Gene Expression In Stem Cells Research Articles

Distinct and overlapping roles of STAG1 and STAG2 in cohesin localization and gene expression in embryonic stem cells

BackgroundThe three-dimensional organization of the genome in the nucleus plays an integral role in many biological processes, including gene expression. The genome is folded into DNA loops that bring together distal regulatory elements and genes. Cohesin, a ring-shaped protein complex, is a major player in the formation of DNA loops. Cohesin is composed of a core trimer and one of two variant STAG subunits, STAG1 or STAG2. It is not understood whether variant STAG proteins give rise to cohesin complexes with distinct functions. Recent studies have begun to characterize the roles of STAG1 and STAG2, with partially contradictory results.ResultsHere, we generate stable single-knockout embryonic stem cell lines to investigate the individual contributions of STAG1 and STAG2 in regulating cohesin chromosomal localization and function. We report both overlapping roles for STAG1 and STAG2 in cohesin localization and somewhat distinct roles in gene expression. STAG1 and STAG2 occupy the same sites across the genome, yet do not exist together in a higher order complex. Despite their shared localization, STAG1 and STAG2 have both distinct and redundant effects on gene expression. Loss of both STAG1 and STAG2 causes widespread transcriptome dysregulation, altered cohesin DNA occupancy, and reduced cell proliferation.ConclusionsTogether, this work reveals the requirement of at least one STAG protein for proper cohesin function. STAG1 and STAG2 have independent roles in cohesin localization and both overlapping and distinct roles in gene expression. The roles of STAG1 and STAG2 in mouse embryonic stem cells may be somewhat different than in other cell types, due to their relative expression levels. These results advance our understanding of the link between mammalian genome organization and gene expression during development and disease contexts.

Abstract A19: Regulation of glioblastoma tumor growth and stem cell properties through Gα12 and tissue factor, upstream and downstream players in YAP signaling

Abstract Glioblastoma multiforme (GBM), the most common and aggressive form of brain tumor, has been associated with the dysregulation of G-protein coupled receptors signaling. Data from The Cancer Genome Atlas demonstrated remarkable upregulation of mRNA for the Gα12 protein, which is elevated in 26% of GBM patient tumors (the highest rate of tumor samples surveyed). The Gα12 protein couples GPCRs to guanine nucleotide exchange factors (GEFs) for RhoA and was originally isolated as an oncogene. Furthermore, the subset of GPCRs that couples to Gα12 to activate RhoA includes S1P, LPA, and thrombin. These are generated, respectively, through sphingosine kinase, autotaxin, and tissue factor, all of which are elevated in glioblastoma tumors or cell lines. Signaling to RhoA leads to activation of the transcriptional co-activator YAP (Yes-associated protein 1). The role of YAP in growth of glioblastoma cells and in vivo GBM tumor development was explored in our previous studies (Yu et al., Oncogene 2018) using human glioblastoma cell lines and GBM stem cells (GSCs) derived from patient-derived xenografts (PDX). We showed that shRNA mediated knockdown of YAP in GSC-23 PDX cells significantly attenuated in vitro self-renewal capability assessed by limiting dilution, neurosphere formation, and stem cell gene expression. Orthotopic xenografts of the YAP knockdown PDX cells formed significantly smaller tumors and were of lower morbidity than wild-type cells. Our current work examines the effects of Gα12 since it transduces effects of multiple GPCRs upstream to RhoA that could be activated in the tumor environment. Targeting Gα12 using shRNA in two GBM cancer stem cell lines, GSC-23 and HK-281, decreased Gα12 mRNA levels by 80% without compensatory upregulation in the expression of the homologous Gα13 protein. Loss of Gα12 attenuated GSC-23 stemness as determined by limiting dilution assay and reduced mRNA expression levels of canonical stem cells genes (CCND1, NANONG, OCT4, SOX2, and NESTIN by approximately 50%). We are currently examining tumor growth of GSC-23 control and Gα12 knockdown cells as orthotopic xenografts. We are also examining the role of specific YAP-regulated target genes identified by RNA-sequencing of S1P-treated control and YAP knockout glioblastoma cells. In particular, one of the genes highly dependent on YAP is F3, which encodes the protein for tissue factor (TF) which is involved not only in coagulation but also in generation of thrombin, a ligand for PAR1, a GPCR that is upregulated in GBM and regulates RhoA signaling. Expression of TF was also selectively decreased in tumors from PDX cells lacking YAP, indicating that this transcriptional pathway is regulated in preclinical GBM models. Thus, Gα12/RhoA-mediated YAP signaling pathway appears to play a pivotal role in GBM tumor growth and progression. Citation Format: Olga M. Chaim, Jinhui Ma, Jacqueline Lara, Frank Furnari, Shigeki Myamoto, Joan H. Brown. Regulation of glioblastoma tumor growth and stem cell properties through Gα12 and tissue factor, upstream and downstream players in YAP signaling [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A19.

