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Background: Cancer stem cell biomarkers SRY (sex-determining region Y)-box 2 (SOX2) and octamer-binding transcription factor 4 (Oct4) account for radioresistance in cervical squamous cell cancers (CSCCs). Their clinical implications are limited and contradictory., Methods: In this prospective cohort study, we recruited patients with FIGO IB2-IVA CSCC treated with primary chemoradiotherapy on regular follow-up. Tissue biopsy specimens were evaluated for SOX2 and Oct4 expression by immunohistochemistry, quantified by a product of proportion and intensity scores., Results: A total of 59 patients were included. Most had a moderately differentiated (81%), keratinizing (59%) CSCC, and ≥FIGO stage IIB disease (95%). SOX2 expression (high:low 21:38 patients) and Oct4 expression (high:low 4:55 patients) had a significant interrelation (p = 0.005, odds ratio (95% CI) - 1.23 (1.004-1.520)). At a median follow-up of 36 months, the 3-year overall survival (OS) was 60% and 53% for low and high SOX2 expression (p = 0.856), and 54% and 100% for low and high Oct4 expression (p = 0.114). The 3-year disease-frese survival (DFS) was 65% and 50% in the low and high SOX2 expression (p = 0.259), and 59% and 75% for low and high Oct4 expression (p = 0.598). SOX2 expression was the only variable significantly associated with a lower OS and DFS on regression analysis., Conclusion: Our study demonstrated a trend toward improved OS and DFS with low SOX2 and high Oct4 expression in CSCC patients undergoing chemoradiotherapy., (© 2024 John Wiley & Sons Australia, Ltd.)

Most high-grade ovarian carcinomas (HGOCs) are sensitive to carboplatin (CBP)-based chemotherapy but frequently recur within 24 months. Recurrent tumors remain CBP-sensitive and acquire resistance only after several treatment rounds. Recurrences arise from a small number of residual tumor cells not amenable to investigation in patients. We developed patient-derived xenografts (PDXs) that allow the study of these different stages of CBP-sensitive recurrence and acquisition of resistance. We generated PDX models from CBP-sensitive and intrinsically resistant HGOC. PDXs were CBP- or mock-treated and tumors were sampled, after treatment and at recurrence. We also isolated models with acquired-resistance from CBP-sensitive PDXs. Tumors were characterized at the histological and transcriptome levels. PDX models reproduced treatment response seen in the patients. CBP-sensitive residual tumors contained nonproliferating tumor cell clusters embedded in a fibrotic mesh. In nontreated PDX tumors and treated CBP-resistant tumors, fibrotic tissue was not prevalent. Residual tumors had marked differences in gene expression when compared to naïve and recurrent tumors, indicating downregulation of the cell cycle and proliferation and upregulation of interferon response and the epithelial-mesenchymal transition. This gene expression pattern resembled that described in embryonal diapause and 'drug-tolerant persister' states. Residual and acquired-resistance tumors share the overexpression of three genes: CEACAM6, CRYAB, and SOX2. Immunostaining analysis showed strong CEACAM6, CRYAB, and SOX2 protein expression in CBP-sensitive residual and acquired-resistance PDX, thus confirming the RNA profiling results. In HGOC PDX, CBP-sensitive recurrences arise from a small population of quiescent, drug-tolerant, residual cells embedded in a fibrotic mesh. These cells overexpress CEACAM6, CRYAB, and SOX2, whose overexpression is also associated with acquired resistance and poor patient prognosis. CEACAM6, CRYAB, and SOX2 may thus serve as a biomarker to predict recurrence and emergence of resistant disease in CBP-treated HGOC patients. © 2022 The Pathological Society of Great Britain and Ireland., (© 2022 The Pathological Society of Great Britain and Ireland.)

