Changes In Gene Signatures Research Articles

Administration of intratumoral GD2-directed interleukin-2 immunocytokine and local radiation therapy to activate immune rejection of spontaneous canine melanoma.

Canine malignant melanoma provides a clinically relevant, large animal parallel patient population to study the GD2-reactive hu14.18-IL-2 immunocytokine as it is similar to human melanoma and expresses GD2. The objectives of this study were to evaluate safety, radiation fractionation, and identify informative biomarkers of an in-situ tumor vaccine involving local radiation therapy plus intratumoral-immunocytokine in melanoma tumor-bearing dogs. Twelve dogs (six dogs/arm) with locally advanced or metastatic melanoma were randomized to receive a single 8 Gy fraction (arm A) or three 8 Gy fractions over 1 week (arm B) to the primary site and regional lymph nodes (when clinically involved) with the single or last fraction 5 days before intratumoral-immunocytokine at 12 mg/m 2 on 3 consecutive days. Serial tumor biopsies were obtained. All 12 dogs completed protocol treatment, and none experienced significant or unexpected adverse events. Evidence of antitumor activity includes one dog with a complete response at day 60, one dog with a partial response at day 60, and four dogs with mixed responses. Histology of serial biopsies shows a variably timed increase in intratumoral lymphocytic inflammation in some dogs. Canine NanoString analyses of serial biopsies identified changes in gene signatures of innate and adaptive cell types versus baseline. There were no significant differences in NanoString results between arm A and arm B. We conclude that intratumoral-immunocytokine in combination with local radiation therapy in canine melanoma is well tolerated and has antitumor activity with the potential to inform clinical development in melanoma patients.

Abstract 7615: Epithelial-mesenchymal-transition gene signature changes and poor oncological outcome in Candida-positive pancreatic ductal adenocarcinoma

Abstract Candida are commonly found in bile collected during surgery in pancreatic ductal adenocarcinoma (PDAC) patients. However, their significance in tumor biology and oncological outcome is unclear. PDAC patients receiving neoadjuvant therapy and pancreatectomy with intraoperative bile fungal culture were identified (N=40) in a prospective single-institution cohort. Tumor regression scores (TRS) were abstracted from pathology reports and correlated with bile fungal cultures. Bulk tumor RNASeq data were obtained from the Oncology Research Information Exchange Network (ORIEN). Tumor microbiome data of non-metastatic PDAC patients with upfront surgery were extracted using the {exotic} tool (Hoyd et al. Cancer Res. Commun., 2023). Gene expression profiles were compared between Candida+ and Candida- tumors, defined by a cutoff microbial count of 0. Overall survival (OS) of resected PDAC patients with Candida+ and Candida- tumors was compared using Kaplan Meier survival analysis. From the single-institution PDAC cohort, Candida+ bile was associated with poor pathological response to neoadjuvant therapy (Table 1). Using the ORIEN cohort, Candida were detected in 67% (106/158) of the PDAC tumors. Patients with Candida+ tumors had worse OS (median 28 vs. 56 months, Log-Rank p<0.008). Gene Set Enrichment Analysis (GSEA) using Hallmark gene sets showed an enrichment of epithelial-mesenchymal-transition (EMT) gene set and a reduction of metabolic and DNA repair gene sets in Candida+ tumors. GSEA for Immune signature and Tumor Immune Microenvironment Deconvolution (TIMEx) cell types showed an enrichment of Stromal-Fibroblast and EMT signature in Candida+ tumors. Further investigations are underway to determine the causal and metabolic relationship between Candida and this tumor microenvironment with EMT and stromal fibroblasts which is a well-known contributor to poor chemotherapy response and oncological outcome of PDAC. Table 1: Pathological Response to Neoadjuvant Therapy for PDAC Bile Culture TRS0/1 TRS2 TRS3 Candida - 5 (25%) 15 (75%) 0 (0%) Candida + 1 (5%) 11 (55%) 8 (40%) TRS: Tumor Regression Score (TRS0: complete response; TRS1: near-complete response; TRS2: partial response; TRS3: no response). Chi-Square Test, P=0.0035. Citation Format: Po Hien Ear, Min Hu, Jonathan S. Shilyansky, Naomi H. Fei, Sam Coleman, Rebecca Hoyd, Caroline E. Wheeler, Kelsey L. Steckly, Michelle L. Churchman, Nicholas Denko, Rebecca D. Dodd, Sheetal Hardikar, Ning Jin, Qin Ma, Martin D. McCarter, Abdul Rafeh Naqash, Afaf E. Osman, Gregory Riedlinger, Lary A. Robinson, Bryan P. Schneider, Eric A. Singer, Ahmad A. Tarhini, Gabriel Tinoco, Cornelia M. Ulrich, Yousef Zakharia, Daniel Spakowicz, Aik Choon Tan, Carlos H. Chan. Epithelial-mesenchymal-transition gene signature changes and poor oncological outcome in Candida-positive pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7615.

