSD and MDD. It also sought to determine whether neuroplasticity-related genes were involved. We compared effects of methylone with MDMA to explore similarities and differences in their brain effects because MDMA-assisted psychotherapy has recently shown benefit in clinical trials for PTSD and methylone is a structural analog of MDMA. Monoamine binding, uptake and release studies were performed and a high-throughput-screen evaluated agonist/antagonist activities at 168 GPCRs in vitro. We used RNA sequencing (RNA-seq) to probe drug-induced gene expression changes in the amygdala and frontal cortex, two brain areas responsible for emotional learning that are affected by PTSD and MDD. Rats were treated with methylone or MDMA (both 10 mg/kg, IP), and their responses were compared with controls. We performed functional enrichment analysis to identify which pathways were regulated by methylone and/or MDMA. We confirmed changes in gene expression using immunohistochemistry. Methylone, a monoamine uptake inhibitor and releaser, demonstrated no off-target effects at 168 GPCRs, unlike MDMA, which showed activity at 5HT2A and 5HT2C receptors. RNA-seq results revealed significant regulation of myelin-related genes in the amygdala, confirmed by immunohistochemistry. In the frontal cortex, methylone significantly upregulated genes implicated in neuroplasticity. Results suggest that (1) methylone is a rapid-acting neuroplastogen that affects key brain substrates for PTSD and MDD and that (2) methylone appears to exhibit higher specificity and fewer off-target effects than MDMA. Together, these results are consistent with the reported clinical experiences of methylone and MDMA and bolster the potential use of methylone in the treatment of PTSD and, potentially, other neuropsychiatric disorders.

Cross-talk between QseBC and PmrAB two-component systems is crucial for regulation of motility and colistin resistance in Enteropathogenic Escherichia coli.

The quorum sensing two-component system (TCS) QseBC has been linked to virulence, motility and metabolism regulation in multiple Gram-negative pathogens, including Enterohaemorrhagic Escherichia coli (EHEC), Uropathogenic E. coli (UPEC) and Salmonella enterica. In EHEC, the sensor histidine kinase (HK) QseC detects the quorum sensing signalling molecule AI-3 and also acts as an adrenergic sensor binding host epinephrine and norepinephrine. Downstream changes in gene expression are mediated by phosphorylation of its cognate response regulator (RR) QseB, and 'cross-talks' with non-cognate regulators KdpE and QseF to activate motility and virulence. In UPEC, cross-talk between QseBC and TCS PmrAB is crucial in the regulation and phosphorylation of QseB RR that acts as a repressor of multiple pathways, including motility. Here, we investigated QseBC regulation of motility in the atypical Enteropathogenic E. coli (EPEC) strain O125ac:H6, causative agent of persistent diarrhoea in children, and its possible cross-talk with the KdpDE and PmrAB TCS. We showed that in EPEC QseB acts as a repressor of genes involved in motility, virulence and stress response, and in absence of QseC HK, QseB is likely activated by the non-cognate PmrB HK, similarly to UPEC. We show that in absence of QseC, phosphorylated QseB activates its own expression, and is responsible for the low motility phenotypes seen in a QseC deletion mutant. Furthermore, we showed that KdpD HK regulates motility in an independent manner to QseBC and through a third unidentified party different to its own response regulator KdpE. We showed that PmrAB has a role in iron adaptation independent to QseBC. Finally, we showed that QseB is the responsible for activation of colistin and polymyxin B resistance genes while PmrA RR acts by preventing QseB activation of these resistance genes.

Transcriptomic Signature and Functional Abnormalities of Bone Marrow Mesenchymal Stromal Cells Mediate Disease Progression of Myelodysplastic/Myeloproliferative Neoplasms