Objectives: Cancer stem cells are a small group of highly tumorigenic cells with the trend of self-renewal, tumor progression, metastasis, recurrence, and therapeutic resistance. SOX2 is an important cancer stem cell marker that is involved in tumorigenesis and correlated with aggressive features in various types of malignancies. The present study was aimed to investigate the expression of this biomarker in neoplastic tissues of oral squamous cell carcinoma (OSCC) to determine whether it has the potential to predict the outcomes and survival of the affected patients., Materials and Methods: The medical records of 73 patients diagnosed with OSCC were retrospectively studied. Clinical and pathologic features included age, sex, tumor size, histologic grade, lymph node involvement, recurrence, metastasis, and follow up. Immunohistochemical analysis for SOX2 protein expression was performed, and its correlations with clinicopathologic features were evaluated., Results: SOX2 was significantly associated with tumor size, lymph node metastasis, and patients' survival. We found no apparent correlation between SOX2 and tumor recurrence, distant metastasis, or differentiation. The multivariate analysis identifies patients' age, sex, and SOX2 expression as independent prognostic factors for overall survival., Conclusion: SOX2 may worsen the prognosis and be a progressive malignant factor, which can help clinicians investigate OSCC patients' survival and plan the appropriate treatment accordingly., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The hair follicle dermal papilla is critical for hair generation and de novo regeneration. When cultured in vitro, dermal papilla cells from different species demonstrate two distinguishable growth patterns under the conventional culture condition: a self-aggregative three dimensional spheroidal (3D) cell pattern and a two dimensional (2D) monolayer cell pattern, correlating with different hair inducing properties. Whether the loss of self-aggregative behavior relates to species-specific differences or the improper culture condition remains unclear. Can the fixed 2D patterned dermal papilla cells recover the self-aggregative behavior and 3D pattern also remains undetected. Here, we successfully constructed the two growth patterns using sika deer ( Cervus nippon ) dermal papilla cells and proved it was the culture condition that determined the dermal papilla growth pattern. The two growth patterns could transit mutually as the culture condition was exchanged. The fixed 2D patterned sika deer dermal papilla cells could recover the self-aggregative behavior and transit back to 3D pattern, accompanied by the restoration of hair inducing capability when the culture condition was changed. In addition, the global gene expressions during the transition from 2D pattern to 3D pattern were compared to detect the potential regulating genes and pathways involved in the recovery of 3D pattern and hair inducing capability.

The course of differentiation of pluripotent stem cells into cardiomyocytes and the intermediate cell types are characterized using molecular markers for different stages of development. These markers have been selected primarily from studies in the mouse and from a limited number of human studies. However, it is not clear how well mouse cardiogenesis compares with human cardiogenesis at the molecular level. We tackle this issue by analyzing and comparing the expression of common cardiomyogenesis markers [platelet-derived growth factor receptor, alpha polypeptide (PDGFR-α), fetal liver kinase 1 (FLK1), ISL1, NK2 homeobox 5 (NKX2.5), cardiac troponin T (CTNT), connexin43 (CX43), and myosin heavy chain 7 (MYHC-B)] in the developing pig heart at embryonic day (E)15, E16, E18, E20, E22, and E24 and in differentiating cardiomyocytes from human induced pluripotent stem cells (hiPSCs). We found that porcine expression of the mesoderm marker FLK1 and the cardiac progenitor marker ISL1 was in line with our differentiating hiPSC and reported murine expression. The cardiac lineage marker NKX2.5 was expressed at almost all stages in the pig and hiPSC, with an earlier onset in the hiPSC compared with reported murine expression. Markers of immature cardiomyocytes, CTNT, and MYHC-B were consistently expressed throughout E16-E70 in the pig, which is comparable with mouse development, whereas the markers increased over time in the hiPSC. However, the commonly used mature cardiomyocyte marker, CX43, should be used with caution, as it was also expressed in the pig mesoderm, as well as hiPSC immature cardiomyocytes, while this has not been reported in mice. Based on our observations in the various species, we suggest to use FLK1/PDGFR-α for identifying cardiac mesoderm and ISL1/NKX2.5 for cardiac progenitors. Furthermore, a combination of two or more of the following, CTNT + /MYHC-B + /ISL1 + could mark immature cardiomyocytes and CTNT + /ISL1 - mature cardiomyocytes. CX43 should be used together with sarcomeric proteins. This knowledge may help improving differentiation of hiPSC into more in vivo-like cardiac tissue in the future.

