Stem Cell Signaling Pathways Research Articles

Proteins of Wnt signaling pathway in cancer stem cells of human glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive primary glial brain tumor. The prognosis for GBM patients is not favorable, with the median survival time being 15 months. Its treatment resistance is associated with GBM cell population having cancer stem cells (CSCs). Wnt/β-catenin signaling pathway is a strategically important molecular mechanism, providing proliferation of stem cells of all types. This study compares the expression levels of signaling pathway proteins in CD133(+) CSCs and CD133(-) differentiated glioblastoma cells (DGCs). the present study used U-87MG cells of human glioblastoma, the material was tested for mycoplasma contamination. High-performance liquid chromatography (HPLC) mass spectrometry was used for proteome analysis. Biological and molecular functions, signaling pathways and protein-protein interactions were analyzed using free-access databases: PubMed, PANTHER, Gene Ontology, Swiss-Prot and KEGG. Protein-protein interactions (PPIs) were analyzed using the STRING database (version 10). There were identified 589 proteins with significantly changed expression in CD133+ CSCs, as compared with CD133-DGCs (P<0.05). Bioinformatics analysis allowed to attribute 134 differentially expressed proteins to 16 signaling pathways. A significant increase in expression of eight Wnt signaling pathway proteins (APC, CSNK1E, CSNK1A, CSNK2A2, CSNK2B, CTNNB1, DVL1, RUVBL) was detected, as well as four proteins of the non-canonical Wnt pathway-RHOA, ROCK2, RAC2, DAAM1. Special attention should be paid to β-catenin (CTNNB1) with more than 13.98-fold increase of expression in CSCs and Disheveled-associated activator of morphogenesis 1 (DAAM1) with 6.15-fold higher upregulation level. proteins of Wnt/β-catenin signaling cascade are a prospective target for regulating CSCs activity.

Decursin and Decursinol Angelate Suppress Adipogenesis through Activation of β-catenin Signaling Pathway in Human Visceral Adipose-Derived Stem Cells.

Visceral adiposity is closely associated with metabolic disorders and cardiovascular diseases. Angelica gigas Nakai (AGN) has been reported to possess anti-obesity effects and higher amounts of coumarin compounds are present in AGN. However, the active compounds suppressing adipogenesis in AGN and mechanisms of action have not been investigated in adipose-derived stem cells (ASCs) isolated from visceral adipose tissue (VAT). Among four coumarin compounds of AGN, decursin (D) and decursinol angelate (DA) significantly inhibited adipocyte differentiation from ASCs. D and DA downregulated CCAAT/enhancer binding protein α (C/EBPα), peroxisome proliferator-activated receptor γ (PPARγ), adipocyte fatty acid binding protein (aP2), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) at both mRNA and protein levels. Next, treatment with adipogenic differentiation medium (ADM) on ASCs downregulated β-catenin expression at protein level, while addition of D and DA could restore protein expression and nuclear translocation of β-catenin suppressed by ADM. D and DA treatment on ADM treated ASCs increased inhibitory phosphorylation of Glycogen synthase kinase (GSK)-3β, thereby preventing β-catenin from degradation. Additionally, si-β-catenin transfection significantly upregulated protein expression of C/EBPα and PPARγ, alleviating the anti-adipogenic effect of D and DA on ADM treated ASCs. Overall, D and DA, active compounds from AGN, suppressed adipogenesis through activation of β-catenin signaling pathway in ASCs derived from human VAT, possibly using as natural anti-visceral adiposity agents.

Sonic Hedgehog, a Novel Endogenous Damage Signal, Activates Multiple Beneficial Functions of Human Endometrial Stem Cells.