Epigenetic switch reshapes epithelial progenitor cell signatures and drives inflammatory pathogenesis in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a complex inflammatory skin disease with undefined mechanistic underpinnings. Here, we investigated HS epithelial cells and demonstrated that HS basal progenitors modulate their lineage restriction and give rise to pathogenic keratinocyte clones, resulting in epidermal hyperproliferation and dysregulated inflammation in HS. When comparing to healthy epithelial stem/progenitor cells, in HS, we identified changes in gene signatures that revolve around the mitotic cell cycle, DNA damage response and repair, as well as cell-cell adhesion and chromatin remodeling. By reconstructing cell differentiation trajectory and CellChat modeling, we identified a keratinocyte population specific to HS. This population is marked by S100A7/8/9 and KRT6 family members, triggering IL1, IL10, and complement inflammatory cascades. These signals, along with HS-specific proinflammatory cytokines and chemokines, contribute to the recruitment of certain immune cells during the disease progression. Furthermore, we revealed a previously uncharacterized role of S100A8 in regulating the local chromatin environment of target loci in HS keratinocytes. Through the integration of genomic and epigenomic datasets, we identified genome-wide chromatin rewiring alongside the switch of transcription factors (TFs), which mediated HS transcriptional profiles. Importantly, we identified numerous clinically relevant inflammatory enhancers and their coordinated TFs in HS basal CD49fhigh cells. The disruption of the S100A enhancer using the CRISPR/Cas9-mediated approach or the pharmacological inhibition of the interferon regulatory transcription factor 3 (IRF3) efficiently reduced the production of HS-associated inflammatory regulators. Our study not only uncovers the plasticity of epidermal progenitor cells in HS but also elucidates the epigenetic mechanisms underlying HS pathogenesis.

PM2.5 promotes pulmonary fibrosis by mitochondrial dysfunction.

Pulmonary fibrosis is known as an incurable lung disorder with irreversible progression of chronic injury, myofibroblast proliferation, extracellular matrix (ECM) accumulation, and tissue scarring. Atmospheric particulate matter 2.5 (PM2.5 ) is implicated as a risk factor of several diseases, especially lung diseases such as pulmonary fibrosis. The molecular mechanism which participates PM2.5 -induced pulmonary fibrosis in type II alveolar cells (AEII) has yet to be determined. Our results proved that short- and long-term exposure to PM2.5 significantly stimulated epithelial-mesenchymal transition (EMT) activity in AEII cells, according to, changes in gene signature analyzed by RNA-seq and cell morphology. Furthermore, Gene Ontology (GO) enrichment analysis also suggested that mitochondrial dysfunction was related to progression of pulmonary fibrosis in AEII after PM2.5 exposure. We observed a marked decline in mitochondria membrane potential (MMP), as well as fragmented mitochondria, in AEII cells exposed to PM2.5 , which suggests that energy metabolism is suppressed after PM2.5 exposure. We also confirmed that PM2.5 exposure could influence the expression levels of Mfn1, Mfn2, and Drp1 in AEII. Pretreatment of mitochondrial fusion promoter M1 was able to reverse mitochondrial dysfunction as well as EMT in AEII. These data suggested the key role of mitochondrial fragmentation in AEII, which was induced by PM2.5 exposure, and participated pathogenesis of pulmonary fibrosis. Finally, we investigated the response of lung tissue exposed to PM2.5 in vivo. The data indicated that the lung tissue exposed to PM2.5 obviously induced collagen accumulation. Moreover, IHC results revealed that PM2.5 enhanced Drp1 expression but suppressed Mfn1 and Mfn2 expression in lung tissue. The current study provides novel insight of pulmonary fibrosis caused by PM2.5 exposure.

