Gene Expression In Stem Cells Research Articles

Opposite effects of low intensity light of different wavelengths on the planarian regeneration rate

Planarian freshwater flatworms have the unique ability to regenerate due to stem cell activity. The process of regeneration is extremely sensitive to various factors, including light radiation. Here, the effect of low-intensity LED light of different wavelengths on regeneration, stem cell proliferation and gene expression associated with these processes was studied. LED matrices with different wavelengths (red (λmax = 635 nm), green (λmax = 520 nm) and blue (λmax = 463 nm), as well as LED laser diodes (red (λmax = 638.5 nm), green (λmax = 533 nm) and blue (λmax = 420 nm), were used in the experiments. Computer-assisted morphometry, whole-mount immunocytochemical study and RT-PCR were used to analyze the biological effects of LED light exposure on the planarian regeneration in vivo. It was found that a one-time exposure of regenerating planarians with low-intensity red light diodes stimulated head blastema growth in a dose-dependent manner (up to 40%). The green light exposure of planarians resulted in the opposite effect, showing a reduced head blastema growth rate by up to 21%. The blue light exposure did not lead to any changes in the rate of head blastema growth. The maximum effects of light exposure were observed at a dose of 175.2 mJ/cm2. No significant differences were revealed in the dynamics of neoblasts' (planarian stem cells) proliferation under red and green light exposure. However, the RT-PCR gene expression analysis of 46 wound-induced genes revealed their up-regulation upon red LED light exposure, and down-regulation upon green light exposure. Thus, we have demonstrated that the planarian regeneration process is rather sensitive to the effects of low-intensity light radiation of certain wavelengths, the biological activity of red and green light being dictated by the different expression of the genes regulating transcriptional activity.

Fenretinide, Tocilizumab, and Reparixin Provide Multifaceted Disruption of Oral Squamous Cell Carcinoma Stem Cell Properties: Implications for Tertiary Chemoprevention.

Locoregional recurrence of oral squamous cell carcinoma (OSCC) dramatically reduces patient survival. Further, as many OSCC recurrences are inoperable, radiotherapy and chemotherapy with or without biological adjuncts are the remaining treatment options. Although the tumors may initially respond, radiotherapy- and chemotherapy-resistant cancer stem cells (CSC) can readily repopulate OSCC tumors. Currently, following the initial OSCC treatment, patients are closely monitored until a recurrence or a second primary is detected. Identification of agents with complementary mechanisms to suppress CSC tumorigenic functions could change this passive approach. The goals of this study were twofold: (1) develop and validate CSC-enriched (CSCE) OSCC cell lines and (2) identify chemopreventive agents that obstruct multiple CSCE protumorigenic pathways. CSCE cultures, which were created by paclitaxel treatment followed by three tumorsphere passes, demonstrated CSC characteristics, including increased expression of stem cell and inflammatory genes, increased aldehyde dehydrogenase (ALDH) activity, and enhanced in vitro/in vivo proliferation and invasion. Three chemopreventives, fenretinide, tocilizumab, and reparixin, were selected due to their distinct and complementary CSC-disruptive mechanisms. The CSCE selection process modulated the cells' intermediate filaments resulting in an epithelial-predominant (enhanced cytokeratin, proliferation, IL6 release) line and a mesenchymal-predominant (upregulated vimentin, invasive, IL8 release) line. Our results confirm that 4HPR binds with appreciably higher affinity than Wnt at the Frizzled binding site and significantly inhibits CSC-enabling Wnt-β-catenin downstream signaling. Notably, combination fenretinide-tocilizumab-reparixin treatment significantly suppressed IL6 and IL8 release, stem cell gene expression, and invasion in these diverse CSCE populations. These promising multiagent in vitro data provide the basis for our upcoming in vivo CSCE tertiary chemoprevention studies.