Local endometrial stem cells play an important role in regulating endometrial thickness, which is an essential factor for successful embryo implantation and pregnancy outcomes. Importantly, defects in endometrial stem cell function can be responsible for thin endometrium and subsequent recurrent pregnancy losses. Therefore, many researchers have directed their efforts toward finding a novel stimulatory factor that can enhance the regenerative capacity of endometrial stem cells. Sonic hedgehog (SHH) is a morphogen that plays a key role in regulating pattern formation throughout embryonic limb development. In addition to this canonical function, we identified for the first time that SHH is actively secreted as a stem cell-activating factor in response to tissue injury and subsequently stimulates tissue regeneration by promoting various beneficial functions of endometrial stem cells. Our results also showed that SHH exerts stimulatory effects on endometrial stem cells via the FAK/ERK1/2 and/or phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways. More importantly, we also observed that endometrial stem cells stimulated with SHH showed markedly enhanced differentiation and migratory capacities and subsequent invivo therapeutic effects in an endometrial ablation animal model.

Recent Advances of the Hippo/YAP Signaling Pathway in Brain Development and Glioma.

The Hippo signaling pathway is highly conserved from Drosophila melanogaster to mammals and plays a crucial role in organ size control, tissue regeneration, and tumor suppression. The Yes-associated protein (YAP) is an important transcriptional co-activator that is negatively regulated by the Hippo signaling pathway. The Hippo signaling pathway is also regulated by various upstream regulators, such as cell polarity, adhesion proteins, and other signaling pathways (the Wnt/β-catenin, Notch, and MAPK pathways). Recently, accumulated evidence suggests that the Hippo/YAP signaling pathway plays important roles in central nervous system development and brain tumor, including glioma. In this review, we summarize the results of recent studies on the physiological effect of the Hippo/YAP signaling pathway in neural stem cells, neural progenitor cells, and glial cells. In particular, we also focus on the expression of MST1/2, LATS1/2, and the downstream effector YAP, in glioma, and offer a review of the latest research of the Hippo/YAP signaling pathway in glioma pathogenesis. Finally, we also present future research directions and potential therapeutic strategies for targeting the Hippo/YAP signaling in glioma.

TGFβ Family Signaling Pathways in Pluripotent and Teratocarcinoma Stem Cells’ Fate Decisions: Balancing Between Self-Renewal, Differentiation, and Cancer

The transforming growth factor-β (TGFβ) family factors induce pleiotropic effects and are involved in the regulation of most normal and pathological cellular processes. The activity of different branches of the TGFβ family signaling pathways and their interplay with other signaling pathways govern the fine regulation of the self-renewal, differentiation onset and specialization of pluripotent stem cells in various cell derivatives. TGFβ family signaling pathways play a pivotal role in balancing basic cellular processes in pluripotent stem cells and their derivatives, although disturbances in their genome integrity induce the rearrangements of signaling pathways and lead to functional impairments and malignant transformation into cancer stem cells. Therefore, the identification of critical nodes and targets in the regulatory cascades of TGFβ family factors and other signaling pathways, and analysis of the rearrangements of the signal regulatory network during stem cell state transitions and interconversions, are key issues for understanding the fundamental mechanisms of both stem cell biology and cancer initiation and progression, as well as for clinical applications. This review summarizes recent advances in our understanding of TGFβ family functions in naїve and primed pluripotent stem cells and discusses how these pathways are involved in perturbations in the signaling network of malignant teratocarcinoma stem cells with impaired differentiation potential.

Investigating the Effect of Ibuprofen on DLL1 and NOTCH1 Expression in Gastric Cancer Stem Cells Derived from MKN-45 Cell Line