Background Mesenchymal stromal cells (MSC) are vital components of the bone marrow (BM) microenvironment niche and regulate hematopoietic stem and progenitor cell (HSPC) activity. While HSPC harbor primary disease driver mutations, MSC in myelodysplastic syndrome and myeloproliferative neoplasm (MDS/MPN) have been shown to influence the emergence and selection of leukemic clones and disease progression. However, it remains unclear if there are specific functional characteristics or genetic signals of MSC that interact with HSPC at different stages of disease progression. We examined BM samples from MDS/MPN patients with high rate of leukemic transformation in less than 2 years (HR) versus those with stable disease for 5 years (SD), to investigate the phenotypic differentiation, functional characteristics, and transcriptomic profiles of MSC and the HSPC counterparts. Methods We obtained BM samples at diagnosis and follow-up from MDS/MPN patients including chronic myelomonocytic leukemia (CMML) treated at Singapore General Hospital. We studied 5 HR and 13 SD patient samples to compare against 5 healthy donor samples. MSC were derived in MesenCult-ACF media (StemCell Technologies), and phenotypic characterization was done after 1-2 passages. HSPC were subjected to co-culture expansion with MSC in MyeloCult-H5100 for 7 days, and long-term culture-initiating cell assays (LTC-IC) using MSC as feeder cells for 6 weeks. Total RNA extracted from MSC and HSPC were subjected to bulk and single-cell RNA sequencing (scRNA-Seq) (10X Genomics platform). Principal component analysis (PCA) was used for the uniform manifold approximation and projection (UMAP). Gene set enrichment analysis (GSEA) was performed on differentially expressed genes to examine canonical signaling pathways. Cellchat algorithm was applied to predict intercellular communication between MSC and HSPC using scRNA-Seq data. Results Baseline clinical variables for the international prognostic scoring system (IPSS) including age at diagnosis, myeloblast percentage and cytogenetics were not significantly different between HR and SD patients. Decreased osteogenic and chondrogenic differentiation, but relatively preserved adipogenic differentiation capacity was observed in all MDS/MPN MSC samples. A significantly lower yield of CD34+ CD38- primitive HSPC with myeloid skewing was observed in the co-culture experiments with MDS/MPN MSC, compared to healthy MSC. Pre-treatment of SD-MSC feeder with the hypomethylating agent 5-azacytidine improved the clonogenic potential of the corresponding co-cultured HSPC, but did not restore the HSPC colony yield with HR-MSC feeder. Unbiased PCA of bulk RNA-Seq data demonstrated greater clustering of HR-MSC, while SD and healthy MSC overlapped significantly. Genes associated with mesenchymal multipotency and differentiation (PODXL20, FOXQ122, SERPINA923) were generally downregulated in MDS/MPN MSC, likely attributed to promoter hypermethylation. Significant differential upregulation of CD74 and IL-6 expression was observed in HR-MSC, which suggested a uniquely enriched inflammatory signature. GSEA results showed that pathways associated with G2M cell cycle checkpoint, DNA repair, glycolysis, and response to azacytidine were suppressed in HR-MSC. UMAP on scRNA-Seq data of longitudinal MSC samples identified distinct clustering of HR-MSC at disease progression. Cell-cell interaction analysis predicted a strong communication between collagen-encoding genes (COL1A1 and COL1A2) in HR-MSC at diagnosis and CD44, a negative regulator of HSPC proliferation, and the interaction was further enhanced at disease progression (Figure 1). Ongoing analysis of downstream gene expression changes and secretome profile of HSPC in MDS/MPN MSC-based in-vivo scaffold models will be presented. Conclusion Together, our data point towards a degree of co-development and communication between MSC and HSPC during disease progression of MDS/MPN. Further characterization of the unique functional and transcriptomic signatures in MSC may help to optimize disease prognostication and identify novel therapeutic targets in MDS/MPN patients.

Amyloid-β Pathology-Specific Cytokine Secretion Suppresses Neuronal Mitochondrial Metabolism

IntroductionNeuroinflammation and metabolic dysfunction are early alterations in Alzheimer’s disease (AD) brain that are thought to contribute to disease onset and progression. Glial activation due to protein deposition results in cytokine secretion and shifts in brain metabolism, which have been observed in AD patients. However, the mechanism by which this immunometabolic feedback loop can injure neurons and cause neurodegeneration remains unclear.MethodsWe used Luminex XMAP technology to quantify hippocampal cytokine concentrations in the 5xFAD mouse model of AD at milestone timepoints in disease development. We used partial least squares regression to build cytokine signatures predictive of disease progression, as compared to healthy aging in wild-type littermates. We applied the disease-defining cytokine signature to wild-type primary neuron cultures and measured downstream changes in gene expression using the NanoString nCounter system and mitochondrial function using the Seahorse Extracellular Flux live-cell analyzer.ResultsWe identified a pattern of up-regulated IFNγ, IP-10/CXCL10, and IL-9 as predictive of advanced disease. When healthy neurons were exposed to these cytokines in proportions found in diseased brain, gene expression of mitochondrial electron transport chain complexes, including ATP synthase, was suppressed. In live cells, basal and maximal mitochondrial respiration were impaired following cytokine stimulation.ConclusionsWe identify a pattern of cytokine secretion predictive of progressing amyloid-β pathology in the 5xFAD mouse model of AD that reduces expression of mitochondrial electron transport complexes and impairs mitochondrial respiration in healthy neurons. We establish a mechanistic link between disease-specific immune cues and impaired neuronal metabolism, potentially causing neuronal vulnerability and susceptibility to degeneration in AD.