Cladophora glomerata methanolic extract promotes chondrogenic gene expression and cartilage phenotype differentiation in equine adipose-derived mesenchymal stromal stem cells affected by metabolic syndrome

BackgroundChondrogenesis represents a highly dynamic cellular process that leads to the establishment of various types of cartilage. However, when stress-related injuries occur, a rapid and efficient regeneration of the tissues is necessary to maintain cartilage integrity. Mesenchymal stem cells (MSCs) are known to exhibit high capacity for self-renewal and pluripotency effects, and thus play a pivotal role in the repair and regeneration of damaged cartilage. On the other hand, the influence of certain pathological conditions such as metabolic disorders on MSCs can seriously impair their regenerative properties and thus reduce their therapeutic potential.ObjectivesIn this investigation, we attempted to improve and potentiate the in vitro chondrogenic ability of adipose-derived mesenchymal stromal stem cells (ASCs) isolated from horses suffering from metabolic syndrome.MethodsCultured cells in chondrogenic-inductive medium supplemented with Cladophora glomerata methanolic extract were experimented for expression of the main genes and microRNAs involved in the differentiation process using RT-PCR, for their morphological changes through confocal and scanning electron microscopy and for their physiological homeostasis.ResultsThe different added concentrations of C. glomerata extract to the basic chondrogenic inductive culture medium promoted the proliferation of equine metabolic syndrome ASCs (ASCsEMS) and resulted in chondrogenic phenotype differentiation and higher mRNA expression of collagen type II, aggrecan, cartilage oligomeric matrix protein, and Sox9 among others. The results reveal an obvious inhibitory effect of hypertrophy and a strong repression of miR-145-5p, miR-146-3p, and miR-34a and miR-449a largely involved in cartilage degradation. Treated cells additionally exhibited significant reduced apoptosis and oxidative stress, as well as promoted viability and mitochondrial potentiation.ConclusionChondrogenesis in EqASCsEMS was found to be prominent after chondrogenic induction in conditions containing C. glomerata extract, suggesting that the macroalgae could be considered for the enhancement of ASC cultures and their reparative properties.

Cerebrospinal Fluid and Photobiomodulation Effects on Neural Gene Expression in Dental Pulp Stem Cells.

Introduction: Dental pulp cells, a unique source of ectomesenchymal pluripotent stem cells, are originated from the skull neural crest. They are considered as one ideal source of cells for the regenerative medicine applications. Cerebrospinal fluid (CSF), a transparent fluid found in the brain and spinal cord, is enriched with electrolytes, proteins, and growth factors such as EGF, bFGF, BDNF, GDNF, and neuropeptides and can be utilized as a trigger in order to induce the neural differentiation. On the other hand, photobiomodulation (PBM), with the ability to prevent cell apoptosis, can induce cell proliferation by means of increasing the ATP synthesis in mitochondria and facilitating the secretion of the growth factors. In this research, we first aimed to isolate and culture the dental pulp stem cells (DPSCs) and subsequently to investigate their potential for neural differentiation. Methods: Human dental pulp stem cells (hDPSCs) were isolated from the pulp tissues using an outgrowth method and subsequently cultured. In order to access the cells' differentiation potential, cells were firstly classified into four groups which were treated with CSF, gallium aluminum arsenide diode laser irradiation (808 nm; 30 mW power output) and a combination of both, while the fourth group was considered as the control. MTT assay was then used to examine the viability of cells following the treatments. After 4, 7, and 14 days the cell morphology in the treated groups was evaluated while RT-PCR was used in order to evaluate the Nestin and β-tubulinIII neural gene marker expressions. Results: It was shown that PBM has the ability to elevate the proliferation of DPSCs. Also, the differentiated morphology was obvious in the CSF treated group, especially on day 14 with the formation of three-dimensional (3D) structures. The results of gene expression analysis showed that on the fourth day of post-treatment, Nestin and β-tubulinIII gene expressions were reduced in all groups while a rising trend in their expression was observed subsequently on days 7 and 14. Conclusion: In accordance with previous studies, including functional and protein base researches, it has been demonstrated that CSF has a direct role in neural induction. Although past works have been significant, none of them shows a 3D structure. In this article, we investigated the dual effect of PBM and CSF. Initial results confirmed the upregulation of neural-related transcription factors. The 3D organization of the formed tissue could imply the initiation of organogenesis which has not been reported before. In sum, the dual effect of CSF and PBM has been shown to have the potential for contributing to the initiation of neurogenesis and organogenesis.

Substrate stiffness affects the morphology and gene expression of epidermal neural crest stem cells in a short term culture.