Background: Cancer stem cells (CSCs) harbor the self-renewal properties of the embryonic stem cells in addition to differentiation. While maintaining the balance between self-renewal and differentiation is required for homeostasis, dysregulation in the stem cell signaling pathways such as Notch signaling increases the proliferation of these cells. As a result, tumorigenic properties are obtained. Therefore, it appears that by targeting the CSCs, the generation of new cells could be reduced and the tumor could be contained. Ibuprofen is one of the nonsteroidal anti-inflammatory drugs that are in use to control pain and inflammation. They inhibit cyclooxygenase 1, 2 (COX1,2) activity and synthesis of prostaglandins. Recent studies have provided some evidence for the anti-tumor activities of NSAIDs through inhibition of cell proliferation. Methods: In this study, we investigate the changes in the expression patterns of two key genes, i.e., DLL1 and NOTCH1, which are involved in the Notch signaling pathway in the MKN-45 derived gastric CSCs treated with ibuprofen. Results: Our results showed that ibuprofen up-regulates the expression of the DLL1 gene (4.1 fold) and reduces 68% the transcript level of the NOTCH1 gene (Pvalue < 0.05). These findings show that in gastric CSCs, NOTCH1 gene may act as an oncogene and, conversely, DLL1 gene may act as a tumor suppressor gene. Conclusion: Our findings suggest that ibuprofen, by targeting CSCs, may be used as an adjuvant chemotherapy drug to improve gastric cancer treatment outcomes.

Identification of new cancer stem cell markers and signaling pathways in HER‑2‑positive breast cancer by transcriptome sequencing.

Human epidermal growth factor receptor (HER)-2-positive breast cancer accounts for ~25% of all breast cancer cases, has a high propensity for relapse, metastasis and drug resistance, and is associated with a poor prognosis. Therefore, it is necessary to develop more effective therapeutic targets for the treatment of HER-2-positive breast cancer. CD44+/CD24−/low is currently the most commonly used marker for breast cancer stem cells (CSCs), which are considered the main cause of drug resistance, relapse and metastasis. In the present study, the ratio of CD44+/CD24−/low cells was almost zero in SK-BR-3 cells; however, it was >90% in MDA-MB-231 cells, as determined by flow cytometry. Since SK-BR-3 and MDA-MB-231 cells both exhibit a strong propensity for invasion and migration, it was hypothesized that there may be other markers of CSCs in SK-BR-3 cells. Therefore, transcriptome sequencing was performed for SK-BR-3 and MDA-MB-231 cells. It was observed that several leukocyte differentiation antigens and other CSC markers were significantly more highly expressed in SK-BR-3 cells. Furthermore, the expression of aldehyde dehydrogenase (ALDH)1A3, CD164 and epithelial cell adhesion molecule (EpCAM) was higher in SK-BR-3 cells compared with in other subtypes of breast cell lines, as determined by reverse transcription-polymerase chain reaction and western blot analysis. In addition, the expression levels of ALDH1A3, ALDH3B2 and EpCAM were higher in HER-2-positive breast cancer compared with in paracancerous tissues and other subtypes of breast cancer, as determined by immunohistochemistry. The expression of β-catenin in the Wnt signaling pathway was lower in SK-BR-3 cells compared with in MDA-MB-231 cells, which may be used as a prognostic indicator for breast cancer. These findings may help identify novel CSC markers and therapeutic targets for HER-2-positive breast cancer.

Alteration of Hippo signaling pathway in the neural stem cells derived from the patients with mental retardation

Alteration of Hippo signaling pathway in the neural stem cells derived from the patients with mental retardation

Osteostimulatory effect of biocomposite scaffold containing phytomolecule diosmin by Integrin/FAK/ERK signaling pathway in mouse mesenchymal stem cells

Non-availability of an ideal alternative for autografts in treating critical-size bone defects is a major challenge in orthopedics. Phytocompounds have been proven to enhance osteogenesis via various osteogenic signaling pathways, but its decreased bioavailability and increased renal clearance limit its application. In this study, we designed a biocomposite scaffold comprising gelatin (Gel) and nanohydroxyapatite (nHAp) incorporated with diosmin (DM) and we investigated its bone forming potential in vitro and in vivo. Physiochemical characterization of the scaffold showed that DM had no effect on altering the material characteristics of the scaffold. The addition of DM enhanced the osteoblast differentiation potential of the scaffold in mouse mesenchymal stem cells at both cellular and molecular levels, possibly via the integrin-mediated activation of FAK and ERK signaling components. Using the rat tibial bone defective model, we identified the effect of DM in Gel/nHAp scaffold on enhancing bone formation in vivo. Based on our results, we suggest that Gel/nHAp/DM can be a potential therapeutic agent in scaffold-mediated bone regeneration.