Abstract 2792: Development of pharmacodynamic assays for quantifying SMARCA2 protein degradation and target gene expression in response to a SMARCA2 degrader (PRT3789)

Abstract The SWI/SNF complex plays an important role in controlling gene expression via chromatin remodeling. One of its catalytic subunits, SMARCA4, is frequently mutated in multiple tumor types, and SMARCA4-deficient cells are highly dependent on the other catalytic subunit, SMARCA2, for survival. Therefore, targeting SMARCA2 with selective protein degraders has therapeutic potential in SMARCA4 deficient human cancers. We have previously described the development of a potent and selective SMARCA2 targeted degrader, PRT3789, that demonstrates robust degradation of SMARCA2 protein and excellent preclinical efficacy in SMARCA4-del models (Hulse, et al. 2022). PRT3789 will be evaluated in a Phase 1 clinical trial in patients with SMARCA4-mutant cancer. In order to assess target engagement following clinical administration of PRT3789, we developed two pharmacodynamic (PD) assays to measure SMARCA2 protein degradation and changes in SMARCA2 target gene expression in human peripheral mononuclear cells (PBMCs). We used the MSD® S-PLEX platform for the development of a sensitive and quantitative, plate-based immunoassay for determining the protein concentration of SMARCA2 in human PBMC lysates. This assay exhibits a wide dynamic range of detection for SMARCA2 with an LLOD at 1.5 pg/mL, as well as exhibiting acceptable spike recovery and minimal cross-reactivity to SMARCA4. In addition, we optimized the PBMC isolation protocol and lysis conditions to enable sensitive detection of SMARCA2 protein. In order to demonstrate downstream gene expression changes as a consequence of SMARCA2 protein degradation, we developed a secondary qPCR assay. PBMCs from healthy human donors were treated ex vivo with PRT3789 for 24 - 48 hours, followed by RNA-sequencing to identify differentially expressed genes. We refined an 8-gene panel that showed robust expression in PBMCs, as well as consistent and dose-dependent changes in response to PRT3789 treatment. We further calculated a differential gene expression (DGE) score based on expression of this gene panel that correlates with SMARCA2 protein degradation. In summary, we describe the development and characterization of two independent clinically relevant PD assays that can be used to evaluate target engagement in blood samples following PRT3789 administration. Citation Format: Andrew Moore, Carly Bachner, Lalitha Srinivasan, Pradeep Kurup, Alex Grego, Caroline Vitkovitsky, Koichi Ito, Neha Bhagwat, Peggy Scherle. Development of pharmacodynamic assays for quantifying SMARCA2 protein degradation and target gene expression in response to a SMARCA2 degrader (PRT3789) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2792.

The Novel RXR Agonist MSU-42011 Differentially Regulates Gene Expression in Mammary Tumors of MMTV-Neu Mice

Retinoid X receptor (RXR) agonists, which activate the RXR nuclear receptor, are effective in multiple preclinical cancer models for both treatment and prevention. While RXR is the direct target of these compounds, the downstream changes in gene expression differ between compounds. RNA sequencing was used to elucidate the effects of the novel RXRα agonist MSU-42011 on the transcriptome in mammary tumors of HER2+ mouse mammary tumor virus (MMTV)-Neu mice. For comparison, mammary tumors treated with the FDA approved RXR agonist bexarotene were also analyzed. Each treatment differentially regulated cancer-relevant gene categories, including focal adhesion, extracellular matrix, and immune pathways. The most prominent genes altered by RXR agonists positively correlate with survival in breast cancer patients. While MSU-42011 and bexarotene act on many common pathways, these experiments highlight the differences in gene expression between these two RXR agonists. MSU-42011 targets immune regulatory and biosynthetic pathways, while bexarotene acts on several proteoglycan and matrix metalloproteinase pathways. Exploration of these differential effects on gene transcription may lead to an increased understanding of the complex biology behind RXR agonists and how the activities of this diverse class of compounds can be utilized to treat cancer.