Investigation of bone reconstruction using an attenuated immunogenicity xenogenic composite scaffold fabricated by 3D printing

Bone is known to have a natural function to heal itself. However, if the bone damage is beyond a critical degree, intervention such as bone grafting may be imperative. In this work, the fabrication of a novel bone scaffold composed of natural bone components and polycaprolactone (PCL) using 3D printing is put forward. α1, 3-galactosyltransferase deficient pigs were used as the donor source of a xenograft. Decellularized porcine bone (DCB) with attenuated immunogenicity was used as the natural component of the scaffold with the aim to promote bone regeneration. The 3D printed DCB-PCL scaffolds combined essential advantages such as uniformity of the interconnected macropores and high porosity and enhanced compressive strength. The biological properties of the DCB-PCL scaffolds were evaluated by studying cell adhesion, viability, alkaline phosphatase activity and osteogenic gene expression of human bone marrow-derived mesenchymal stem cells. The in vitro results demonstrated that the DCB-PCL scaffolds exhibit an enhanced performance in promoting bone differentiation, which is correlated to the DCB content. Furthermore, critical-sized cranial rat defects were used to assess the effect of DCB-PCL scaffolds on bone regeneration in vivo. The results confirm that in comparison with PCL scaffolds, the DCB-PCL scaffolds can significantly improve new bone formation in cranial defects. Thus, the proposed 3D printed DCB-PCL scaffolds emerge as a promising regeneration alternative in the clinical treatment of large bone defects.

Quiescent stem cell marker genes in glioma gene networks are sufficient to distinguish between normal and glioblastoma (GBM) samples

Grade 4 glioma or GBM has poor prognosis and is the most aggressive grade of glioma. Accurate diagnosis and classification of tumor grade is a critical determinant for development of treatment pathway. Extensive genomic sequencing of gliomas, different cell types, brain tissue regions and advances in bioinformatics algorithms, have presented an opportunity to identify molecular markers that can complement existing histology and imaging methods used to diagnose and classify gliomas. ‘Cancer stem cell theory’ purports that a minor population of stem cells among the heterogeneous population of different cell types in the tumor, drive tumor growth and resistance to therapies. However, characterization of stem cell states in GBM and ability of stem cell state signature genes to serve as diagnostic or prognostic molecular markers are unknown. In this work, two different network construction algorithms, Weighted correlation network analysis (WGCNA) and Multiscale Clustering of Geometric Network (MEGENA), were applied on publicly available glioma, control brain and stem cell gene expression RNA-seq datasets, to identify gene network regulatory modules associated with GBM. Both gene network algorithms identified consensus or equivalent modules, HuAgeGBsplit_18 (WGCNA) and c1_HuAgeGBsplit_32/193 (MEGENA), significantly associated with GBM. Characterization of HuAgeGBsplit_18 (WGCNA) and c1_HuAgeGBsplit_32/193 (MEGENA) modules showed significant enrichment of rodent quiescent stem cell marker genes (GSE70696_QNPbyTAP). A logistic regression model built with eight of these quiescent stem cell marker genes (GSE70696_QNPbyTAP) was sufficient to distinguish between control and GBM samples. This study demonstrates that GBM associated gene regulatory modules are characterized by diagnostic quiescent stem cell marker genes, which may potentially be used clinically as diagnostic markers and therapeutic targets in GBM.

Combined Therapy of AXL and HDAC Inhibition Reverses Mesenchymal Transition in Diffuse Intrinsic Pontine Glioma.