According to the intrinsic plasticity of stem cells, controlling their fate is a critical issue in cell-based therapies. Recently, a growing body of evidence has suggested that substrate stiffness can affect the fate decisions of various stem cells. Epidermal neural crest stem cells as one of the main neural crest cell derivatives hold great promise for cell therapies due to presenting a high level of plasticity. This study was conducted to define the influence of substrate stiffness on the lineage commitment of these cells. Here, four different polyacrylamide hydrogels with elastic modulus in the range of 0.7-30 kPa were synthesized and coated with collagen and stem cells were seeded on them for 24 hr. The obtained data showed that cells can attach faster to hydrogels compared with culture plate and cells on <1 kPa stiffness show more neuronal-like morphology as they presented several branches and extended longer neurites over time. Moreover, the transcription of actin downregulated on all hydrogels, while the expression of Nestin, Tubulin, and PDGFR-α increased on all of them and SOX-10 and doublecortin gene expression were higher only on <1 kPa. Also, it was revealed that soft hydrogels can enhance the expression of glial cell line-derived neurotrophic factor, neurotrophin-3, and vascular endothelial growth factor in these stem cells. On the basis of the results, these cells can respond to the substrate stiffness in the short term culture and soft hydrogels can alter their morphology and gene expression. These findings suggested that employing proper substrate stiffness might result in cells with more natural profiles similar to the nervous system and superior usefulness in therapeutic applications.

Histone 1 Mutations Drive Lymphomagenesis By Inducing Primitive Stem Cell Functions and Epigenetic Instructions through Profound 3D Re-Organization of the B-Cell Genome

Melnick: Janssen: Research Funding; Epizyme: Consultancy; Constellation: Consultancy.

DNA Methylation-Based Classification Highlights the Role of the JAK-STAT Pathway in Acute Myeloid Leukemia

DNA Methylation-Based Classification Highlights the Role of the JAK-STAT Pathway in Acute Myeloid Leukemia

28P - GLPG 1790, a new selective EPHA2 inhibitor, is active in colorectal cancer cell lines belonging to the CMS4/mesenchymal-like subtype

BackgroundEPHA2 tyrosine kinase receptor is implicated in tumor progression, stemness phenotype and resistance to treatment in a wide range of cancers. We investigated the effects of GLPG1790, a new selective Eph receptor inhibitor, in colorectal cancer (CRC) across the 4 consensus molecular subtypes (CMS). MethodsWe tested the antiproliferative effect of GLPG 1790 used alone or in combination with either 5-fluorouracil, oxaliplatin or SN38 (the active metabolite of irinotecan) in a panel of 11 CRC cell lines encompassing the 4 consensus molecular subtypes (CMS). Cell cycle analysis was performed in order to understand possible cell cycle perturbation after treatment. Pathway analysis using western blot (WB) was also performed. We then evaluated the expression of stemness genes upon treatment using qRT-PCR. ResultsGLPG 1790 is active in CRC cell lines, with the strongest activity in the cell lines from the CMS4/mesenchymal-like cluster. Combination with chemotherapeutics is not synergistic according to Chou-Talalay model. The selective inhibitor elicits a persistent inactivation of EPHA2 receptor, associated to G0-G1 cell cycle block in the sensitive cell lines. Furthermore, GLPG 1790 is able to decrease the expression of cancer stem cell genes in cell lines belonging to the CMS4 group. ConclusionsEPHA2 blockade using the selective inhibitor GLPG 1790 has a strong antiproliferative effect in the chemorefractory subgroup of CMS4/mesenchymal-like CRC cell lines, associated to a G0-G1 cell cycle arrest.The stronger efficacy of GLPG1790 on the mesenchymal-like subtype is probably due to the impairment of cancer cell stemness and induction of cell differentiation after treatment. Legal entity responsible for the studyThe authors. FundingUniversità della Campania "Luigi Vanvitelli"; Galapagos NV (drug supply); Associazione Italiana per la Ricerca sul Cancro (AIRC). DisclosureP.P. Vitiello: Travel/Accommodation/Expenses: Amgen; Research grant/Funding (institution): Bayer; Research grant/Funding (institution): Merck; Research grant/Funding (institution): Roche; Research grant/Funding (institution): Servier; Research grant/Funding (institution): Ipsen; Travel/Accommodation/Expenses: BMS; Travel/Accommodation/Expenses: Sanofi. C. Cardone: Research grant/Funding (institution): Amgen; Research grant/Funding (institution): Bayer; Research grant/Funding (institution): Ipsen; Research grant/Funding (institution): Merck; Research grant/Funding (institution): Roche. D. Ciardiello: Travel/Accommodation/Expenses: Sanofi. L. Poliero: Travel/Accommodation/Expenses: BMS. C. Borrelli: Travel/Accommodation/Expenses: BMS. N. Zanaletti: Travel/Accommodation/Expenses: BMS. P. Vitale: Travel/Accommodation/Expenses: BMS. T. Troiani: Honoraria (self), Research grant/Funding (institution): Roche; Honoraria (self), Research grant/Funding (institution): Merck; Honoraria (self), Research grant/Funding (institution): Bayer; Honoraria (self), Travel/Accommodation/Expenses: Amgen; Travel/Accommodation/Expenses: Servier; Travel/Accommodation/Expenses: Sanofi; Travel/Accommodation/Expenses: Novartis. F. Ciardiello: Advisory/Consultancy, Research grant/Funding (institution): Amgen; Advisory/Consultancy, Research grant/Funding (institution): Bayer; Advisory/Consultancy, Research grant/Funding (institution): Roche; Advisory/Consultancy, Research grant/Funding (institution): Merck; Advisory/Consultancy: Servier; Advisory/Consultancy: Pfizer; Research grant/Funding (institution): Ipsen. E. Martinelli: Honoraria (self), Research grant/Funding (institution): Amgen; Honoraria (self), Research grant/Funding (institution): Merck; Honoraria (self), Research grant/Funding (institution): Bayer; Honoraria (self), Research grant/Funding (institution): Roche; Honoraria (self), Honoraria (institution): Servier. All other authors have declared no conflicts of interest.