Background: Small cell neuroendocrine carcinoma of the uterine cervix (SCNEC) is a rare cancer involving the human papilloma virus (HPV), and has few available treatments. The present work aimed to assess the feasibility of SOX2 and HPV statuses as predictive indicators of SCNEC prognosis., Methods: The associations of SOX2 and/or high-risk (HR)-HPV RNA in situ hybridization (RISH) levels with clinicopathological characteristics and prognostic outcomes for 88 neuroendocrine carcinoma (NEC) cases were analyzed., Results: Among these patients with SCNEC, SOX2, P16 INK4A and HR-HPV RISH expression and SOX2/HR-HPV RISH co-expression were detected in 68(77.3%), 76(86.4%), 73(83.0%), and 48(54.5%), respectively. SOX2-positive and HR-HPV RISH-positive SCNEC cases were associated with poorer overall survival (OS, P = 0.0170, P = 0.0451) and disease-free survival (DFS, P = 0.0334, P = 0.0309) compared with those expressing low SOX2 and negative HR-HPV RISH. Alternatively, univariate analysis revealed that SOX2 and HR-HPV RISH expression, either separately or in combination, predicted the poor prognosis of SCNEC patients. Multivariate analysis revealed that the co-expression of SOX2 with HR-HPV RISH may be an independent factor of OS [hazard ratio = 3.597; 95% confidence interval (CI): 1.085-11.928; P = 0.036] and DFS [hazard ratio = 2.880; 95% CI: 1.199-6.919; P = 0.018] prediction in SCNEC., Conclusions: Overall, the results of the present study suggest that the co-expression of SOX2 with HR-HPV RISH in SCNEC may represent a specific subgroup exhibiting remarkably poorer prognostic outcomes compared with the expression of any one marker alone.

Background: Sex-determining region Y-box 2 (SOX2) is a transcriptional factor that drives embryonic stem cells to neuroendocrine cells in lung development and is highly expressed in small-cell lung cancer (SCLC). However, the prognostic role of SOX2 and its relationship with tumor-infiltrating lymphocytes (TILs) has not been determined in SCLC. Herein, we assessed the expression of SOX2 and CD8+ TILs to obtain insights into the prognostic role of SOX2 and CD8+ TILs in limited-stage (LS)-SCLC., Methods: A total of 75 patients with LS-SCLC was enrolled. The SOX2 expression and CD8+ TILs were evaluated by immunohistochemistry., Results: High SOX2 and CD8+ TIL levels were identified in 52 (69.3%) and 40 (53.3%) patients, respectively. High SOX2 expression was correlated with increased density of CD8+ TILs (p = 0.041). Unlike SOX2, high CD8+ TIL numbers were associated with significantly longer progression-free survival (PFS; 13.9 vs. 8.0 months, p = 0.014). Patients with both high SOX2 expression and CD8+ TIL numbers (n = 29, 38.7%) had significantly longer PFS and overall survival (OS) compared to those from the other groups (median PFS 19.3 vs. 8.4 months; p = 0.002 and median OS 35.7 vs. 17.4 months; p = 0.004, respectively). Multivariate Cox regression analysis showed that the combination of high SOX2 expression and CD8+ TIL levels was an independent good prognostic factor for OS (HR = 0.471, 95% CI, 0.250-0.887, p = 0.02) and PFS (HR = 0.447, 95% CI, 0.250-0.801, p = 0.007) in SCLC., Conclusions: Evaluation of the combination of SOX2 and CD8+ TIL levels may be of a prognostic value in LS-SCLC., (© 2021 The Author(s) Published by S. Karger AG, Basel.)