Amentoflavone inhibits tumorsphere formation by regulating the Hedgehog/Gli1 signaling pathway in SUM159 breast cancer stem cells

• Amentoflavone inhibited tumorsphere formation of basal-like breast cancer stem cells (CSCs). • Amentoflaovne regulated the expression of CSC markers, CD44, CD24, ALDH1, OCT4 and NANOG in SUM159 breast CSCs. • Amentoflavone suppressed Hedgehog signaling mediators, Gli1 and Smo, leading to inhibition of breast CSCs stemness. A novel anticancer approach is to target cancer stem cells (CSCs), a rare subpopulation potentially associated with metastasis, resistance to chemotherapy, and recurrence of cancers. This study aimed to investigate the effects of amentoflavone, a natural compound identified from Chamaecyparis obtusa , on stemness of basal-like SUM159 breast CSCs, and its underlying mechanism of action. We found that amentoflavone suppressed tumorsphere formation and regulated the expression of the stem cell markers CD44, CD24, and ALDH1 in a dose dependent manner, and inhibited Hedgehog (Hh) signaling by down-regulating Smo, Gli1, OCT4, and NANOG. Knock-down of Gli1 by siRNA significantly affected the expression of CD44, CD24, ALDH1, OCT4, and NANOG, and reduced tumorsphere formation efficiency in SUM159 breast CSCs. Our findings indicate that amentoflavone inhibits SUM159 breast cancer stemness by suppressing Hh/Gli1 signaling, and is a potent agent for targeting breast CSCs.

Role of Wnt Signaling Pathway in Stem Cells Derived Retinal Regeneration

Human optic nerve injury and its associated neurodegenerative diseases are often followed by permanent vision loss.Stem cell therapy has long been considered a promising mode to treat retinal degenerative diseases.Recent studies revealed that there are silent retinal stem cells in the eyes of mammals and even humans.These stem cells can be activated again under certain conditions and differentiate into retinal neurons to repair the damaged retina.Wnt signaling pathway plays a crucial role in conducting growth-stimulating signals and regulates cell proliferation,differentiation and apoptosis.This article review the regulatory effect of Wnt signaling pathway on stem cells-based retinal regeneration and the sources of retinal stem cells.

Generation of pig induced pluripotent stem cells using an extended pluripotent stem cell culture system

BackgroundPigs have emerged as one of the most popular large animal models in biomedical research, which in many cases is considered as a superior choice over rodent models. In addition, transplantation studies using pig pluripotent stem (PS) cell derivatives may serve as a testbed for safety and efficacy prior to human trials. Recently, it has been shown that mouse and human PS cells cultured in LCDM (recombinant human LIF, CHIR 99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride) medium exhibited extended developmental potential (designated as extended pluripotent stem cells, or EPS cells), which could generate both embryonic and extraembryonic tissues in chimeric mouse conceptus. Whether stable pig induced pluripotent stem (iPS) cells can be generated in LCDM medium and their chimeric competency remains unknown.MethodsiPS cells were generated by infecting pig pericytes (PC) and embryonic fibroblasts (PEFs) with a retroviral vector encoding Oct4, Sox2, Klf4, and cMyc reprogramming factors and subsequently cultured in a modified LCDM medium. The pluripotency of PC-iPS and PEF-iPS cells was characterized by examining the expression of pluripotency-related transcription factors and surface markers, transcriptome analysis, and in vitro and in vivo differentiation capabilities. Chimeric contribution of PC-iPS cells to mouse and pig conceptus was also evaluated with fluorescence microscopy, flow cytometry, and PCR analysis.ResultsIn this study, using a modified version of the LCDM medium, we successfully generated iPS cells from both PCs and PEFs. Both PC-iPS and PEF-iPS cells maintained the stable “dome-shaped” morphology and genome stability after long-term culture. The immunocytochemistry analyses revealed that both PC-iPS and PEF-iPS cells expressed OCT4, SOX2, and SALL4, but only PC-iPS cells expressed NANOG and TRA-1-81 (faint). PC-iPS and PEF-iPS cells could be differentiated into cell derivatives of all three primary germ layers in vitro. The transcriptome analysis showed that PEF-iPS and PC-iPS cells clustered with pig ICM, Heatmap and volcano plot showed that there were 1475 differentially expressed genes (DEGs) between PC-iPS and PEF-iPS cells (adjusted p value < 0.1), and the numbers of upregulated genes and downregulated genes in PC-iPS cells were 755 and 720, respectively. Upregulated genes were enriched with GO terms including regulation of stem cell differentiation, proliferation, development, and maintenance. And KEGG pathway enrichment in upregulated genes revealed Wnt, Jak-STAT, TGF-β, P53, and MAPK stem cell signaling pathways. Fluorescence microscopy and genomic PCR analyses using pig mtDNA-specific and GFP primers showed that the PC-iPS cell derivatives could be detected in both mouse and pig pre-implantation blastocysts and post-implantation conceptuses. Quantitative analysis via flow cytometry revealed that the chimeric contribution of pig PC-iPS cells in mouse conceptus was up to 0.04%.ConclusionsOur findings demonstrate that stable iPS cells could be generated in LCDM medium, which could give rise to both embryonic and extraembryonic cells in vivo. However, the efficiency and level of chimeric contribution of pig LCDM-iPS cells were found low.