Migration Gene Expression of Human Umbilical Cord Mesenchymal Stem Cells: A Comparison between Monophosphoryl Lipid A and Supernatant of Lactobacillus acidophilus.

The capacity of human umbilical cord mesenchymal stem cells (hUMSCs) for migration and homing is very important in regenerative medicine. A detoxified derivative of lipopolysaccharides (LPS) that lacks many of the endotoxic properties of LPS is monophosphoryl lipid A (MPLA). Effects of MPLA on the induction of MSCs migration, have not yet been studied. Also, studies have shown that supernatant of Lactobacillus acidophilus (SLA) culture medium, can stimulate the proliferation of macrophages and lymphocytes in vitro by affecting the properties of the chemotaxis and angiogenesis. Our present study aimed to understanding of the migration of hUMSCs during treatment with MPLA and SLA, separately. HUMSCs were isolated from human umbilical cord and were characterized by investigating surface markers (CD105, CD90, anti-CD29, CD45, and CD34) and their differentiation into adipogenic and osteogenic lineages. HUMSCs were treated with MPLA (10-3 µg/ml) and SLA (3 µl/ml). The morphological changes were not observed in both treated MSCs. Expression levelsof migrationmarkers were determined by quantitative reverse transcription PCR analysis on 2, 4, 6 days after treatment. Results showed that VEGF and MMP-2 but not CXCR-4 was increased in the presence of both treatments. Also, SLA led to a decrease and increase of the expression of VLA-4 and VCAM-1, respectively, while MPLA increased both VLA-4 and VCAM-1 expression.Therefore, it can be suggested that MPLA has more prominent results than SLA, but both treatments can probably be considered as an inducing factor in migration.

Surface Pre-Reacted Glass Filler Contributes to Tertiary Dentin Formation through a Mechanism Different Than That of Hydraulic Calcium-Silicate Cement.

The induction of tissue mineralization and the mechanism by which surface pre-reacted glass-ionomer (S-PRG) cement influences pulpal healing remain unclear. We evaluated S-PRG cement-induced tertiary dentin formation in vivo, and its effect on the pulp cell healing process in vitro. Induced tertiary dentin formation was evaluated with micro-computed tomography (μCT) and scanning electron microscopy (SEM). The distribution of elements from the S-PRG cement in pulpal tissue was confirmed by micro-X-ray fluorescence (μXRF). The effects of S-PRG cement on cytotoxicity, proliferation, formation of mineralized nodules, and gene expression in human dental pulp stem cells (hDPSCs) were assessed in vitro. μCT and SEM revealed that S-PRG induced tertiary dentin formation with similar characteristics to that induced by hydraulic calcium-silicate cement (ProRoot mineral trioxide aggregate (MTA)). μXRF showed Sr and Si ion transfer into pulpal tissue from S-PRG cement. Notably, S-PRG cement and MTA showed similar biocompatibility. A co-culture of hDPSCs and S-PRG discs promoted mineralized nodule formation on surrounding cells. Additionally, S-PRG cement regulated the expression of genes related to osteo/dentinogenic differentiation. MTA and S-PRG regulated gene expression in hDPSCs, but the patterns of regulation differed. S-PRG cement upregulated CXCL-12 and TGF-β1 gene expression. These findings showed that S-PRG and MTA exhibit similar effects on dental pulp through different mechanisms.