Diffuse intrinsic pontine glioma (DIPG) is an incurable type of pediatric brain cancer, which in the majority of cases is driven by mutations in genes encoding histone 3 (H3K27M). We here determined the preclinical therapeutic potential of combined AXL and HDAC inhibition in these tumors to reverse their mesenchymal, therapy-resistant, phenotype. We used public databases and patient-derived DIPG cells to identify putative drivers of the mesenchymal transition in these tumors. Patient-derived neurospheres, xenografts, and allografts were used to determine the therapeutic potential of combined AXL/HDAC inhibition for the treatment of DIPG. We identified AXL as a therapeutic target and regulator of the mesenchymal transition in DIPG. Combined AXL and HDAC inhibition had a synergistic and selective antitumor effect on H3K27M DIPG cells. Treatment of DIPG cells with the AXL inhibitor BGB324 and the HDAC inhibitor panobinostat resulted in a decreased expression of mesenchymal and stem cell genes. Moreover, this combination treatment decreased expression of DNA damage repair genes in DIPG cells, strongly sensitizing them to radiation. Pharmacokinetic studies showed that BGB324, like panobinostat, crosses the blood-brain barrier. Consequently, treatment of patient-derived DIPG xenograft and murine DIPG allograft-bearing mice with BGB324 and panobinostat resulted in a synergistic antitumor effect and prolonged survival. Combined inhibition of AXL and HDACs in DIPG cells results in a synergistic antitumor effect by reversing their mesenchymal, stem cell-like, therapy-resistant phenotype. As such, this treatment combination may serve as part of a future multimodal therapeutic strategy for DIPG.

HOXA GENE EXPRESSION IMPLICATED IN TREATMENT RESISTANCE AND POOR PROGNOSIS IN GLIOBLASTOMA

This review brings into view the prognosis attributable to glioblastoma (GBM) and resistance to treatment, surgical interventions and chemotherapy seem ineffective at procuring a better prognosis for patients with the disease. Albeit there exist varying interventions for GBM, the median survival still comes to 12 to 15 months for afflicted patients, this has aroused the need for improvement in treatment success The principal goal is to create a better prognosis and have a decline in treatment resistance invariably leading to better survival rates via adequate treatment for GBM. A relationship exists between HOX genes (homeobox genes) and glioblastoma as is evident from literature. Treatment resistance has been observed in overexpression of HOX genes, the effectiveness of treatment could result from silencing these genes A series of studies have highlighted the role that HOX genes play in glioblastoma prognosis. Promotion of human glioblastoma initiation, aggressiveness, and resistance to Temozolomide has been associated with HOXA9 as shown by Pojo et al. The role of HOX gene expression in cancer stem cells should be studied as it could provide a means of designing CSC-targeted therapies, as CSCs play a part in initiation and progression of solid tumors.

The transcriptional corepressor CBFA2T3 inhibits all-trans-retinoic acid–induced myeloid gene expression and differentiation in acute myeloid leukemia

CBFA2/RUNX1 partner transcriptional co-repressor 3 (CBFA2T3, also known as MTG16 or ETO2) is a myeloid translocation gene family protein that functions as a master transcriptional corepressor in hematopoiesis. Recently, it has been shown that CBFA2T3 maintains leukemia stem cell gene expression and promotes relapse in acute myeloid leukemia (AML). However, a role for CBFA2T3 in myeloid differentiation of AML has not been reported. Here, we show that CBFA2T3 represses retinoic acid receptor (RAR) target gene expression and inhibits all-trans-retinoic acid (ATRA)-induced myeloid differentiation of AML cells. ChIP-Seq revealed that CBFA2T3 targets the RARα/RXRα cistrome in U937 AML cells, predominantly at myeloid-specific enhancers associated with terminal differentiation. CRISPR/Cas9-mediated abrogation of CBFA2T3 resulted in spontaneous and ATRA-induced activation of myeloid-specific genes in a manner correlated with myeloid differentiation. Importantly, these effects were reversed by CBFA2T3 re-expression. Mechanistic studies showed that CBFA2T3 inhibits RAR target gene transcription by acting at an early step to regulate histone acetyltransferase recruitment, histone acetylation, and chromatin accessibility at RARα target sites, independently of the downstream, RAR-mediated steps of transcription. Finally, we validated the inhibitory effect of CBFA2T3 on RAR in multiple AML subtypes and patient samples. To our knowledge, this is the first study to show that CBFA2T3 down-regulation is both necessary and sufficient for enhancing ATRA-induced myeloid gene expression and differentiation of AML cells. Our findings suggest that CBFA2T3 can serve as a potential target for enhancing AML responsiveness to ATRA differentiation therapies.

Tremella-Like ZnO@Col-I-Decorated Titanium Surfaces with Dual-Light-Defined Broad-Spectrum Antibacterial and Triple Osteogenic Properties.