Abstract C054: Characterizing differences in the chromatin accessibility landscape by donor ancestry in human triple-negative breast cancer cell lines

Abstract African American (AA) women are at increased risk of developing and dying from Triple-Negative Breast Cancer (TNBC), an aggressive breast cancer subtype, compared to European American (EA) women in the United States. In addition to social determinants, further investigation into biologic factors that contribute to these disparities is needed to fully understand this multi-factorial problem. In particular, the epigenetics of racial/population diversity and its influence on breast cancer incidence and outcomes remain underexplored. Using ATAC-sequencing, we characterized differences in chromatin accessibility between EA-derived versus AA-derived breast cancer cell lines across a range of breast cancer subtypes. Interestingly, our principal component analysis of the top 50,000 peaks with the most variance revealed separation of chromatin profiles by genetic ancestry specifically in TNBC cell lines (N=10). Digital footprinting analysis of differentially open chromatin regions using TOBIAS revealed significant differences in transcription factor (TF) binding by ancestry (61 TFs with a differential binding score > 0.25 or < -0.25 and FDR < 0.01). AA TNBC cell lines exhibited increased binding of transcription factors associated with epithelial-to-mesenchymal transition (ZEB1, SNAI1, GRHL2), cancer stemness and chemotherapeutic resistance (TFAP2C, NRF1), and others such as KAISO, whose aberrant expression has been previously linked to a distinct biology and poor outcomes in AA breast and prostate cancer patients. Pathway analysis of genes located within promoters of differentially open chromatin regions reveals enrichment of the following KEGG pathways in AA TNBC cells: inflammatory mediator regulation of TRP channels (p-adj = 0.009), Hippo signaling (p-adj = 0.006), Wnt signaling (p-adj = 0.031), and Rap-1 signaling (p-adj = 0.046). Together, these data reveal a differential chromatin landscape associated with the aberrant activity of critical TFs and downstream gene expression changes that may contribute to worsened TNBC biology in women of African ancestry. Additionally, as many of these cell lines are used routinely in biomedical research, these findings also indicate that the ancestral origin of patient derived cell lines matters and may contribute to biologic variation in experimental data, suggesting that inclusion of diversely sourced cell lines should be considered in experimental design. Citation Format: Alexandra R. Harris, Vishal Koparde, Gatikrushna Panigrahi, Maeve Bailey-Whyte, Tiffany Dorsey, Stefan Ambs. Characterizing differences in the chromatin accessibility landscape by donor ancestry in human triple-negative breast cancer cell lines [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C054.

EKLF/Klf1 regulates erythroid transcription by its pioneering activity and selective control of RNA Pol II pause-release.

EKLF/Klf1 is a zinc-finger transcription activator essential for erythroid lineage commitment and terminal differentiation. Using ChIP-seq, we investigate EKLF DNA binding and transcription activation mechanisms during mouse embryonic erythropoiesis. We utilize the Nan/+ mouse that expresses the EKLF-E339D (Nan) variant mutated in its conserved zinc-finger region and address the mechanism of hypomorphic and neomorphic changes in downstream gene expression. First, we show that Nan-EKLF limits normal EKLF binding to a subset of its sites. Second, we find that ectopic binding of Nan-EKLF occurs largely at enhancers and activates transcription through pioneering activity. Third, we find that for a subset of ectopic targets, gene activation is achieved in Nan/+ only by Nan-EKLF binding to distal enhancers, leading to RNA polymerase II pause-release. These results have general applicability to understanding how a DNA binding variant factor confers dominant disruptive effects on downstream gene expression even in the presence of its normal counterpart.

Molecular Mechanisms of Psilocybin and Implications for the Treatment of Depression.