Characterization and radiosensitivity of HPV-related oropharyngeal squamous cell carcinoma patient-derived xenografts

Background: Oropharyngeal squamous cell carcinomas (OPSCC) are rising rapidly in incidence due to Human Papillomavirus (HPV) and/or tobacco smoking. Prognosis is better for patients with HPV-positive disease, but may also be influenced by tobacco smoking and other factors. There is a need to individualize treatment to minimize morbidity and improve prognosis. Patient-derived xenografts (PDX) is an emerging pre-clinical research model that may more accurately reflect the human disease, and is an attractive platform to study disease biology and develop treatments and biomarkers. In this study we describe the establishment of PDX models, compare PDX tumors to the human original, and assess the suitability of this model for radiotherapy research and biomarker development.Material and methods: Tumor biopsies from 34 patients with previously untreated OPSCC were implanted in immunodeficient mice, giving rise to 12 squamous cell carcinoma PDX models (7 HPV+, 5 HPV-). Primary and PDX tumors were characterized extensively, examining histology, immunohistochemistry, cancer gene sequencing and gene expression analysis. Radiosensitivity was assessed in vivo in a growth delay assay.Results: Established PDX models maintained histological and immunohistochemical characteristics as well as HPV-status of the primary tumor. Important cancer driver gene mutations, e.g., in TP53, PIK3CA and others, were preserved. Gene expression related to cancer stem cell markers and gene expression subtype were preserved, while gene expression related to hypoxia and immune response differed. Radiosensitivity studies showed high concordance with clinical observations.Conclusion: PDX from OPSCC preserves important molecular characteristics of the human primary tumor. Radiosensitivity were in accordance with clinically observed treatment response. The PDX model is a clinically relevant surrogate model of head and neck cancer. Perspectives include increased understanding of disease biology, which could lead to development of novel treatments and biomarkers.

The Trends in Global Gene Expression in Mouse Embryonic Stem Cells During Spaceflight.

The environment in space differs greatly from the environment on the ground. Spaceflight causes a number of physiological changes in astronauts, such as bone loss and immune system dysregulation. These effects threaten astronauts’ space missions, and understanding the underlying cellular and molecular mechanisms is important to manage the risks of space missions. The biological effects of spaceflight on mammalian cells, especially with regards to DNA damage, have attracted much attention. Rad9 −/− mouse embryonic stem cells (mESCs) are known to be extremely sensitive to DNA damage agents. In this study, a project of the SJ-10 satellite programme, we investigated the gene expression profiles of both Rad9 −/− mESCs and Rad9 +/+ (wild-type) mESCs in space with a focus on genes critical for inducing, preventing, or repairing genomic DNA lesions. We found that spaceflight downregulated more genes than it upregulated in both wild-type and Rad9 −/− mESCs, indicating a suppressive effect of spaceflight on global gene expression. In contrast, Rad9 deletion upregulated more genes than it downregulated. Of note, spaceflight mainly affected organ development and influenced a wide range of cellular functions in mESCs, while Rad9 deletion mainly affected the development and function of the hematological system, especially the development, differentiation and function of immune cells. The patterns of gene expression in mouse embryonic stem cells in space is distinct from those in other types of cells. In addition, both spaceflight and Rad9 deletion downregulated DNA repair genes, suggesting a possibility that spaceflight has negative effects on genome for embryonic stem cells and the effects are likely worsen when the genome maintenance mechanism is defective.