FAK mediates BMP9-induced osteogenic differentiation via Wnt and MAPK signaling pathway in synovial mesenchymal stem cells

Objective Focal adhesion kinase (FAK) has critical functions in proliferation and differentiation of many cell types, however, the role of FAK on BMP9-induced osteogenic differentiation in SMSCs has not been characted. The purpose of current study is to explore the mechanism of FAK on the BMP9-induced osteogenesis of SMSCs in vitro and in vivo. Methods The optimal dose of BMP9 was determined by incubation in different BMP9 concentrations, then cells were transfected with siRNA-induced FAK knockdown in BMP9-induced osteogenesis. Cell proliferation, migration, the osteogenic capacity, and the underlying mechanism were further detected in vitro. Imaging and pathological examination were conducted to observe the bone formation in vivo. Results Our findings suggested that BMP9 could obviously promote FAK phosphorylation in osteogenic conditions. In contrast, FAK knockdown significantly decreased the cell proliferation, migration, the osteogenic capacity of SMSCs. To be specific, FAK knockdown could markedly inhibit the Wnt and MAPK signal pathway of SMSCs induced by BMP9. Besides, FAK knockdown could also effectively inhibit BMP-9-induced bone formation in vivo. Conclusion FAK plays a pivotal role in promoting BMP9-induced osteogenesis of SMSCs, which is probably via activating Wnt and MAPK pathway.

The role of innate immunity in MDS pathogenesis.

The role of innate immunity in MDS pathogenesis.

Targeting the BRAF Signaling Pathway in CD133pos Cancer Stem Cells of Anaplastic Thyroid Carcinoma

Background:Cancer stem cells (CSCs) with a self-renewal ability in tumor cells population, execute a pivotal function in tumorigenesis, retrogression, and metastasis of malignant cancers such as anaplastic thyroid carcinoma (ATC).Materials and Methods:In this study, we isolated CSCs subpopulation with CD133 surface marker from three ATC cell lines by magnetic cell sorting assay. After confirming the segregation by the flow cytometry method, BRAF and sodium-iodide symporter (NIS) genes were investigated in them before and after incubation with BRAF inhibitor. Also, we evaluated the NIS protein expression and localization.Results:Established upon q-RT PCR data, when compared to human normal thyrocytes, the BRAFV600E gene was over-expressed in CD133pos cells (>1705.99 ± 55.55 fold, Mean ± SEM, n=3, P- value<0.05), whilst the expression of NIS gene was very restricted (< 0.0008 ± 5.43 fold, Mean ± SEM, n=3, P- value<0.05) in them. Also, our results showed that BRAF inhibition affected NIS protein expression and localization.Conclusions:Current study showed that the differentiate genes/proteins expression can be induced in the CSCs via focus on signal transduction pathways and targeting their molecules, that are involved in expression of these genes/proteins. Therefore, attention to targeting CSCs along with routine thyroid cancer therapy, can help to ATC treatment.