Therapeutic deficiencies with monoaminergic antidepressants invites the need to identify and develop novel rapid-acting antidepressants. Hitherto, ketamine and esketamine are identified as safe, well-tolerated rapid-acting antidepressants in adults with treatment-resistant depression, and also mitigate measures of suicidality. Psilocybin is a naturally occurring psychoactive alkaloid and non-selective agonist at many serotonin receptors, especially at serotonin 5-HT2A receptors, and is found in the Psilocybe genus of mushrooms. Preliminary studies with psilocybin have shown therapeutic promise across diverse populations including major depressive disorder. The pharmacodynamic mechanisms mediating the antidepressant and psychedelic effects of psilocybin are currently unknown but are thought to involve the modulation of the serotonergic system, primarily through agonism at the 5-HT2A receptors and downstream changes in gene expression. It is also established that indirect effects on dopaminergic and glutamatergic systems are contributory, as well as effects at other lower affinity targets. Along with the direct effects on neurochemical systems, psilocybin alters neural circuitry and key brain regions previously implicated in depression, including the default mode network and amygdala. The aim of this review is to synthesize the current understanding of the receptor pharmacology and neuronal mechanisms underlying the psychedelic and putative antidepressant properties of psilocybin.

Renal Cell Carcinoma Associated with Germline Mutations in the Krebs Cycle

Germline mutations in the fumarate hydratase (FH) and succinate dehydrogenase (SDH) genes lead to hereditary leiomyomatosis and RCC (HLRCC) and hereditary paraganglioma and pheochromocytoma, respectively. The renal cell carcinomas that arise in these conditions are characterized by dysregulated Krebs cycles, accumulation of oncometabolites, downstream changes in gene expression, and epigenetic modifications that carry unique therapeutic implications. In this review, we evaluate the current literature on these tumors, including the epidemiology, clinical course, screening guidelines, and management of localized and metastatic disease.

Comparable Initial Engagement of Intracellular Signaling Pathways by Parathyroid Hormone Receptor Ligands Teriparatide, Abaloparatide, and Long-Acting PTH.

ABSTRACTMultiple analogs of parathyroid hormone, all of which bind to the PTH/PTHrP receptor PTH1R, are used for patients with osteoporosis and hypoparathyroidism. Although ligands such as abaloparatide, teriparatide (hPTH 1‐34 [TPTD]), and long‐acting PTH (LA‐PTH) show distinct biologic effects with respect to skeletal and mineral metabolism endpoints, the mechanistic basis for these clinically‐important differences remains incompletely understood. Previous work has revealed that differential signaling kinetics and receptor conformation engagement between different PTH1R peptide ligands. However, whether such acute membrane proximal differences translate into differences in downstream signaling output remains to be determined. Here, we directly compared short‐term effects of hPTH (1‐34), abaloparatide, and LA‐PTH in multiple cell‐based PTH1R signaling assays. At the time points and ligand concentrations utilized, no significant differences were observed between these three ligands at the level of receptor internalization, β‐arrestin recruitment, intracellular calcium stimulation, and cAMP generation. However, abaloparatide showed significantly quicker PTH1R recycling in washout studies. Downstream of PTH1R‐stimulated cAMP generation, protein kinase A regulates gene expression via effects on salt inducible kinases (SIKs) and their substrates. Consistent with no differences between these ligands on cAMP generation, we observed that hPTH (1‐34), abaloparatide, and LA‐PTH showed comparable effects on SIK2 phosphorylation, SIK substrate dephosphorylation, and downstream gene expression changes. Taken together, these results indicate that these PTH1R peptide agonists engage downstream intracellular signaling pathways to a comparable degree. It is possible that differences observed in vivo in preclinical and clinical models may be related to pharmacokinetic factors. It is also possible that our current in vitro systems are insufficient to perfectly match the complexities of PTH1R signaling in bona fide target cells in bone in vivo. © 2020 American Society for Bone and Mineral Research © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Model-based pathway enrichment analysis applied to the TGF-beta regulation of autophagy in autism

To differentiate between conditions of health and disease, current pathway enrichment analysis methods detect the differential expression of distinct biological pathways. System-level model-driven approaches, however, are lacking. Here we present a new methodology that uses a dynamic model to suggest a unified subsystem to better differentiate between diseased and healthy conditions. Our methodology includes the following steps: 1) detecting connections between relevant differentially expressed pathways; 2) construction of a unified in silico model, a stochastic Petri net model that links these distinct pathways; 3) model execution to predict subsystem activation; and 4) enrichment analysis of the predicted subsystem. We apply our approach to the TGF-beta regulation of the autophagy system implicated in autism. Our model was constructed manually, based on the literature, to predict, using model simulation, the TGF-beta-to-autophagy active subsystem and downstream gene expression changes associated with TGF-beta, which go beyond the individual findings derived from literature. We evaluated the in silico predicted subsystem and found it to be co-expressed in the normative whole blood human gene expression data. Finally, we show our subsystem’s gene set to be significantly differentially expressed in two independent datasets of blood samples of ASD (autistic spectrum disorders) individuals as opposed to controls. Our study demonstrates that dynamic pathway unification can define a new refined subsystem that can significantly differentiate between disease conditions.