The growing population of peri-implant diseases (PIDs) has become a public obsession, mainly due to the lack of antibacterial ability and osteogenic promotion of titanium (Ti) implants. Herein, inspired by tremella, we reported zinc oxide (ZnO)@collagen type I (Col-I)-decorated Ti for PIDs treatments. Compared with pure Ti implants, ZnO@Col-I-decorated Ti could be activated by a safe visible yellow light and showed excellent broad-spectrum antibacterial properties. The proliferation and osteogenic gene expression of bone marrow mesenchymal stem cells (BMSCs) indicated that the triple osseointegration of implants was realized through (I) the remarkedly improved surface hydrophilicity of ZnO@Col-I-decorated Ti, (II) the function of Col-I, and (III) the excellent near-infrared (NIR)-induced photothermal performance of ZnO. Collectively, the proposed dual-light-defined ZnO@Col-I coating was a promising implant surface modification system to provide customized treatments for each PID patient.

The lncRNA LAMP5-AS1 drives leukemia cell stemness by directly modulating DOT1L methyltransferase activity in MLL leukemia

BackgroundMixed-lineage leukemia (MLL) gene rearrangements trigger aberrant epigenetic modification and gene expression in hematopoietic stem and progenitor cells, which generates one of the most aggressive subtypes of leukemia with an apex self-renewal. It remains a challenge to directly inhibit rearranged MLL itself because of its multiple fusion partners and the poorly annotated downstream genes of MLL fusion proteins; therefore, novel therapeutic targets are urgently needed.MethodsqRT-PCR, receiver operating characteristic (ROC), and leukemia-free survival analysis were used to validate LAMP5-AS1 (LAMP5 antisense 1) expression and evaluate its clinical value. We performed in vitro and in vivo experiments to investigate the functional relevance of LAMP5-AS1 in MLL leukemia progression and leukemia cell stemness. RNA electrophoretic mobility shift assays (EMSA), histone methyltransferase assay, RNA pull-down assay, and RNA fluorescence in situ hybridization (FISH) were used to validate the relationship between LAMP5-AS1 and the methyltransferase activity of DOT1L. The downstream ectopic target genes of LAMP5-AS1/DOT1L were validated by the chromatin immunoprecipitation (ChIP) and western blot.ResultsWe discovered that a long noncoding RNA (lncRNA) LAMP5-AS1 can promote higher degrees of H3K79 methylation, followed by upregulated expression of the self-renewal genes in the HOXA cluster, which are responsible for leukemia stemness in context of MLL rearrangements. We found that LAMP5-AS1 is specifically overexpressed in MLL leukemia patients (n = 58) than that in the MLL-wt leukemia (n = 163) (p < 0.001), and the patients with a higher expression level of LAMP5-AS1 exhibited a reduced 5-year leukemia-free survival (p < 0.01). LAMP5-AS1 suppression significantly reduced colony formation and increased differentiation of primary MLL leukemia CD34+ cells. Mechanistically, LAMP5-AS1 facilitated the methyltransferase activity of DOT1L by directly binding its Lys-rich region of catalytic domain, thus promoting the global patterns of H3K79 dimethylation and trimethylation in cells. These observations supported that LAMP5-AS1 upregulated H3K79me2/me3 and the transcription of DOT1L ectopic target genes.ConclusionsThis is the first study that a lncRNA regulates the self-renewal program and differentiation block in MLL leukemia cells by facilitating the methyltransferase activity of DOT1L and global H3K79 methylation, showing its potential as a therapeutic target for MLL leukemia.

Blood Serum Stimulates p38-Mediated Proliferation and Changes in Global Gene Expression of Adult Human Cardiac Stem Cells.

During aging, senescent cells accumulate in various tissues accompanied by decreased regenerative capacities of quiescent stem cells, resulting in deteriorated organ function and overall degeneration. In this regard, the adult human heart with a generally low regenerative potential is of extreme interest as a target for rejuvenating strategies with blood borne factors that might be able to activate endogenous stem cell populations. Here, we investigated for the first time the effects of human blood plasma and serum on adult human cardiac stem cells (hCSCs) and showed significantly increased proliferation capacities and metabolism accompanied by a significant decrease of senescent cells, demonstrating a beneficial serum-mediated effect that seemed to be independent of age and sex. However, RNA-seq analysis of serum-treated hCSCs revealed profound effects on gene expression depending on the age and sex of the plasma donor. We further successfully identified key pathways that are affected by serum treatment with p38-MAPK playing a regulatory role in protection from senescence and in the promotion of proliferation in a serum-dependent manner. Inhibition of p38-MAPK resulted in a decline of these serum-mediated beneficial effects on hCSCs in terms of decreased proliferation and accelerated senescence. In summary, we provide new insights in the regulatory networks behind serum-mediated protective effects on adult human cardiac stem cells.