Generation of a nitric oxide signaling pathway in mesenchymal stem cells promotes endothelial lineage commitment.

Enhancing differentiation of mesenchymal stem cells (MSCs) to endothelial cells may improve their ability to vascularize tissue and promote wound healing. This study describes a novel role for nitric oxide (NO) in reprogramming MSCs towards an endothelial lineage and highlights the role of Wnt signaling and epigenetic modification by NO. Rat MSCs were transduced with lentiviral vectors expressing endothelial nitric oxide synthase (pLV-eNOS) and a mutated caveolin gene (pLV-CAV-1F92A ) to enhance NO generation resulting in increased in vitro capillary tubule formation and endothelial marker gene expression. An exogenous source of NO could also stimulate CD31 expression in MSCs. NO was associated with an arterial-specific endothelial gene expression profile of Notch1, Dll4, and Hey2 and significantly reduced expression of venous markers. Wnt signaling associated with NO was evident through increased gene expression of Wnt3a and β-catenin protein, and expression of the endothelial marker Pecam-1 could be significantly reduced by treatment with the Wnt signaling inhibitor Dkk-1. The role of NO as an epigenetic modifier was evident with reduced gene expression of the methyltransferase, DNMT1, and bisulfite sequencing of the endothelial Flt1 promoter region in NO-producing MSCs showed significant demethylation compared to control cells. Finally, subcutaneous implantation of NO-producing MSCs seeded in a biomaterial scaffold (NovoSorb®) resulted in survival of transplanted cells and the formation of blood vessels. In summary, this study describes, NO as a potent endothelial programming factor which acts as an epigenetic modifier in MSCs and may provide a novel platform for vascular regenerative therapy.

Sinapic acid-loaded chitosan nanoparticles in polycaprolactone electrospun fibers for bone regeneration in vitro and in vivo

Sinapic acid (SA) is a plant-derived phenolic compound known for its multiple biological properties, but its role in the promotion of bone formation is not yet well-studied. Moreover, the delivery of SA is hindered by its complex hydrophobic nature, limiting its bioavailability. In this study, we fabricated a drug delivery system using chitosan nanoparticles (nCS) loaded with SA at different concentrations. These were incorporated into polycaprolactone (PCL) fibers via an electrospinning method. nCS loaded with 50 μM SA in PCL fibers promoted osteoblast differentiation. Furthermore, SA treatment activated the osteogenesis signaling pathways in mouse mesenchymal stem cells. A critical-sized rat calvarial bone defect model system identified that the inclusion of SA into PCL/nCS fibers accelerated bone formation. Collectively, these data suggest that SA promoted osteoblast differentiation in vitro and bone formation in vivo, possibly by activating the TGF-β1/BMP/Smads/Runx2 signaling pathways, suggesting SA might have therapeutic benefits in bone regeneration.

Impact of miR-223-3p and miR-2909 on inflammatory factors IL-6, IL-1ß, and TNF-α, and the TLR4/TLR2/NF-κB/STAT3 signaling pathway induced by lipopolysaccharide in human adipose stem cells.