Background: Stemness factors associated with tumorigenesis in different types of cancers have not been specifically studied in oral tongue SCC (OTSSC). Here, we aimed to quantify expression levels and distribution of KLF4 and SOX2, two relevant stemness factors, in oral SCC including OTSCC samples from different subsites., Methods and Results: We determined KLF4 and SOX2 expression levels by immunostaining 35 biopsies of OSCC. Stained wholeslide images were digitized and subjected to automatic cell detection and unbiased quantification using Qupath software. We found statistically significant reduction in KLF4 positive cells density (p= 0.024), and fraction (p= 0.022) in OTSCC from tongue borders compared with other tongue subsites. Instead, quantitative SOX2 analysis did not show differences in expression levels between OTSCC from the borders versus OTSCC developed in others subsites. Notably SOX2 expression was revealed increased in moderately and poorly differentiated OSCC compared with well differentiated ones (positive cells density p= 0.025, fraction p= 0.006). No significant correlation between KLF4 and SOX2 expression was observed, neither in OSCC nor in OTSCC., Conclusions: KLF4 and SOX2 exhibit opposite expression profiles regarding subsite localization and differentiation level in OSCC. Our study prompts future OTSCC prospective studies looking for clinical prognosis to incorporate detailed subsite information in the analysis.

Background: Characterized by abnormal lung growth or maturation, congenital diaphragmatic hernia (CDH) affects 1:3000 live births. Cellular studies report proximal (SOX2 + ) and distal (SOX9 + ) progenitor cells as key modulators of branching morphogenesis and epithelial differentiation, whereas transcriptome studies demonstrate ROBO/SLIT as potential therapeutic targets for diaphragm defect repair in CDH. In this study, we tested the hypothesis that (a) experimental-CDH could changes the expression profile of ROBO1, ROBO2, SOX2 and SOX9; and (b) ROBO1 or ROBO2 receptors are regulators of branching morphogenesis and SOX2/SOX9 balance., Methods: The expression profile for receptors and epithelial progenitor markers were assessed by Western blot and immunohistochemistry in a nitrofen-induced CDH rat model. Immunohistochemistry signals by pulmonary structure were also quantified from embryonic-to-saccular stages in normal and hypoplastic lungs. Ex vivo lung explant cultures were harvested at E13.5, cultures during 4 days and treated with increasing doses of recombinant rat ROBO1 or human ROBO2 Fc Chimera proteins for ROBO1 and ROBO2 inhibition, respectively. The lung explants were analyzed morphometrically and ROBO1, ROBO2, SOX2, SOX9, BMP4, and β-Catenin were quantified by Western blot., Results: Experimental-CDH induces distinct expression profiles by pulmonary structure and developmental stage for both receptors (ROBO1 and ROBO2) and epithelial progenitor markers (SOX2 and SOX9) that provide evidence of the impairment of proximodistal patterning in experimental-CDH. Ex vivo functional studies showed unchanged branching morphogenesis after ROBO1 inhibition; increased fetal lung growth after ROBO2 inhibition in a mechanism-dependent on SOX2 depletion and overexpression of SOX9, non-phospho β-Catenin, and BMP4., Conclusions: These studies provided evidence of receptors and epithelial progenitor cells which are severely affected by CDH-induction from embryonic-to-saccular stages and established the ROBO2 inhibition as promoter of branching morphogenesis through SOX2/SOX9 balance.