Promising Novel Biomarkers and Candidate Drugs or Herbs in Osteoarthritis: Evidence from Bioinformatics Analysis of High‐throughput Data

Background: The most common joint illness is osteoarthritis (OA). The goal of this study was to find changes in gene signatures between normal knee joints and OA tissue samples and look for prospective gene targets for OA. Methods: The gene expression profiles of GSE12021, GSE51588, and GSE55457 were downloaded from Gene Expression Omnibus (GEO). A total of 64 samples (40 OA and 24 standard control samples) were used. The limma program was used to find differentially expressed genes (DEGs) in OA versus NC. Functional annotation and protein-protein interaction (PPI) network construction of OA-specific DEGs were performed. Finally, the candidate drugs and herbs as potential drugs to treat OA were predicted in the DGIdb and TCMIO databases. Results: A total of 19 upregulated and 27 downregulated DEGs between OA and NC samples were identified. DEGs, such as PTN, COMP, NELL1, and MN1, have shown a significant correlation with OA and are expected to become new biomarkers. Cellular senescence, positive regulation of ossification, and Vascular endothelial growth factor (VEGF) were significantly enriched for OA‐specific DEGs. In cell composition analysis, DEGs were also found to be highly enriched in the cytosol. We identified a total of 68 types of drugs or molecular compounds that are promising to reverse OA-related DEGs. Honeycomb and cinnamon oil have the possibility of treating OA. Conclusion: Our findings suggest new biomarkers that can be used to diagnose OA. Furthermore, we tried to find drugs and traditional Chinese medicine that may improve the progress of OA. This research may improve the identification and treatment of these uncontrollable chronic diseases.

T-cell infiltrates and microglia adopt long-term gene signature changes leading to age-specific responses to traumatic brain injury in mice

Abstract Aged traumatic brain injury (TBI) patients suffer increased mortality and long-term neurocognitive/neuropsychiatric morbidity than younger patients. Microglia, the resident macrophages of the brain, are complicit in both. We hypothesized that aged microglia would fail to return to a homeostatic state after TBI and adopt a long-term, injury-associated state within aged brains compared to young brains after TBI. Young and aged male C57BL/6 mice underwent TBI via controlled cortical impact vs. sham injury and were sacrificed four months post-TBI. We utilized single-cell RNA sequencing to examine age-associated cellular responses after TBI. Brains were harvested with CD45+ cells isolated via florescence-activated cell sorting. cDNA libraries were prepared via the 10x Genomics Chromium Single Cell 3’ Reagent Kit, followed by sequencing on a HiSeq 4000 instrument and computation analyses. Post-injury, aged mice demonstrated a proportional decrease in homeostatic microglia, and greater increased infiltrating T cells compared to young-adult mice. Of note, aged mice post-injury had a subpopulation of age-specific, immune-inflammatory microglia resembling gene profiles of neurodegenerative disease-associated microglia with enriched pathways involving in leukocyte recruitment. Contrastingly, post-injury, aged mice demonstrate a heterogenous T-cell infiltration with gene profiles corresponding to CD8 effector memory, CD8 native-like, CD4, and double-negative T cells and enriched pathways such as macromolecule synthesis. Taken together, our data showed that age-specific gene signature changes in the T-cell infiltrates and the microglial subpopulation contribute to increased vulnerability of the aged brain to TBI. Supported by R01 GM130662

Regulation of stress-induced hematopoiesis.