MicroRNAs (miRNAs) are small non-coding RNA molecules that play an important role in the regulation of gene expression related to inflammatory responses. Human adipose stem cells are characterized by pluripotent differentiation potential and isolated from adipose tissues. These cells regulate inflammation mainly by interacting with immune cells and affecting the secretion of immune factors; details of this interaction are currently unknown. In the current study, we successfully established an acute inflammation model and a chronic inflammation model involving adipose stem cells. We used high-throughput miRNA microarray analysis to identify miRNAs that were significantly (p < 0.05) differentially expressed during both acute and chronic inflammation. Lipopolysaccharide (LPS) significantly (p < 0.05) reduced the expression of miR-223-3P and miR-2909, while promoting the production of pro-inflammatory cytokines, interleukin (IL) 6, IL-1β, and tumor necrosis factor (TNF)-α via the Toll-like receptor (TLR) 4/TLR2/nuclear factor (NF)-κB/signal transducer and activator of transcription (STAT) 3 signaling pathway in human adipose stem cells. Further, miR-223-3P expression was significantly (p < 0.05) reduced in human adipose stem cells during activation by IL-6 stimulation. The inducible down-regulation of miR-223-3P resulted in the activation of STAT3, which was directly targeted by miR-223-3P. STAT3 directly targeted TLR4 and TLR2, promoting the production of the pro-inflammatory cytokine, IL-6, and formed a positive feedback loop to regulate IL-6 levels. Similarly, TNF-α significantly (p < 0.05) increased the expression of miR-223-3p, with LPS and TLR4/TLR2/NF-κB/STAT3 forming a negative feedback loop to regulate TNF-α levels. In addition, miR-2909, which depends on NF-κB, targeted Krueppel-like factor (KLF) 4 to regulate the levels of pro-inflammatory cytokines, IL-6, IL-1β, and TNF-α. We conclude that miR-223-3p and miR-2909 form a complex regulatory network with pro-inflammatory factors and signaling pathways in adipose stem cells stimulated by LPS. These findings will inform the development of therapies against autoimmune and inflammatory diseases.

Abstract P1-09-06: Gene-set enrichment analysis (GSEA) of breast tissue from healthy women with less than six months history of breastfeeding shows enrichment in Hedgehog signaling, notch signaling and luminal progenitor gene signatures

Abstract Introduction: Multiple epidemiological studies have shown that prolonged breastfeeding is associated with a reduced risk of developing triple negative/basal-like breast cancer (TN/BLBC). We have modeled abrupt involution (AI) due to lack of breastfeeding and gradual involution (GI) of the mammary gland that occurs over time upon prolonged breastfeeding in wild-type FVB/N mice and discovered prominent histological and molecular changes in the AI glands over time. Our studies revealed for the first time a clear and persistent expansion of mammary luminal progenitor (LP) epithelial cells in AI glands (AACR abstract#2242, 2018). Here, we corroborate animal studies using normal human breast tissue obtained from a reduction mammoplasty tissue collection study (OSU-2011C0094). Methods: Breast tissue obtained from parous premenopausal women with no history of breast cancer who breastfed for ≥6 months (GI, n=16) versus those who breastfed for &amp;lt;6 months (AI, n=16) (OSU-2011C0094) was used for gene expression analysis. RNA isolated from these normal mammary tissues was analyzed using Affymatrix Gene ChIP Human Transcriptome array 2.0; Gene Set Enrichment Analysis (GSEA) was used to analyze the microarray data. Molecular Signatures Database was used in GSEA querying C2 curated gene sets, Hallmark gene sets, and Lim-Mammary-Luminal-Progenitor gene sets. H&amp;E sections of the breast tissue were used to assess lobular type by counting number of ductules per terminal ductal lobular unit (TDLU). False discovery rate (FDR) q-values and p-values were used for multiple comparison adjustment. Results: GSEA revealed that breast tissue obtained from women in the AI cohort exhibited strong positive enrichment for Notch and Hedgehog Signaling (Hhg) pathways (FDR q-value= 0.20 and 0.12, respectively). In GI women, GSEA showed an overall trend towards enrichment in metabolic pathways and immune system functions. Moreover, there was non-significant trend towards positive enrichment of mouse LP gene signature in AI women only (FDR q-value= 0.30). Age and BMI were not statistically different between AI and GI cohorts. Analysis of TDLU, the primary anatomical source of most breast cancers, revealed that breast tissue from AI women had proportionally higher lobular type 1 only epithelium than GI women who exhibited more differentiated lobular epithelium (p-value= 0.049). Conclusion: We report here for the first time that mammary glands from women who breastfed &amp;lt;6 months were enriched for stem-cell signaling pathways and LP gene signature. This reflects some similarity to BRCA1 mutation carriers, who demonstrate expanded luminal progenitor population. In addition, higher Type 1 TDLU's are seen in breast tissue from parous women who breastfed &amp;lt;6 months. Together, these data demonstrate features for TN/BLBC precursors enriched in patients who breastfed for &amp;lt;6 months. Understanding this mechanistic link will help in developing prevention strategies, particularly for African-American women who have lower prevalence of breastfeeding and higher incidence of TN/BLBC. Citation Format: Basree MM, Shinde N, Palettas M, Weng D, Stover DG, Sizemore GM, Shields P, Majumder S, Ramaswamy B. Gene-set enrichment analysis (GSEA) of breast tissue from healthy women with less than six months history of breastfeeding shows enrichment in Hedgehog signaling, notch signaling and luminal progenitor gene signatures [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-09-06.