TNF‑α promotes the malignant transformation of intestinal stem cells through the NF‑κB and Wnt/β‑catenin signaling pathways.

Cancer stem cells are responsible for tumorigenesis, progression, recurrence and metastasis. Intestinal stem cells (ISCs) are regarded as the origin of intestinal neoplasia. Inflammation also serves an important role in intestinal neoplasia. To explore the molecular mechanisms underlying the inflammation-mediated induction of intestinal tumorigenesis, the present study investigated the function of tumor necrosis factor (TNF)-α in the malignant transformation of ISCs. NCM460 spheroid (NCM460s) cells with higher expression of stem cell genes, such as Oct4, Nanog, Sox2 and Lgr5, and with a higher ratio of CD133+, were obtained from NCM460 cells in serum-free medium. TNF-α accelerated cell proliferation, migration and invasion, induced chemotherapy resistance and the epithelial-mesenchymal transition. NF-κB and Wnt/β-catenin pathways were activated in TNF-α-induced inflammatory responses, leading to the nuclear translocation of p65 and β-catenin, as well as promoter activity of NF-κB and TCF/LEF transcription factors. It was further demonstrated that TNF-α-induced activation of the NF-κB and Wnt/β-catenin signaling pathways, as well as the upregulation of proinflammatory cytokines, were significantly suppressed by p65-knockdown. Notably, PDTC, an inhibitor of NF-κB signaling, reversed TNF-α-induced activation of the NF-κB and Wnt/β-catenin pathways. A similar role was observed for IWP-2, an inhibitor of Wnt/β-catenin signaling. Collectively, these results demonstrated that the NF-κB and Wnt/β-catenin pathways were activated to promote TNF-α-induced malignant transformation of ISCs, in which these two pathways cross-regulated each other.

Differential regulation of lineage commitment in human and mouse primed pluripotent stem cells by the nucleosome remodelling and deacetylation complex.

Differentiation of mammalian pluripotent cells involves large-scale changes in transcription and, among the molecules that orchestrate these changes, chromatin remodellers are essential to initiate, establish and maintain a new gene regulatory network. The Nucleosome Remodelling and Deacetylation (NuRD) complex is a highly conserved chromatin remodeller which fine-tunes gene expression in embryonic stem cells. While the function of NuRD in mouse pluripotent cells has been well defined, no study yet has defined NuRD function in human pluripotent cells. Here we find that while NuRD activity is required for lineage commitment from primed pluripotency in both human and mouse cells, the nature of this requirement is surprisingly different. While mouse embryonic stem cells (mESC) and epiblast stem cells (mEpiSC) require NuRD to maintain an appropriate differentiation trajectory as judged by gene expression profiling, human induced pluripotent stem cells (hiPSC) lacking NuRD fail to even initiate these trajectories. Further, while NuRD activity is dispensable for self-renewal of mESCs and mEpiSCs, hiPSCs require NuRD to maintain a stable self-renewing state. These studies reveal that failure to properly fine-tune gene expression and/or to reduce transcriptional noise through the action of a highly conserved chromatin remodeller can have different consequences in human and mouse pluripotent stem cells.

Abstract IA06: The tissue-specific microenvironment in primary tumors and metastases

Abstract Cancer cells within tumors exist in specialized niches in the microenvironment. The tumor cells are heterogeneous and can have properties as divergent as self-renewal, tumor initiation and repopulation potential, dormancy, evasion of cell death, and metastasis. Metastasis is the major cause of poor outcome in breast cancer. The microenvironment is critical for regulating multiple aspects of tumor progression and metastasis. Understanding the processes involved in metastasis is critical for developing new preventative and therapeutic strategies. We have analyzed the matrix environment and its function in tumor progression using in vivo imaging, genetic models of breast cancer, patient-derived xenografts, organotypic cultures, and patient samples. We have found that the matrix environment and the innate immune cells responding to the primary tumor and infiltrating the metastatic sites are critical for regulating tumor invasion and metastasis. Indeed, the inflammatory microenvironment of metastatic sites was distinct from that of the parental tumor and the normal tissue, even before metastasis takes hold. Interplay between nonmetastatic primary breast cancer and innate immune response acts to control metastatic progression. At the single-cell level, early-stage metastatic cells possess a distinct stemlike gene expression signature. The metastatic cells and the microenvironment are distinct in different metastatic sites and from the primary tumor. Micrometastatic cells are distinct due to their increased expression of basal, stem cell, prosurvival, and dormancy-associated genes. In contrast, cells from macrometastases are similar to primary tumor cells, which are more differentiated and heterogeneous. We validated our findings with a large primary breast cancer tissue microarray and pleural effusions from breast cancer patients. These results suggest that novel cancer immunotherapies that activate and augment the function of antitumoral myeloid subpopulations could have therapeutic potential and be clinically important for patients with advanced breast cancer. Citation Format: Zena Werb, Hugo Gonzalez, Juliane Winkler, Eric Berens. The tissue-specific microenvironment in primary tumors and metastases [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr IA06.