The hematopoietic compartment is tasked with the establishment and maintenance of the entire blood program in steady-state and in response to stress. Key to this process are hematopoietic stem cells (HSCs), which possess the unique ability to self-renew and differentiate to replenish blood cells throughout an organism's lifetime. Though tightly regulated, the hematopoietic system is vulnerable to both intrinsic and extrinsic factors that influence hematopoietic stem and progenitor cell (HSPC) fate. Here, we review recent advances in our understanding of hematopoietic regulation under stress conditions such as inflammation, aging, mitochondrial defects, and damage to DNA or endoplasmic reticulum. Recent studies have illustrated the vast mechanisms involved in regulating stress-induced hematopoiesis, including cytokine-mediated lineage bias, gene signature changes in aged HSCs associated with chronic inflammation, the impact of clonal hematopoiesis and stress tolerance, characterization of the HSPC response to endoplasmic reticulum stress and of several epigenetic regulators that influence HSPC response to cell cycle stress. Several key recent findings have deepened our understanding of stress hematopoiesis. These studies will advance our abilities to reduce the impact of stress in disease and aging through clinical interventions to treat stress-related outcomes.

Analysis of OX40 agonist antibody (PF-04518600) in patients with hepatocellular carcinoma.

523 Background: PF-04518600 (PF-8600) is a humanized agonist IgG2 monoclonal antibody to the tumor necrosis factor superfamily receptor OX40. PF-8600 was given to patients (pts) with advanced/metastatic hepatocellular carcinoma (HCC) in dose expansion of a phase 1 study (NCT02315066). Safety and tolerability were primary endpoints and exploratory endpoints included biomarker analyses. Methods: Pts received either 30 mg (Arm 1) or 250 mg (Arm 2) of PF-8600 intravenously Q2W. Pts had pathologic diagnosis of advanced HCC, a Child-Pugh score of A or B7, and had ≤2 prior lines of therapy, or if treatment naïve, had declined standard of care. Radiological tumor assessments were conducted Q8W. Biopsy samples collected at baseline and wk 6 were analyzed by immunohistochemistry and RNA sequencing for pharmacodynamic (PD) analyses. Whole blood samples were collected longitudinally for DNA extraction for high-throughput sequencing of the T cell receptor β-chain. Results: Arm 1 enrolled 16 pts (mean age 65.6 yrs; range 54-81 yrs; prior PD-1/PD-L1: 5 pts) and Arm 2 enrolled 19 pts (mean age 61.7 yrs; range 26-79 yrs; prior PD-1/PD-L1: 3 pts). All grade treatment related adverse events (TRAEs) occurred in 69% of pts in Arm 1 and 58% of pts in Arm 2. The rate of ≥Grade 3 TRAEs was 31% and 16% in Arms 1 and 2, respectively. For both arms combined, the most common (≥10%) TRAEs were rashes and pruritus. In Arm 1, 8 pts (50%) and in Arm 2, 10 pts (53%) achieved stable disease (SD), with a mean duration of 18.4 (range: 14.0-30.3 wks) and 17.4 wks (range: 8.0-31.9 wks), respectively. PD effects were more evident in Arm 1 than Arm 2, including increased OX40 tumor expression and positive changes in gene signatures, reflecting an active anti-tumor immune response. Conclusions: PF-8600 was generally well tolerated and provided meaningful disease control. While safety and efficacy were not significantly different between the 2 doses, there were potential differences in the PD data. The safety and relative durability of SD in HCC pts may provide a rationale for exploration of combination therapy in this pt population. Clinical trial information: NCT02315066.

Polarization of Adipose‐Derived Mesenchymal Stem/Stromal Cells by Toll‐Like Receptor Priming