Paradoxical Role of Glypican-1 in Prostate Cancer Cell and Tumor Growth

Recent studies suggest that glypican-1 (GPC-1) is a biomarker for prostate cancer, but there are few studies elucidating the role of GPC-1 in prostate cancer progression. We observed high expression of GPC-1 in more aggressive prostate cancer cell lines such as PC-3 and DU-145. While inhibition of GPC-1 expression in PC-3 cells decreased cell growth and migration in vitro, it surprisingly increased cell proliferation and migration in DU-145 cells, suggesting that the role of GPC-1 is cell type-dependent. Further, GPC-1 inhibition increased PC-3 tumor size in NCr nude mice xenografts. We hypothesized that the discrepancy between the in vitro and in vivo data is mediated by stromal cells in the tumor microenvironment. Thus, we tested the effect of tumor conditioned media (TCM) on gene expression in human mesenchymal stem cells and fibroblasts. Treatment of stromal cells with TCM from PC-3 cells transfected with GPC-1 shRNA increased the expression of migration markers, endocrine/paracrine biomolecules, and extracellular matrix components. Additionally, the decreased cell growth in GPC-1 knockdown PC-3 cells was rescued by coculturing with stromal cells. These data demonstrate the paradoxical role that GPC-1 plays in prostate cancer cell growth by interacting with stromal cells and through ECM remodeling and endocrine/paracrine signaling.

Typical halogenated flame retardants affect human neural stem cell gene expression during proliferation and differentiation via glycogen synthase kinase 3 beta and T3 signaling

2′,2′,4,4′-tetrabromo diphenyl ether (BDE-47), one of the most abundant congeners of commercial pentaBDE utilized as flame retardants, has been phased out of production due to its potential neural toxicity and endocrine disrupting activities, and yet still present in the environment. Several alternatives to BDE-47, including tetrabromobisphenol A (TBBPA), tetrabromobisphenol S (TBBPS), tetrachlorobisphenol A (TCBPA) and decabromodiphenyl ether (BDE-209), are presently employed without restrictions and their potential toxic effects on human neural development are still unclear. In this study, we utilized a human neural stem cell (hNSC)-based system to evaluate the potential developmental neurotoxic effects of the above-mentioned five chemicals, at environment and human exposure relevant concentrations. We found that those compounds slightly altered the expression of hNSC identity markers (SOX2, SOX3 and NES), without impairing cell viability or proliferation, in part by either modulating glycogen synthase kinase 3 beta (GSK3β) signaling (TBBPS, TCBPA and BDE-47), and slightly disturbing the NOTCH pathway (TBBPA, TBBPS and TCBPA). Moreover, the five chemicals seemed to alter hNSC differentiation by perturbing triiodothyronine (T3) cellular signaling. Thus, our findings suggest that the five compounds, especially TBBPS, TCBPA, and BDE-47, may affect hNSC self-renewal and differentiation abilities and potentially elicit neural developmental toxicity.

Interleukin-33/ST2 Signaling Promotes Hepatocellular Carcinoma Cell Stemness Expansion Through Activating c-Jun N-terminal Kinase Pathway

BackgroundInterleukin-33 (IL-33) has a variety of biological activities in different pathological models. However, the underlying effects of IL-33 on hepatocellular carcinoma (HCC) have not been fully elucidated. Therefore, we focused on investigating the biological effects of IL-33 on HCC stemness expansion. Materials and MethodsIL-33 expression in clinical tissue specimens were evaluated using immunohistochemical technology. Western blotting, flow cytometry, and quantitative real-time polymerase chain reaction were used to detect protein expressions in HCC cell lines. ST2 expression was downregulated by utilizing the synthetic siRNA sequence that specifically targets ST2. The transfection of ST2 siRNAs and control siRNAs into HCC cells was performed with Lipofectamine RNAi MAX (Life Technologies) according to the manufacturer's protocol. ResultsOur results demonstrated that IL-33 is expressed both in cancer cells and stromal cells of the HCC microenvironment, and that IL-33 expression in cancer cells, but not in stromal cells, was negatively associated with survival of HCC patients. IL-33 promotes HCC stemness expansion, including upregulating core stem cell gene expression, inducing cell sphere formation and preventing chemotherapy-induced apoptosis in vitro. Mechanistically, IL-33 binds to its receptor ST2 and induces phosphorylation of c-Jun N-terminal kinase activation (JNK), which leads to HCC stemness expansion. ConclusionsIL-33/ST2 signaling might be potential prognostic markers and therapeutic targets for HCC patients.

ELK1 has a dual activating and repressive role in human embryonic stem cells.