Prenatal dexamethasone exposure induces nonalcoholic fatty liver disease in male rat offspring via the miR-122/YY1/ACE2-MAS1 pathway

Epidemiological studies have shown that nonalcoholic fatty liver disease (NAFLD) has an intrauterine developmental origin. We aimed to demonstrate that NAFLD is caused by prenatal dexamethasone exposure (PDE) in adult male rat offspring and to investigate the intrauterine programming mechanism. Liver samples were obtained on gestational day (GD) 21 and postnatal week (PW) 28. The effects and epigenetic mechanism of dexamethasone were studied with bone marrow mesenchymal stem cells (BMSCs) hepatoid differentiated cells and other cell models. In the PDE group, lipid accumulation increased, triglyceride synthesis-related gene expression increased, and oxidation-related gene expression decreased in livers of adult male rat offspring. In utero, hepatic triglyceride synthesis increased and oxidative function decreased in PDE fetal male rats. Moreover, low hepatic miR-122 expression, high Yin Yang-1 (YY1) expression and angiotensin-converting enzyme 2 (ACE2)-Mas receptor (MAS1) signaling pathway inhibition were observed before and after birth. At the cellular level, dexamethasone (100–2500 nM) elevated the intracellular triglyceride content, increased triglyceride synthesis-related gene expression and decreased oxidation-related gene expression. Dexamethasone treatment also decreased miR-122 expression, increased YY1 expression and inhibited the ACE2-MAS1 signaling pathway. Interference or overexpression of glucocorticoid receptor (GR), miR-122, YY1 and ACE2 could reverse the changes in downstream gene expression. In summary, PDE could induce NAFLD in adult male rat offspring. The programming mechanism included inhibition of miR-122 expression after GR activation, and dexamethasone increased hepatocyte YY1 expression; these effects resulted in ACE2-MAS1 signaling pathway inhibition, which led to increased hepatic triglyceride synthesis and decreased oxidative function. The increased triglyceride synthesis and decreased oxidative function of hepatocytes caused by low miR-122 expression due to dexamethasone could continue postnatally, eventually leading to NAFLD in adult rat offspring.

GINS2 attenuates the development of lung cancer by inhibiting the STAT signaling pathway.

GINS complex subunit 2 (GINS2) controls DNA replication. GINS2 expression is upregulated in several kinds of aggressive tumors. However, the effect of GINS2 in lung cancer remains unclear. We performed TCGA database analysis to confirm the clinical significance of GINS2 in lung cancer. After silencing GINS2 in A549 cells, we performed MTT assays, flow cytometry assays, colony formation assays, cell cycle analyses and RNA sequence analysis to elucidate the effect of GINS2 on lung cancer. Moreover, we assessed tumor growth and analyzed body fluorescence in mice as a measure of tumor burden. The TCGA database analysis demonstrated that GINS2 mRNA and protein was highly expressed in three kinds of lung cancer tissues. Subsequently, knockdown of GINS2 inhibited cell proliferation, colony formation, cell cycle arrest and apoptosis in A549 cells. On the other hand, we also investigated the effect of GINS2 on tumor formation in vivo. The analysis of nude mouse tumors showed that the tumor volume and weight of shGINS2 mice were significantly smaller than those of the control mice. To reveal the mechanism of GINS2 in lung cancer, we collected A549 cells with GINS2 knockdown to examine the downstream gene expression changes. The results showed that STAT1 and STAT2 mRNA and protein expression were significantly upregulated after GINS2 knockdown in A549 cells. Our results suggest that GINS2 inhibits the proliferation of lung cancer cells by inhibiting the STAT signaling pathway, which may be a potential biomarker for the diagnosis or prognosis of lung cancer.