P122 EPITHELIAL TLR4 PROMOTES OXIDATIVE STRESS VIA NADPH OXIDASE ACTIVATION AND INDUCES REDOX-SENSITIVE PATHWAYS DURING TUMORIGENESIS

Dysbiosis and accumulation of reactive oxygen species (ROS) are hallmarks of ulcerative colitis (UC) and have been associated with tumor progression. We have previously shown that UC patient biopsies and colorectal adenomas overexpress toll-like receptor 4 (TLR4) in intestinal epithelial cells (IECs). A potential mechanism of TLR4-mediated tumorigenesis is by providing cancer stem cells with redox signals that they efficiently use to outgrow their non-transformed counterparts. We test the hypothesis that epithelial signaling via TLR4 induces the production of ROS through the NADPH oxidases Nox1 and Duox2. We also explore the main redox-sensitive cascades that participate in TLR4-driven tumorigenesis. Colonoids from wild-type (WT), TLR4-KO, and Nox1-KO mice were stimulated with LPS and tested for H2O2 production in the presence of the NADPH oxidase inhibitor, diphenyleneiodonium. IECs isolated from villin-TLR4 mice (which have constitutive activation of epithelial TLR4) and their WT littermates were analyzed for H2O2 production (Amplex Red) and expression of the NADPH oxidases Nox1 and Duox2 (qPCR). Villin-TLR4 and their littermates underwent a model of CAC by azoxymethane injection followed by 2 cycles of 3% dextran sulfate sodium (1.5% in villin-TLR4 mice). Cancer stem cells (aldehyde dehydrogenase positive) were sorted for RNA sequencing. LPS incubation enhanced the expression of Nox1 and Duox2 in WT but not TLR4-KO colonoids, demonstrating that these oxidative enzymes are regulated by TLR4. Consistently, LPS stimulation elicited H2O2 release by WT organoids in a NADPH oxidase-dependent manner. Nox1-KO colonoids showed attenuated production of H2O2 upon LPS stimulation, confirming the participation of this NADPH oxidase in TLR4-mediated production of ROS. Constitutive activation of TLR4 induced the production of H2O2 in IECs, which was accompanied by a marked upregulation of Nox1, Duox2, and DuoxA2. RNAseq done on cancer stem cells shows that cancer stem cells of villin-TLR4 tumors have increased expression of NADPH oxidases, as well as enrichment of redox-sensitive pathways, including PI3K, myc, Rac1, and NF-κB. All of these pathways have been implicated in the promotion of proliferative and antiapoptotic signals. TLR4 controls the expression and function of oxidative enzymes in IECs, enhancing redox signals in cancer stem cells upon bacterial challenge. In the context of inflammation-associated dysbiosis, we speculate that overexpression of TLR4 provides additional activation of proliferative and antiapoptotic pathways, accelerating tumor development and growth. The results of our studies highlight how we might target critical signaling pathways in inflammatory-driven cancer stem cells.