Sharks and rays (elasmobranchs) have the remarkable capacity to continuously regenerate their teeth. The polyphyodont system is considered the ancestral condition of the gnathostome dentition. Despite this shared regenerative ability, sharks and rays exhibit dramatic interspecific variation in their tooth morphology. Ray (batoidea) teeth typically constitute crushing pads of flattened teeth, whereas shark teeth are pointed, multi-cuspid units. Although recent research has addressed the molecular development of the shark dentition, little is known about that of the ray. Furthermore, how dental diversity within the elasmobranch lineage is achieved remains unknown. Here, we examine dental development and regeneration in two Batoid species: the thornback skate (Raja clavata) and the little skate (Leucoraja erinacea). Using in situ hybridization and immunohistochemistry, we examine the expression of a core gnathostome dental gene set during early development of the skate dentition and compare it to development in the shark. Elasmobranch tooth development is highly conserved, with sox2 likely playing an important role in the initiation and regeneration of teeth. Alterations to conserved genes expressed in an enamel knot-like signalling centre may explain the morphological diversity of elasmobranch teeth, thereby enabling sharks and rays to occupy diverse dietary and ecological niches., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The cancer stem cells deliver uncontrolled proliferative capacity within the tumor imparting to increasing size while epithelial mesenchymal transition adds to the invasive potential. Studies using specific markers elucidating the role of these phenomena may bring advancement in the targeted therapy of tumor. SOX2 and OCT4 are two among few stem cell markers indicative of proliferative potential and WNT5A is an epithelial mesenchymal transition marker indicative of invasive potential. We aimed to determine the association between expression of SOX2, OCT4 and WNT5A in oral epithelial dysplasia, oral squamous cell carcinoma and normal oral mucosa. 20 cases of oral squamous cell carcinoma, 20 cases of oral epithelial dysplasia (leukoplakia with dysplasia) and 25 normal oral mucosa tissues specimens were immunohistochemically stained to assess SOX2, OCT4 and WNT5A expression. SOX2 expression was higher in oral squamous cell carcinoma than in oral epithelial dysplasia and very low in normal oral mucosa. OCT4 was very low in oral squamous cell carcinoma and oral epithelial dysplasia when compared to SOX2, while negative in normal tissues. Co-expression of SOX2 and OCT4 showed statistically non-significant difference for tumor proliferation. WNT5A expression was found to be increasing from normal oral mucosa to oral epithelial dysplasia and oral squamous cell carcinoma. In conformity with present study, SOX2 itself can act as a potential marker for proliferation in tumor cells while OCT4 has non-significant role in regulation of tumor behavior in oral squamous cell carcinoma as well as in oral epithelial dysplasia. WNT5A can be a putative marker in studying invasive potential of oral squamous cell carcinoma.

Background: Although androgen deprivation therapy (ADT) is the initial treatment strategy for prostate cancer (PCa), recurrent castration-resistant prostate cancer (CRPC) eventually ensues. In this study, cancer-derived immunoglobulin G (CIgG) is found to be induced after ADT, identifying CIgG as a potential CRPC driver gene., Methods: The expression of CIgG and its clinical significance in PCa tissue was analyzed by The Cancer Genome Atlas database and immunohistochemistry. Subsequently, the sequence features of prostate cell line VHDJH rearrangements were analyzed. We also assessed the effect of CIgG on the migratory, invasive and proliferative abilities of PCa cells in vitro and vivo. Suspended microsphere, colony formation and drug-resistant assays were performed using PC3 cells with high CIgG expression (CIgG high ) and low CIgG expression (CIgG -/low ), and A nonobese diabetic/severe combined immunodeficiency mouse tumor xenograft model was developed for the study of the tumorigenic effects of the different cell populations. The SOX2-CIgG signaling pathway was validated by immunohistochemistry, immunofluorescence, quantitative reverse transcription-polymerase chain reaction, Western blot, luciferase, and chromatin immunoprecipitation assays and bioinformatics analyses. Finally, we investigated the effect of RP215 inhibition on the progression of PCa in vivo using a Babl/c nude mouse xenograft model., Results: CIgG is frequently expressed in PCa and associated with clinicopathological characteristics, moreover, CIgG transcripts with unique patterns of VHDJH rearrangements are found in PCa cells. Functional analyses identified that CIgG was induced by ADT and upregulated by SOX2 (SRY (sex determining region Y)-box 2) in PCa, promoting the development of PCa. In addition, our findings underscore a novel role of CIgG signaling in the maintenance of stemness and the progression of cancer through mitogen activated protein kinase/extracellular-signal-regulated kinase and AKT in PCa. In vivo experiments further demonstrated that depleting CIgG significantly suppressed the growth of PCa cell xenografts. Furthermore, a CIgG monoclonal antibody named RP215 exhibits tumor inhibitory effect as well., Conclusion: Our data suggests that CIgG could be a driver of PCa development, and that targeting the SOX2-CIgG axis may therefore inhibit PCa development after ADT., (© 2020 Wiley Periodicals LLC.)