Age-related hearing loss: Why we need to think about sex as a biological variable.

It has long been known that age-related hearing loss (ARHL) is more common, more severe, and with an earlier onset in men compared to women. Even in the absence of confounding factors such as noise exposure, these sexdifferences in susceptibility to ARHL remain. In the last decade, insight into the pleiotrophic nature by which estrogen signaling can impact multiple signaling mechanisms to mediate downstream changes in gene expression and/or elicit rapid changes in cellular function has rapidly gathered pace, and a role for estrogen signaling in the biological pathways that confer neuroprotection is becoming undeniable. Here I review the evidence why we need to consider sex as a biological variable (SABV) when investigating the etiology of ARHL. Loss of auditory function with aging is frequency-specific and modulated by SABV. Evidence also suggests that differences in cochlear physiology between women and men are already present from birth. Understanding the molecular basis of these sex differences in ARHL will accelerate the development of precision medicine therapies for ARHL.

Mass Spectrometry Untangles Plant Membrane Protein Signaling Networks.

Plasma membranes (PMs) act as primary cellular checkpoints for sensing signals and controlling solute transport. Membrane proteins communicate with intracellular processes through protein interaction networks. Deciphering these signaling networks provides crucial information for elucidating in vivo cellular regulation. Large-scale proteomics enables system-wide characterization of the membrane proteome, identification of ligand-receptor pairs, and elucidation of signals originating at membranes. In this review we assess recent progress in the development of mass spectrometry (MS)-based proteomic pipelines for determining membrane signaling pathways. We focus in particular on current techniques for the analysis of membrane protein phosphorylation and interaction, and how these proteins may be connected to downstream changes in gene expression, metabolism, and physiology.

Abstract 5506: NOTCH1 activation in head and neck squamous cell carcinoma leads to growth inhibition, changes in gene expression associated with early differentiation, and acquisition of stem cell-like properties

Abstract Background: The NOTCH1 gene functions as either an oncogene or tumor suppressor in cancer depending upon the tumor type. Our group previously characterized the genomic alterations in head and neck squamous cell carcinoma (HNSCC), discovering that NOTCH1 is frequently altered with a pattern of inactivating mutations suggesting it is a tumor suppressor in this cancer type. However, recent work by others suggests NOTCH1 signaling plays a more complex role, possibly promoting a more aggressive phenotype or cancer stem cell-like properties in HNSCCs with wild type NOTCH1. Our present study aimed to systematically compare the phenotypic consequences of NOTCH1 signaling in HNSCC to better understand its function in cancer, and identify targets downstream of NOTCH1 signaling. Methods: Established HNSCC cell lines wild type for NOTCH1 (PJ34, FADU) or harboring an inactivating mutation (UMSCC22A) were engineered to express activated cleaved NOTCH1 (cl-NOTCH1) from a doxycycline-inducible promoter. In vitro cell growth was measured with clonogenic assays. Stem cell-like properties were measured by orosphere formation and anoikis resistance. Stem cell markers for HNSCC including Aldehyde dehydrogenase activity (ALDH), CD133, and CD44 expression were measured by flow cytometry. NOTCH1-regulated downstream gene expression changes were examined by RNA-seq and qRT-PCR. Results: Activation of NOTCH1 inhibited clonogenic growth of all three cell lines, regardless of original NOTCH1 gene status. Growth inhibition was frequently accompanied by spontaneous formation of spheroid-like structures, characteristic of stem cells. NOTCH1 activation in UMSCC22A and FADU cells promoted orosphere formation and anoikis resistance, conveying some stem cell-like properties. However, classical stem cell markers including ALDH activity, CD133, and CD44 expression were not affected by NOTCH1 activation. Furthermore, RNA-seq demonstrated that critical cancer-associated pathways, including proliferation, differentiation, and migration, were regulated by NOTCH1. NOTCH1 activation downregulated gene expression of ITGA3, ITGA4, ITGB1, ITGB6, and LAMC2, which are key adhesion molecules that human basal keratinocytes use for attachment to the basement membrane and maintenance of the stem cell compartment. Concomitantly, NOTCH1 activation increased the basal/superbasal marker SOX2, but also the early differentiation markers KRT4 and KRT13. SiRNA-mediated SOX2 silencing blocked NOTCH1-promoted anoikis resistance. Conclusion: NOTCH1 activation inhibits in vitro growth regardless of mutational status. We hypothesize that stem cell-like properties associated with NOTCH1 activation in HNSCC may be a consequence of pathways that recapitulate early differentiation, rather than true stem cell maintenance. Citation Format: Chenfei Huang, Shhyam Moorthy, Qiuli Li, Rami Saade, Jiping Wang, Xiayu Rao, Noriaki Tanaka, Jiexin Zhang, Lin Tang, Curtis R. Pickering, Patrick A. Zweidler-McKay, Abdullah A. Osman, Tong-Xin Xie, Eve Shinbrot, Liu Xi, David Wheeler, Adel K. El-Naggar, Jing Wang, Jeffrey N. Myers, Mitchell J. Frederick. NOTCH1 activation in head and neck squamous cell carcinoma leads to growth inhibition, changes in gene expression associated with early differentiation, and acquisition of stem cell-like properties [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 5506.