Mesenchymal stem/stromal cells (MSCs) are a multipotent stromal cell population of interest for their potential use in cell therapy and immunotherapy due to many interesting traits, such as: their ability to home to injury sites, their ability to differentiate into cells of non‐hematopoietic lineages, and their secretion of paracrine factors with roles in tissue repair and immunomodulation. Similarly to how they home to injury sites, MSCs can also home to tumors, and there exert modulatory functions in the tumor microenvironment. Contrasting MSC effects on tumor progression have been reported in the literature, without great insight into what leads MSCs to exert one function or the other. Manipulating MSCs to exert an anti‐tumorigenic response represents a potential tool for the development of cancer therapeutics utilizing this cell population. In the past, it was shown that MSCs derived from human bone marrow (hBM‐MSCs) can be “polarized”, by toll‐like receptor (TLR) priming, into two phenotypes with opposite effects in tumor progression. In this model, TLR4 priming leads to an immune activating MSC1 phenotype with roles in tumor repression, whereas TLR3 priming induces a stronger immune suppressive MSC2 phenotype that promotes tumor progression. Human adipose‐derived MSCs (hASCs) are an alternative to hBM‐MSCs for therapeutic development, with the advantages of a relative ease of isolation and higher abundance in comparison to their bone marrow counterpart. Although the MSC1 and MSC2 polarization have not been reported in hASCs, we hypothesize that hASCs can also undergo MSC1/MSC2 polarization upon short term and low level TLR stimulation in a similar way to hBM‐MSCs. To test our hypothesis, we primed hASCs with TLR3 or TLR4 agonists and evaluated gene expression changes expected to occur, based on the previously described hBM‐MSCs polarization phenotypes. In our preliminary data, we have been able to observe, consistent with the literature, an upregulation of IL8 in TLR4 primed hASCs, and upregulation of CCL5, IP10 and IDO in TLR3 primed hASCs. On the other hand, gene expression remained unchanged for IL6, TGFβ1, and other associated genes. These results suggest that hASCs can polarize, but there are some differences in the gene signature changes induced by polarization of hBM‐MSCs and hASCs. In addition, some differences have been observed across different donor cells and priming conditions, which could contribute to the immunomodulatory inconsistencies reported in the literature. Current and future experiments aim to further characterize the gene expression signature changes on hASCs upon polarization, and to determine if these polarized phenotypes can, similarly to polarized hBM‐MSCs, differentially modulate tumor progression.Support or Funding InformationPurdue Doctoral Fellowship and Basic Medical Sciences Department, College of Veterinary Medicine, Purdue UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Association between changes in gene signatures expression and disease activity among patients with systemic lupus erythematosus

BackgroundWe assessed the stability of BAFF, interferon, plasma cell and LDG neutrophil gene expression signatures over time, and whether changes in expression coincided with changes in SLE disease activity.MethodsTwo hundred forty-three patients with SLE were evaluated for disease activity, serological parameters and peripheral blood gene signatures in clinic visits (2 or more per patient) that occurred between 2009 and 2012. Levels of the BAFF gene transcript, plasma cell signature, Interferon (IFN) signature and the low density granulocytes (LDG)-associated neutrophil gene signature were assessed in PAX-gene-preserved peripheral blood by global microarray. The stability of repeated measures of gene expression was quantified using intra-class correlation coefficients. SLE disease activity was measured using the Physicians Global Assessment and the SELENA-SLEDAI index and its components. Using a mixed effects regression model we assessed: 1) the association between a patient’s average gene signature expression over time and disease activity, and 2) the association between a patient’s changes in gene expression over time and changes in disease activity.ResultsGene expression signatures showed more within-person stability than systolic blood pressure. The IFN signature exhibited the most stability. Patients with high levels of BAFF and IFN transcripts tended to have significantly higher levels of musculoskeletal disease, skin disease, anti-dsDNA, and erythrocyte sedimentation rate, and lower levels of complement. However, changes in BAFF or IFN gene signatures were not associated with changes in disease activity. Similar associations were seen between the LDG gene signature and disease activity. However, when LDG increased, complement tended to increase. Patients with high levels of plasma cell gene signature tended to have higher levels of anti-dsDNA and lower levels of complement. However, unlike the other gene signatures, changes in plasma cell gene signature significantly coincided with changes in anti-dsDNA and complement.ConclusionsThe gene expression signatures were relatively stable within patients over time. BAFF and interferon gene expression were markers of patients with generally higher disease activity, but changes in these gene signatures did not coincide with changes in disease activity. Plasma Cell gene signature expression tracked with the traditional SLE serologic markers of anti-dsDNA and complement.

Correlation of cutaneous disease activity with type 1 interferon gene signature and interferon β in dermatomyositis.