Stemness and reprogramming involve transcriptional master regulators that suppress cell differentiation while promoting self-renewal. A distinguished example thereof is SOX2, a high mobility group (HMG)-box transcription factor (TF), whose subcellular localization and turnover regulation in embryonic, induced-pluripotent, and cancer stem cells (ESCs, iPSCs, and CSCs, respectively) is mediated by the PI3K/AKT/SOX2 axis, a stem cell-specific branch of the PI3K/AKT signaling pathway. Further effector functions associated with PI3K/AKT induction include cell cycle progression, cellular (mass) growth, and the suppression of apoptosis. Apoptosis, however, is a central element of DNA damage response (DDR), where it provides a default mechanism for cell clearance when DNA integrity cannot be maintained. A key player in DDR is tumor suppressor p53, which accumulates upon DNA-damage and is counter-balanced by PI3K/AKT enforced turnover. Accordingly, stemness sustaining SOX2 expression and p53-dependent DDR mechanisms show molecular-functional overlap in PI3K/AKT signaling. This constellation proves challenging for stem cells whose genomic integrity is a functional imperative for normative ontogenesis. Unresolved mutations in stem and early progenitor cells may in fact provoke transformation and cancer development. Such mechanisms are also particularly relevant for iPSCs, where genetic changes imposed through somatic cell reprogramming may promote DNA damage. The current review aims to summarize the latest advances in the understanding of PI3K/AKT/SOX2-driven stemness and its intertwined relations to p53-signaling in DDR under conditions of pluripotency, reprogramming, and transformation.

Aim: In this study, we aimed to develop a polycationic non-viral carrier for the delivery of the reprogramming factors to the L929 fibroblast cell. Methods: We have prepared (3-hydroxybutyrate- co -3-hydroxyhexanoate) PHBHHx-based nanoparticles with the solvent diffusion method. Cytotoxicity of PXNs was determined via MTT assay. Transfection efficiency was evaluated via screening GFP expression by fluorescence microscopy. The expression of reprogramming factors (Oct4, Klf4, and Sox2) was determined by RT-qPCR. Results: PXNs with 32.9 ± 0.41 mV zeta potential and 177.6 ± 0.80 nm size were used for transfection of L929 Fbroblast cells. The percentage of cell viability of PXN were between 91.8%(±2.9) and 42.1%(±1.3). The transfection efficiency was found as 71.6%(±3,5). According to RT-qPCR data, the rate of transfection factors was significantly increased after the 11th cycle compared to non-transfected cells. Based on these results, it can be concluded that newly developed PXN is thought to be an effective tool for reprogramming cells.

Background: Many studies have been done to reported the value of SRY-related HMG-box Gene 2 (SOX2) in prognosis of solid tumors. But results were not particularly consistent among these studies because of the limitations of the small sample data., Methods: We searched relevant studies published before November 2018 by PubMed, Web of Science and EMBASE. In this meta-analysis, hazard ratio (HR) values for overall survival (OS) were cumulatively pooled and quantitatively analyzed., Results: A meta-analysis based on 12 studies with 3318 patients was conducted to assess the potential correlation between SOX2 overexpression and OS in human solid tumors. A total of 12 studies (n = 3318) were assessed in the meta-analysis. It suggested that the high expression of SOX2 obviously indicates poor survival and prognosis in both univariate and multivariate analysis. In the univariate analysis, the combined HR for OS was 1.66 (95% confidence interval [CI]: 1.46-1.89, P < .001). The pooled HR of multivariate analysis for OS was 1.51 (95% confidence interval [CI]: 1.32-1.71, P < .001)., Conclusions: This meta-analysis indicated that the high expression level of SOX2 is significantly associated with a decline in survival of human with solid tumors. On the basis of the expression level in solid tumors, SOX2 is expected to be a meaningful prognostic biomarker and effective therapeutic target.