Background: The ERK MAPK pathway plays a pivotal role in regulating numerous cellular processes during normal development and in the adult but is often deregulated in disease scenarios. One of its key nuclear targets is the transcription factor ELK1, which has been shown to play an important role in controlling gene expression in human embryonic stem cells (hESCs). ELK1 is known to act as a transcriptional activator in response to ERK pathway activation but repressive roles have also been uncovered, including a putative interaction with the PRC2 complex. Methods: Here we probe the activity of ELK1 in hESCs by using a combination of gene expression analysis in hESCs and during differentiation following ELK1 depletion and also analysis of chromatin occupancy of transcriptional regulators and histone mark deposition that accompany changes in gene expression. Results: We find that ELK1 can exert its canonical activating activity downstream from the ERK pathway but also possesses additional repressive activities. Despite its co-binding to PRC2 occupied regions, we could not detect any ELK1-mediated repression at these regions. Instead, we find that ELK1 has a repressive role at a subset of co-occupied SRF binding regions. Conclusions: ELK1 should therefore be viewed as a dichotomous transcriptional regulator that can act through SRF to generate both activating and repressing properties at different genomic loci.

Abstract 3670: Transcriptional co-activators TAZ/YAP are novel regulators of PAX3-FOXO1 transcriptional programing and fusion-positive rhabdomyosarcoma cancer cell stemness

Abstract Introduction: Fusion Positive Rhabdomyosarcoma (FP-RMS), a soft tissue sarcoma of adolescents and young adults, is driven by the oncogenic transcription factor PAX3-FOXO1 (P3F). Although most patients with FP-RMS initially respond to therapy, tumors frequently become refractory or relapse, underscoring a need to target resistance mechanisms and cancer stem cells. Because P3F is inherently disordered with no viable drug binding sites, it is currently not amendable to direct inhibition. Our prior work demonstrated that transcriptional co-activators TAZ and YAP are highly abundant in human FP-RMS and mediate several cancer phenotypes including apoptosis, differentiation, proliferation, and xenograft tumor growth. TAZ/YAP are essential co-activators for TEADs and AP1, which are among the top enriched transcription factor motifs at P3F binding sites. Here, we show that TAZ/YAP complex with P3F to positively regulate P3F transcriptional activity and that TAZ/YAP mediate FP-RMS stemness. Methods: Functional interactions of TAZ/YAP and P3F were investigated using P3F reporters, immunoblots, and co-immunoprecipitation (co-IP); while co-IP-coupled mass spectrometry (IP-MS) to identify the TAZ/YAP/P3F interactome is currently underway. For reporter assays, gain- and loss-of-function vectors expressing control, wild-type TAZ/YAP (WT), constitutively active TAZ/YAP (S89A/S127A), or shRNA knockdown (KD) were used. Epitope-tagged TAZ, YAP, and P3F were used for co-IPs and IP-MS. To investigate the role of TAZ/YAP in stemness, we evaluated the expression of stem cell genes in FP-RMS 3D-spheres expressing vector, WT, S89A/S127A, or KD. We are now performing RNA-Seq as an unbiased approach to examine genes and pathways regulating FP-RMS stemness. Results: TAZ/YAP functionally augment P3F transcriptional activity in reporter assays and also regulate expression of P3F targets. Immunoblotting of 293T cells co-expressing epitope-tagged proteins demonstrate an interaction between TAZ/YAP and P3F. A TAZ/YAP/P3F complex is also seen in co-IPs of endogenous proteins in FP-RMS cells. Serial passaging of spheres increases TAZ/YAP expression, suggesting these may be important for stem cell enrichment. Indeed, expression of SOX2, OCT4, and NANOG are TAZ/YAP-dependent, whereby their expression decreases with KD and increases with S89A/S127A. Functionally, TAZ/YAP are required for FP-RMS sphere formation in limiting dilution assays. Conclusions: These studies expand on prior work showing TAZ/YAP are potent oncoproteins in FP-RMS. We identify a novel complex between TAZ/YAP and P3F, show TAZ/YAP are positive regulators of P3F transcriptional activity, and demonstrate that TAZ/YAP regulate FP-RMS cancer cell stemness. Thus, targeting TAZ/YAP could be a vulnerability of P3F and for inhibiting the cancer stem cell population. Citation Format: Breanne A. Burgess, Katherine K. Slemmons, Corinne M. Linardic, Michael D. Deel. Transcriptional co-activators TAZ/YAP are novel regulators of PAX3-FOXO1 transcriptional programing and fusion-positive rhabdomyosarcoma cancer cell stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3670.

Monomethylfumarate protects against ovariectomy-related changes in body composition.