Dermatomyositis (DM) is an autoimmune disease primarily affecting skin and muscle. The purpose of this study was to determine whether an association exists between clinical skin disease activity as measured by the validated Cutaneous Dermatomyositis Disease Area and Severity Index (CDASI) and type 1 interferon (IFN) pathway biomarkers in the blood of patients with DM. Forty-two patients with DM and 25 healthy volunteers were prospectively enrolled. CDASI scores were obtained, and serum and blood RNA were isolated from all participants. Associations between CDASI activity and type 1 IFN-inducible gene signature were assessed cross-sectionally in all patient samples and longitudinally on 13 paired visits via transcriptional profiling analyses. By RNAseq analysis, type 1 IFN-inducible genes were the most highly differentially regulated. A CDASI activity threshold of 12 was correlated with an elevated type 1 IFN gene signature and with serum IFN-β, but not with IFN-α protein. Expression analysis showed that all patients with mild disease activity had a low type 1 IFN gene signature, while 93% of patients with moderate-to-high disease activity had elevated gene signature. In longitudinal analysis, changes in CDASI activity showed nonsignificant trends with concordant directional changes in gene signature. A type 1 IFN pathway signature biomarker in blood is highly correlated with CDASI activity scores in DM, and may be a promising surrogate clinical trial end point. The correlation of serum IFN-β, but not IFN-α, with both a gene signature and CDASI suggests that IFN-β drives disease activity in DM.

Astrocyte-specific overexpressed gene signatures in response to methamphetamine exposure in vitro

BackgroundAstrocyte activation is one of the earliest findings in the brain of methamphetamine (Meth) abusers. Our goal in this study was to identify the characteristics of the astrocytic acute response to the drug, which may be critical in pathogenic outcomes secondary to the use.MethodsWe developed an integrated analysis of gene expression data to study the acute gene changes caused by the direct exposure to Meth treatment of astrocytes in vitro, and to better understand how astrocytes respond, what are the early molecular markers associated with this response. We examined the literature in search of similar changes in gene signatures that are found in central nervous system disorders.ResultsWe identified overexpressed gene networks represented by genes of an inflammatory and immune nature and that are implicated in neuroactive ligand-receptor interactions. The overexpressed networks are linked to molecules that were highly upregulated in astrocytes by all doses of methamphetamine tested and that could play a role in the central nervous system. The strongest overexpressed signatures were the upregulation of MAP2K5, GPR65, and CXCL5, and the gene networks individually associated with these molecules. Pathway analysis revealed that these networks are involved both in neuroprotection and in neuropathology. We have validated several targets associated to these genes.ConclusionsGene signatures for the astrocytic response to Meth were identified among the upregulated gene pool, using an in vitro system. The identified markers may participate in dysfunctions of the central nervous system but could also provide acute protection to the drug exposure. Further in vivo studies are necessary to establish the role of these gene networks in drug abuse pathogenesis.

Antiviral Nucleotide Incorporation by Recombinant Human Mitochondrial RNA Polymerase Is Predictive of Increased In Vivo Mitochondrial Toxicity Risk.

Nucleoside or nucleotide inhibitors are a highly successful class of antivirals due to selectivity, potency, broad coverage, and high barrier to resistance. Nucleosides are the backbone of combination treatments for HIV, hepatitis B virus, and, since the FDA approval of sofosbuvir in 2013, also for hepatitis C virus (HCV). However, many promising nucleotide inhibitors have advanced to clinical trials only to be terminated due to unexpected toxicity. Here we describe the in vitro pharmacology of compound 1, a monophosphate prodrug of a 2'-ethynyluridine developed for the treatment of HCV. Compound 1 inhibits multiple HCV genotypes in vitro (50% effective concentration [EC50], 0.05 to 0.1 μM) with a selectivity index of >300 (50% cytotoxic concentration [CC50], 30 μM in MT-4 cells). The active triphosphate metabolite of compound 1, compound 2, does not inhibit human α, β, or γ DNA polymerases but was a substrate for incorporation by the human mitochondrial RNA polymerase (POLRMT). In dog, the oral administration of compound 1 resulted in elevated serum liver enzymes and microscopic changes in the liver. Transmission electron microscopy showed significant mitochondrial swelling and lipid accumulation in hepatocytes. Gene expression analysis revealed dose-proportional gene signature changes linked to loss of hepatic function and increased mitochondrial dysfunction. The potential of in vivo toxicity through mitochondrial polymerase incorporation by nucleoside analogs has been previously shown. This study shows that even moderate levels of nucleotide analog incorporation by POLRMT increase the risk of in vivo mitochondrial dysfunction. Based on these results, further development of compound 1 as an anti-HCV compound was terminated.