LncRNA, PVT-1, controls tumorigenesis and cancer stem-like phenotypes in osteosarcoma through PI3K/TSC2.

e23512 Background: Osteosarcoma is the most common pediatric bone cancer and a key genetic characteristic of this particular malignancy is its complex karyotype. Specifically it has been reported that 40% of osteosarcoma patients’ present with 8q24 amplification. The presence of this specific amplification has been previously associated with a high rate of relapse and poor prognosis for osteosarcoma patients. Within this amplicon resides, a long non-coding RNA gene, PVT-1. Prior studies indicates that PVT-1 has pro-oncogenic properties however the function of PVT-1 in osteosarcoma is not well characterized. Methods: To understand PVT-1 copy number, Fluorescent In Situ Hybridization was performed on both osteosarcoma cell lines and osteosarcoma patient-derived xenografts. In addition the PVT-1 RNA level is elevated in a majority of osteosarcoma samples compared to normal bone. To test PVT-1 pro-oncogenic role in osteosarcoma, several functional assays were performed. Results: Our studies demonstrated that overexpression of PVT-1 in osteosarcoma cell lines promotes multiple tumorigenic behaviors including enhanced proliferation, migration, invasion and chemotherapeutic resistance to cisplatin. PVT-1’s ability to mediate metastasis and contribute to chemotherapeutic sensitivity is a shared phenotype of cancer stem cells. Based on this observation, we hypothesize targeting PVT-1 will reduce cancer stem-cell properties. Osteosarcoma lines with increased levels of PVT-1 exhibited higher expression of cancer stem cell genes: Nanog, SOX2, c-Myc, and Oct4 at both the transcriptomic and proteomic level. In Vitro and In Vivo self-renewal capacity studies showed enhanced osteosarcoma cell self-renewal in the PVT-1 overexpression cohort. Additional molecular studies were performed in order to gain additional insights into potential mechanism of action for PVT-1 including Reverse Phase Protein Array. Initial analysis suggest a role for PVT-1 in regulating the PI3K-AKT-TSC2 pathway. Conclusions: This suggests a potential oncogenic pathway in which PVT-1 enhances cancer stem cell phenotypes. On-going investigations are addressing potential PI3K/TSC2 pathway inhibitors, BEZ-2335 and LY3023414, which could be utilized to regulate PVT-1 mediated tumorigenic roles and cancer stem-like properties.

Role of Chromatin Remodeling Genes and TETs in the Development of Human Midbrain Dopaminergic Neurons.

Understanding epigenetic regulation in the differentiation and maturation of dopaminergic neurons is critical to improve and develop new medications for Parkinson's disease (PD). To explore the role of ten-eleven translocation (TETs) family of dioxygenases and chromatin remodeling genes in the development of human midbrain dopaminergic (mDA) neurons, we globally analyze the epigenetic regulation of gene expression in human induced pluripotent stem cells (iPSCs) and iPSCs-derived mDA neurons. During the conversion of iPSCs into neuronal lineages of dopaminergic progenitors and mDA neurons, the expression patterns of epigenetic genes in multiple sets alter significantly. Vitamin C, an activator of TET enzymes, increases hydroxymethylcytosine (5hmC) level along with a higher yield of mDA neurons. Additionally, vitamin C treatment elevates gene expressions of TET2/3 and vitamin C transporters. Importantly, functional arrays indicate that vitamin C can promote neuronal maturation, synaptic activity, and dopamine release. Collectively, our study demonstrates that chromatin remodeling genes and the TET-5hmC pathway, which is regulated by vitamin C, are critical for the vital developmental stages of human mDA neurons.

Immunomodulatory Effect of Adipose-Derived Stem Cells: The Cutting Edge of Clinical Application.