Osteoporosis, low bone mass that increases fracture susceptibility, affects approximately 75 million individuals in the United States, Europe and Japan, with the number of osteoporotic fractures expected to increase by more than 3-fold over the next 50 years. Bone mass declines with age, although the mechanisms for this decrease are unclear. Aging enhances production of reactive oxygen species, which can affect bone formation and breakdown. The multiple sclerosis drug Tecfidera contains dimethylfumarate, which is rapidly metabolized to monomethylfumarate (MMF); MMF is thought to function through nuclear factor erythroid-derived-2-like-2 (Nrf2), a transcription factor activated by oxidative stress which induces the expression of endogenous anti-oxidant systems. We hypothesized that MMF-elicited increases in anti-oxidants would inhibit osteopenia induced by ovariectomy, as a model of aging-related osteoporosis and high oxidative stress. We demonstrated that MMF activated Nrf2 and induced anti-oxidant Nrf2 target gene expression in bone marrow-derived mesenchymal stem cells. Sham-operated or ovariectomized adult female mice were fed chow with or without MMF and various parameters monitored. Ovariectomy produced the expected effects, decreasing bone mineral density and increasing body weight, fat mass, bone marrow adiposity and serum receptor activator of nuclear factor-kappa-B ligand (RANKL) levels. MMF decreased fat but not lean mass. MMF improved trabecular bone microarchitecture after adjustment for body weight, although the unadjusted data showed few differences; MMF also tended to increase adjusted cortical bone and to reduce bone marrow adiposity and serum RANKL levels. Because these results suggest the possibility that MMF might be beneficial for bone, further investigation seems warranted.

Overexpression of Nanog in amniotic fluid\u2013derived mesenchymal stem cells accelerates dermal papilla cell activity and promotes hair follicle regeneration

Alopecia, one of the most common chronic diseases, can seriously affect a patient’s psychosocial life. Dermal papilla (DP) cells serve as essential signaling centers in the regulation of hair growth and regeneration and are associated with crosstalk between autocrine/paracrine factors and the surrounding environment. We previously demonstrated that amniotic fluid–derived mesenchymal stem cell–conditioned medium (AF-MSC-CM) accelerates hair regeneration and growth. The present study describes the effects of overexpression of a reprogramming factor, Nanog, on MSC properties, the paracrine effects on DP cells, and in vivo hair regrowth. First, we examined the in vitro proliferation and lifespan of AF-MSCs overexpressing reprogramming factors, including Oct4, Nanog, and Lin28, alone or in combination. Among these factors, Nanog was identified as a key factor in maintaining the self-renewal capability of AF-MSCs by delaying cellular senescence, increasing the endogenous expression of Oct4 and Sox2, and preserving stemness. Next, we evaluated the paracrine effects of AF-MSCs overexpressing Nanog (AF-N-MSCs) by monitoring secretory molecules related to hair regeneration and growth (IGF, PDGF, bFGF, and Wnt7a) and proliferation of DP cells. In vivo studies revealed that CM derived from AF-N-MSCs (AF-N-CM) accelerated the telogen-to-anagen transition in hair follicles (HFs) and increased HF density. The expression of DP and HF stem cell markers and genes related to hair induction were higher in AF-N-CM than in CM from AF-MSCs (AF-CM). This study suggests that the secretome from autologous MSCs overexpressing Nanog could be an excellent candidate as a powerful anagen inducer and hair growth stimulator for the treatment of alopecia.

Fetal and trophoblast PI3K p110α have distinct roles in regulating resource supply to the growing fetus in mice.

Studies suggest that placental nutrient supply adapts according to fetal demands. However, signaling events underlying placental adaptations remain unknown. Here we demonstrate that phosphoinositide 3-kinase p110α in the fetus and the trophoblast interplay to regulate placental nutrient supply and fetal growth. Complete loss of fetal p110α caused embryonic death, whilst heterozygous loss resulted in fetal growth restriction and impaired placental formation and nutrient transport. Loss of trophoblast p110α resulted in viable fetuses, abnormal placental development and a failure of the placenta to transport sufficient nutrients to match fetal demands for growth. Using RNA-seq we identified genes downstream of p110α in the trophoblast that are important in adapting placental phenotype. Using CRISPR/Cas9 we showed loss of p110α differentially affects gene expression in trophoblast and embryonic stem cells. Our findings reveal important, but distinct roles for p110α in the different compartments of the conceptus, which control fetal resource acquisition and growth.