Gene expression profiles of HTR8-S/Vneo cells after changes in ABCA1 expression.

ABCA1 is expressed in placental trophoblasts, such that when the expression level of ABCA1 changes, the function of trophoblasts dramatically changes. However, the mechanism by which ABCA1 affects the function of trophoblast cells remains unclear. Here, we used biochemical and transcriptomic to uncover the potential mechanism of the effect of ABCA1 on trophoblast function. HTR8/SVneo cells were either treated with the agonist T0901317 or transfected with siRNA to regulate ABCA1 expression levels. A human gene expression microarray was used to analyze the expression spectrum of ABCA1. Microarray results were confirmed by Western blotting and RT-PCR. Immunofluorescence allowed detection of the cellular localization of ABCA1, CCL8, CXCL10, CXCL11, and S1PR1 in HTR8/SVneo cells. Co-immunoprecipitation was used to test interactions among these proteins. Concomitant with an increase in ABCA1 expression, S1PR1 expression increased, whereas expression of CCL8, CXCL10, and CXCL11 decreased significantly; opposite effects were observed with a decrease in ABCA1 expression. Thus, changes in ABCA1 expression may lead to changes in downstream gene expression. Whereas the interaction between ABCA1 and S1PR1 was direct, interactions among ABCA1 and CCL8, CXCL10, and CXCL11 were indirect. We propose that, in conjunction with S1PR1, ABCA1 regulates expression levels of CCL8, CXCL10, and CXCL11; this may lead to changes in the immune function of trophoblastic cells. Thus, we suspect that the effect of ABCA1 on trophoblast function may involve many biological processes, molecular function changes, and the activation of multiple signaling pathways.

Long-term photoreceptor rescue in two rodent models of retinitis pigmentosa by adeno-associated virus delivery of Stanniocalcin-1

Retinal degenerations, including age-related macular degeneration and the retinitis pigmentosa family of diseases, are among the leading causes of legal blindness in the United States. We previously found that Stanniocalcin-1 (STC-1) reduced photoreceptor loss in the S334ter-3 and Royal College of Surgeons rat models of retinal degeneration. The results were attributed in part to a reduction in oxidative stress. Herein, we tested the hypothesis that long-term delivery of STC-1 would provide therapeutic rescue in more chronic models of retinal degeneration. To achieve sustained delivery, we produced an adeno-associated virus (AAV) construct to express STC-1 (AAV-STC-1) under the control of a retinal ganglion cell targeting promoter human synapsin 1 (hSYN1). AAV-STC-1 was injected intravitreally into the P23H-1 and S334ter-4 rhodopsin transgenic rats at postnatal day 10. Tissues were collected at postnatal day 120 for confirmation of STC-1 overexpression and histologic and molecular analysis. Electroretinography (ERG) was performed in a cohort of animals at that time. Overexpression of STC-1 resulted in a significant preservation of photoreceptors as assessed by outer nuclear thickness in the P23H-1 (P < 0.05) and the S334ter-4 (P < 0.005) models compared to controls. Additionally, retinal function was significantly improved in the P23H-1 model with overexpressed STC-1 as assessed by ERG analysis (scotopic b-wave P < 0.005 and photopic b-wave P < 0.05). Microarray analysis identified common downstream gene expression changes that occurred in both models. Genes of interest based on their function were selected for validation by quantitative real-time PCR and were significantly increased in the S334ter-4 model.