Adipose-derived stem cells (ASCs) represent a promising tool for soft tissue engineering as well as for clinical treatment of inflammatory and autoimmune pathologies. The well-characterized multi-differentiation potential and self-renewal properties of ASCs are coupled with their immunomodulatory ability in providing therapeutic efficacy. Yet, their impact in immune or inflammatory disorders might rely both on cell contact-dependent mechanisms and paracrine effects, resulting in the release of various soluble factors that regulate immune cells functions. Despite the widespread use of ASCs in clinical trials addressing several pathologies, the pathophysiological mechanisms at the basis of their clinical use have been not yet fully investigated. In particular, a thorough analysis of ASC immunomodulatory potential is mandatory. Here we explore such molecular mechanisms involved in ASC immunomodulatory properties, emphasizing the relevance of the milieu composition. We review the potential clinical use of ASC secretome as a mediator for immunomodulation, with a focus on in vitro and in vivo environmental conditions affecting clinical outcome. We describe some potential strategies for optimization of ASCs immunomodulatory capacity in clinical settings, which act either on adult stem cells gene expression and local microenvironment. Finally, we discuss the limitations of both allogeneic and autologous ASC use, highlighting the issues to be fixed in order to significantly improve the efficacy of ASC-based cell therapy.

Regulation of glioblastoma tumor growth and stem cell properties through Gα12: an upstream player in YAP signaling

Glioblastoma multiforme (GBM), the most common and aggressive form of brain tumor, has been associated with the dysregulation of G‐protein coupled receptor signaling. Data from the Cancer Genome Atlas demonstrate remarkable upregulation of mRNA for the Gα12 protein, which is elevated in 26% of GBM patient tumors (the highest rate for all tumor samples surveyed). The Gα12 protein couples GPCRs to guanine nucleotide exchange factors (GEFs) for RhoA and was originally isolated as an oncogene. Furthermore, the subset of GPCRs that couples to Gα12 to activate RhoA includes S1P, LPA, and thrombin, inflammatory mediators that would be generated in the tumor cell environment. GPCR signaling to RhoA leads to activation of the transcriptional co‐activator YAP (Yes‐associated protein 1 gene). The role of YAP in growth of glioblastoma cells and in vivo GBM tumor development was explored in our previous studies (Yu et al. Oncogene 7(41): 5492–5507, 2018) using human glioblastoma cell lines and glioma stem cells (GSCs) derived from patient derived xenografts (PDX). We showed that shRNA mediated knockdown of YAP in GSC‐23 PDX cells significantly attenuated in vitro self‐renewal capability assessed by limiting dilution, oncogene expression, neurosphere formation, and stem cell gene expression. Orthotopic xenografts of the YAP knockdown PDX cells formed significantly smaller tumors and were of lower morbidity than wild‐type cells. Our current work examines the effects of Gα12, which would transduce effects of known and orphan GPCRs upstream that might be activated in the tumor environment to RhoA. We knocked down Gα12 using shRNA in two GBM cancer stem cell lines, GSC‐23 and HK‐281, decreasing Gα12 mRNA levels by 80% without compensatory upregulation in the expression of the homologous Gα13 protein. Loss of Gα12 attenuated GSC‐23 stemness as determined by limiting dilution assay and reduced mRNA levels for canonical stem cells genes (CCND1, NANONG, OCT4, SOX2 and NESTIN) by 35% to 60%. We are currently examining growth of GSC‐23 control and Gα12 knockdown cells following orthotopic injection into mouse brain. Histopathological findings revealed that the deletion of Gα12 reduced tumor‐derived cell migration and invasiveness. Gα12 knockdown tumors also presented with increased areas of necrosis. In vitro transwell assays confirmed that Gα12 knockdown reduced agonist‐stimulated GSC‐23 cell migration. We are exploring the role of YAP‐dependent genes (e.g. Thbs‐1, Dusp‐1, and CCN1) which were selectively decreased in tumors from PDX cells lacking Gα12 to determine their role in tumor cell migration and survival.Support or Funding InformationNIH grant CA218859

PIM protein kinases regulate the level of the long noncoding RNA H19 to control stem cell gene transcription and modulate tumor growth.