Papillary thyroid carcinoma (PTC) is one of the most prevalent tumors in endocrine system. CircRNAs (circular RNAs) are widely known as critical regulators in tumorigenesis of papillary thyroid carcinoma (PTC). The present study focused on the functional investigation and potential molecular mechanism toward circ&#95;0005273 in PTC progression. Gene Expression Omnibus datasets (GSE93522) and qRT-PCR (quantitative real-time PCR) analyses showed that circ&#95;0005273 were upregulated in PTC tissues and cell lines. Moreover, circ&#95;0005273 was located in the cytoplasm of PTC cells and suggested poor prognosis in PTC patients. In vivo and in vitro functional assays indicated that knockdown of circ&#95;0005273 inhibited PTC tumor growth and progression, respectively. Mechanistically, miR-1183 was identified as functional target of circ&#95;0005273, and circ&#95;0005273 could directly bind to miR-1138 and relieve inhibition of SRY (sex-determining region Y)-box 2 (SOX2). Data from Cell Counting Kit-8, colony formation assays and transwell assays revealed that the oncogenic role of circ&#95;0005273 on PTC progression dependent on miR-1183-mediated SOX2 expression. In conclusion, circ&#95;0005273 functioned as a tumor promoter of PTC via circ&#95;0005273/miR-1183/SOX2 axis, suggesting a novel biomarker and therapeutic target for PTC.

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers in China, accompanied by an extremely high mortality rate. Chlorogenic acid (CGA) is a small-molecule compound, that has been shown to have a wide range of biological activities, including antitumor. However, the efficacy and molecular mechanism of CGA on ESCC remains unknown. In this study, we confirmed the inhibition of proliferation by CGA in ESCC cells, as well as the reduction of ESCC xenograft volume by CGA in vivo. In addition, CGA also suppressed both the migration and invasion of ESCC cells in vitro. In a carcinogen-induced murine model of ESCC, hyperplasia of the esophagus was slowed by CGA, while mice suffering from ESCC that were treated with CGA had longer survival times than mice in the control group. The measurement of pluripotency factors (BMI1, SOX2, OCT4 and Nanog) that are related to poor prognosis revealed reduced expression of both BMI1 and SOX2, but not of OCT4 or Nanog, in ESCC cells, in both a dose- and time-dependent manner. Together, our initial findings demonstrate that CGA suppresses ESCC progression, downregulates the expression of BMI1 and SOX2, and provide an anti-tumor candidate for ESCC therapy., (Copyright © 2019 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Purpose : This study aims to determine the expression patterns of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), proliferating cell nuclear antigen (PCNA) and SOX2 in lens epithelial cells (LEC) of cataract patients with pseudoexfoliation syndrome (PEX), and to determine the effect of apoptosis, proliferative activity and stem/progenitor cells on cataract formation in patients with PEX. This is a prospective, randomized clinical trial. Materials and Methods : Setting: institutional. 50 eyes of 50 patients were included. Anterior capsule samples were obtained during phacoemulsification surgery. The specimens of LEC were also examined using the TUNEL, PCNA and SOX2 immunohistochemical staining method. To detect the number of immunopositive cells, the total number of cells in a 3 mm 2 area was counted using a microscope under x20 magnification and the percentage of cells stained positive was determined. Results : In Group 1, increased expression was observed with TUNEL, while decreased expression was detected with PCNA ( p = .008, p = .015). The average percentage of TUNEL immunopositive cells was significantly higher in Group 1 than in Group 2, but there was no statistically meaningful SOX2 expression in Group 1 and Group 2 ( P = .44). Apoptosis rates were 61.75 ± 14.5% and 36.91 ± 14.6% in Groups 1 and 2, respectively. Proliferation rates were 40.96 ± 16.8% and 65.45 ± 16.9% in Groups 1 and 2, respectively. Conclusion : We found increased apoptosis and decreased proliferation of LECs in cataract patients with PEX. We suspected that this could be related to oxidative stress.