Pyranocoumarin Tissue Distribution, Plasma Metabolome and Prostate Transcriptome Impacts of Sub-Chronic Exposure to Korean Angelica Supplement in Mice.

Herbal products containing Korean Angelica gigas Nakai (AGN) root extract are marketed as dietary supplements for memory enhancement, pain killing, and female menopausal symptom relief. We have shown the anticancer activities of AGN supplements in mouse models. To facilitate human anticancer translational research, we characterized the tissue distribution of AGN marker pyranocoumarin compounds decursin (D) and decursinol angelate (DA) ([Formula: see text]% in AGN) and their metabolite decursinol (DOH), assessed the safety of sub-chronic AGN dietary exposure in mice, and explored its impact on plasma aqueous metabolites and the prostate transcriptome. The data show that after a gavage dose, plasma contained readily detectable DOH, but little D and DA, mirroring patterns in the liver. Extra-hepatic tissues retained greater levels of DA and D than the liver did. For sub-chronic exposures, male mice were provided ad libitum AIN93M-pellet diets with 0.5 and 1% AGN for six weeks. No adverse effects were observed on the plasma biochemistry markers of liver and kidney integrity in spite of their enlargement. Histopathological examinations of the liver, kidney and other visceral organs did not reveal tissue abnormalities. Metabolomic assessment of plasma from mice fed the 1%-AGN diet suggested metabolic shifts of key amino acids especially in the methionine-cysteine cycle, purine cycle, and glycolysis-citrate cycle. Prostate transcriptomic profiling identified gene signature changes in the metabolisms of drugs, lipids and cellular energetics, neuro-muscular features, immunity and inflammation, and tumor suppressor/oncogene balance. The safety profile was corroborated with a daily [Formula: see text] injection of AGN extract (100-300[Formula: see text]mg/kg) for four weeks, which resulted in much greater systemic pyranocoumarin exposure than the dietary route did.

Transcriptome Dynamics of Developing Photoreceptors in Three-Dimensional Retina Cultures Recapitulates Temporal Sequence of Human Cone and Rod Differentiation Revealing Cell Surface Markers and Gene Networks.

The derivation of three‐dimensional (3D) stratified neural retina from pluripotent stem cells has permitted investigations of human photoreceptors. We have generated a H9 human embryonic stem cell subclone that carries a green fluorescent protein (GFP) reporter under the control of the promoter of cone‐rod homeobox (CRX), an established marker of postmitotic photoreceptor precursors. The CRXp‐GFP reporter replicates endogenous CRX expression in vitro when the H9 subclone is induced to form self‐organizing 3D retina‐like tissue. At day 37, CRX+ photoreceptors appear in the basal or middle part of neural retina and migrate to apical side by day 67. Temporal and spatial patterns of retinal cell type markers recapitulate the predicted sequence of development. Cone gene expression is concomitant with CRX, whereas rod differentiation factor neural retina leucine zipper protein (NRL) is first observed at day 67. At day 90, robust expression of NRL and its target nuclear receptor NR2E3 is evident in many CRX+ cells, while minimal S‐opsin and no rhodopsin or L/M‐opsin is present. The transcriptome profile, by RNA‐seq, of developing human photoreceptors is remarkably concordant with mRNA and immunohistochemistry data available for human fetal retina although many targets of CRX, including phototransduction genes, exhibit a significant delay in expression. We report on temporal changes in gene signatures, including expression of cell surface markers and transcription factors; these expression changes should assist in isolation of photoreceptors at distinct stages of differentiation and in delineating coexpression networks. Our studies establish the first global expression database of developing human photoreceptors, providing a reference map for functional studies in retinal cultures. Stem Cells 2015;33:3504–3518