The proviral integration site for Moloney murine leukemia virus (PIM) serine/threonine kinases have an oncogenic and prosurvival role in hematological and solid cancers. However, the mechanism by which these kinases drive tumor growth has not been completely elucidated. To determine the genes controlled by these protein kinases, we carried out a microarray analysis in T‐cell acute lymphoblastic leukemia (T‐ALL) comparing early progenitor (ETP‐ALL) cell lines whose growth is driven by PIM kinases to more mature T‐ALL cells that have low PIM levels. This analysis demonstrated that the long noncoding RNA (lncRNA) H19 was associated with increased PIM levels in ETP‐ALL. Overexpression or knockdown of PIM in these T‐ALL cell lines controlled the level of H19 and regulated the methylation of the H19 promoter, suggesting a mechanism by which PIM controls H19 transcription. In these T‐ALL cells, the expression of PIM1 induced stem cell gene expression (SOX2, OCT‐4, and NANOG) through H19. Identical results were found in prostate cancer (PCa) cell lines where PIM kinases drive cancer growth, and both H19 and stem cell gene levels. Small molecule pan‐PIM inhibitors (PIM‐i) currently in clinical trials reduced H19 expression in both of these tumor types. Importantly, the knockdown of H19 blocked the ability of PIM to induce stem cell genes in T‐ALL cells, suggesting a novel signal transduction cascade. In PCa, increases in SOX2 levels have been shown to cause both resistance to the androgen deprivation therapy (ADT) and the induction of neuroendocrine PCa, a highly metastatic form of this disease. Treatment of PCa cells with a small molecule pan‐PIM‐i reduced stem cell gene transcription and enhanced ADT, while overexpression of H19 suppressed the ability of pan‐PIM‐i to regulate hormone blockade. Together, these results demonstrate that the PIM kinases control the level of lncRNA H19, which in turn modifies stem cell gene transcription regulating tumor growth.

An anti-oxidative cell culture dish inhibits intracellular reactive oxygen species accumulation and modulates pluripotency-associated gene expression in mesenchymal stem cells.

Maintenance of the pluripotent state of mesenchymal stem cells (MSCs) during in vitro expansion is an important factor for the successful proliferation of MSCs possessing high differentiation capacity. However, the differentiation potential of MSCs can easily be lost during in vitro expansion, particularly at late passages. Reactive oxygen species (ROS) are signaling molecules that help to maintain MSC function; however, excessive ROS generation can induce senescence and impair both the differentiation capacity and proliferation of MSCs. In this study, we have designed an amphiphilic block copolymer (redox copolymer), which possesses ROS scavenging capacity in the hydrophobic site. When this redox copolymer was coated on cell culture dishes coupled with human E-cadherin chimeric antibody (hE-cad-Fc), it had an antioxidative effect on cultured MSCs. We also confirmed that the redox polymer construct poly(ethylene glycol) tethered chain on the surface prevented nonspecific cell binding, whereas the co-immobilized surface allowed high adhesion of E-cadherin-positive MSCs. Interestingly, the intracellular ROS level was significantly decreased by the prepared cell culture dish, despite ROS being scavenged only on the surface of the dish, on the cell exterior. Consequently, the cultured MSCs retained high expression levels of pluripotency-associated genes, including SOX2.

Transcription Factor Binding in Embryonic Stem Cells Is Constrained by DNA Sequence Repeat Symmetry

Transcription factor (TF) recognition is dictated by the underlying DNA motif sequence specific for each TF. Here, we reveal that DNA sequence repeat symmetry plays a central role in defining TF-DNA-binding preferences. In particular, we find that different TFs bind similar symmetry patterns in the context of different developmental layers. Most TFs possess dominant preferences for similar DNA repeat symmetry types. However, in some cases, preferences of specific TFs are changed during differentiation, suggesting the importance of information encoded outside of known motif regions. Histone modifications also exhibit strong preferences for similar DNA repeat symmetry patterns unique to each type of modification. Next, using an in vivo reporter assay, we show that gene expression in embryonic stem cells can be positively modulated by the presence of genomic and computationally designed DNA oligonucleotides containing identified nonconsensus-repetitive sequence elements. This supports the hypothesis that certain nonconsensus-repetitive patterns possess a functional ability to regulate gene expression. We also performed a solution NMR experiment to probe the stability of double-stranded DNA via imino proton resonances for several double-stranded DNA sequences characterized by different repetitive patterns. We suggest that such local stability might play a key role in determining TF-DNA binding preferences. Overall, our findings show that despite the enormous sequence complexity of the TF-DNA binding landscape in differentiating embryonic stem cells, this landscape can be quantitatively characterized in simple terms using the notion of DNA sequence repeat symmetry.