Selenoglycoproteins attenuate adhesion of tumor cells to the brain microvascular endothelium via a process involving NF-κB activation

Selenium-containing compounds and selenized yeast have anticancer properties. In order to address possible mechanisms involved in these effects, selenoglycoproteins (SGPs) were extracted from selenium-enriched yeast at pH 4.0 and 6.5 (the fractions are called SGP40 and SGP65, respectively), followed by evaluation of their impact on the interactions of lung and breast tumor cells with human brain microvascular endothelial cells (HBMECs). Extracted SGPs, especially SGP40, significantly inhibited adhesion of tumor cells to HBMECs and their transendothelial migration. Because the active components of SGPs are unknown, small selenium-containing compounds [leucyl-valyl-selenomethionyl-arginine (LVSe-MR) and methylselenoadenosine (M-Se-A)], which are normally present in selenized yeast, were introduced as additional treatment groups. Treatment of HBMECs with SGP40, LVSe-MR and M-Se-A induced changes in gene signatures, which suggested a central involvement of nuclear factor (NF)-κB-dependent pathway. These observations were confirmed in the subsequent analysis of NF-κB DNA binding activity, quantitative measurements of the expression of selected genes and proteins, and tumor cell adhesion assay with a specific NF-κB inhibitor as the additional treatment factor. These findings indicate that specific organic selenium-containing compounds have the ability to inhibit tumor cell adhesion to brain endothelial cells via down-regulation of NF-κB. SGPs appear to be more effective than small selenium-containing compounds, suggesting the role of not only selenium but also the glycoprotein component in the observed protective impact.

Abstract 3492: γ-secretase inhibitor PF-03084014 diminishes the tumor-initiating cells and demonstrates synergy with docetaxel in breast cancer xenograft models

Abstract Notch signaling is known to be a survival pathway for tumor-initiating cells. In this report, we demonstrate that the γ-secretase inhibitor PF-03084014 significantly enhances the antitumor activity of docetaxel in multiple xenograft models of triple-negative breast cancer. Mechanistic evaluation revealed that PF-03084014 perturbs the Notch signaling pathway and suppresses the function of tumor initiating cells (TIC). In MDA-MB-231Luc model, treatment of docetaxel led to a significant increase of CD133+/CD44+ and ALDH+ subpopulations by FACS analysis. In combination with PF-03084014, these two unique cell subpopulations were significantly diminished. Correspondingly, the functional analyses by tumor re-implant and mammosphere-forming efficiency assays revealed that docetaxel-therapy promoted the tumor initiating capability of the remaining cells, in which an increased stem cell property and Notch pathway activation were observed through gene signature changes. In contrast, PF-03084014 co-treatment with docetaxel substantially hampered the self-renewal ability of these cells. Notch target gene analysis demonstrated the biological relevance of PF-03084014-induced activity. To characterize the function of CD133+/CD44+ subpopulation, MDA-MB-231Luc tumors were de-bulked by the treatment with docetaxel. Subsequently, the CD133+/CD44+ and CD133-/CD44- subpopulatons were isolated and re-implanted in SCID-bg mice using a limiting dilution approach. The results showed that CD133+/CD44+ cells gave rise to tumors with a 100 % take rate (10/10), whereas CD133-/CD44- cells were not tumorgenic (0/10). In addition, CD133+/CD44+ cells exhibited much higher tumorigenicity compared with the respective adherent parental cell line. PF-03084014 treatment caused a significant delay of CD133+/CD44+ tumor growth. The ability of PF-03084014 to suppress TICs was also observed in other breast cancer xenografts, including patient derived models. This data suggests that anti-TIC is one of the contributing mechanisms for the synergistic activities of PF-03084014 in combination with docetaxel. Our work provides potential therapeutic opportunities for PF-03084014 to improve conventional cytotoxic therapy by inhibiting Notch signaling in tumor-initiating cells and other bulk tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3492. doi:1538-7445.AM2012-3492