Recent studies have shown that re-expression of stem cell factors contribute to pathogenesis, therapy resistance, and recurrent disease in ovarian carcinomas. In this study, we compare the expression and co-expression of stem cell markers ALDH1 and SOX2 in different types of serous ovarian tumors. A total of 215 serous ovarian tumors (161 high-grade serous carcinomas (HGSC), 17 low-grade serous carcinomas (LGSC), 37 atypical proliferative serous tumors (APST)), and 10 cases of serous tubal intraepithelial carcinoma (STIC) were analyzed. Double immunostaining experiments addressed the association of cell proliferation (Ki67) with ALDH1 and the potential co-expression of SOX2 and ALDH1. The prognostic effect was analyzed in the cohort of HGSC. Expression of ALDH1and/or SOX2 was detected with increased frequency in HGSC (88.8%), compared with LGSC (70.5%) and APST (36.4%), while ALDH1 alone was significantly more frequently expressed in LGSC. The majority of all tumor types showed expression of ALDH1 and SOX2 in different cells. Only a minority of HGSC (4.6%) and STIC (20%) showed SOX2/ALDH1 co-expression in > 10% of tumor cells. Double staining also revealed that ALDH1 is associated with the non-proliferating Ki67-negative fraction consistent with a stem cell phenotype. Co-expression of ALDH1 and SOX2 or ALDH1 and Ki67 has no effect on survival. Expression of stem cell factors ALDH1 and/or SOX2 shows increased frequency in high-grade serous ovarian carcinomas compared to low-grade carcinomas and borderline tumors, supporting the concept that stem cell markers play different biological roles in low-grade versus high-grade serous neoplasia of the ovary.

Osimertinib, a third-generation epidermal growth factor receptor - tyrosine kinase inhibitor (EGFR-TKI), shows great efficacy in EGFR-mutant non-small cell lung cancer (NSCLC); however, the resistance is inevitable. Osimertinib induces autophagy in NSCLC cells, but the role of autophagy in osimertinib resistance is not clear. We discovered that enhanced autophagy is associated with osimertinib resistance in vitro and in vivo. Inhibition of autophagy enhanced osimertinib cytotoxicity in both osimertinib-resistant and sensitive cells. Moreover, osimertinib-resistant cells exhibited stem cell-like properties, whereas autophagy inhibition decreased the stemness by downregulating the expression of SOX2 and ALDH1A1. Further, we found that knockdown of Beclin-1 inhibited the stem cell-like properties and restored osimertinib cytotoxicity. Osimertinib combined with chloroquine inhibited tumor growth more effectively than alone in xenograft mice. These results reveal that autophagy plays an adverse role in osimertinib cytotoxicity through inducing stem cell-like properties. Combination therapy of EGFR-TKI and autophagy inhibitor could provide a promising strategy to improve osimertinib cytotoxicity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Lumen integrity in vascularization requires fully differentiated endothelial cells (ECs). Here, we report that endothelial-mesenchymal transitions (EndMTs) emerged in ECs of cerebral arteriovenous malformation (AVMs) and caused disruption of the lumen or lumen disorder. We show that excessive Sry-box 2 (Sox2) signaling was responsible for the EndMTs in cerebral AVMs. EC-specific suppression of Sox2 normalized endothelial differentiation and lumen formation and improved the cerebral AVMs. Epigenetic studies showed that induction of Sox2 altered the cerebral-endothelial transcriptional landscape and identified jumonji domain-containing protein 5 (JMJD5) as a direct target of Sox2. Sox2 interacted with JMJD5 to induce EndMTs in cerebral ECs. Furthermore, we utilized a high-throughput system to identify the β-adrenergic antagonist pronethalol as an inhibitor of Sox2 expression. Treatment with pronethalol stabilized endothelial differentiation and lumen formation, which limited the cerebral AVMs.

Chronic pain is one of the most prevalent chronic diseases in the world. The plastic changes of sensory neurons in dorsal root ganglia (DRG) have been extensively studied as the underlying periphery mechanism. Recent studies revealed that satellite cells, the major glial cells in DRG, also played important roles in the development/modulation of chronic pain. Whether DRG satellite glial cells generate new neurons as their counterparts in enteric nerve ganglia and carotid body do under pathological conditions remains poorly investigated. Here, we report that chronic pain induces proliferation and upregulation of progenitor markers in the sex-determining region Y-box 2 (Sox2)- and platelet-derived growth factor receptor alpha (PDGFRα)-positive satellite glial cells. BrdU incorporation assay revealed the generation of IB4- and CGRP-positive neurons, but not NF200-positive neurons in DRG ipsilateral to injury. Genetic fate tracings showed that PDGFRα-positive cells did not generate neurons, whereas Sox2-positive cells produced both IB4- and CGRP-positive neurons. Interestingly, glial fibrillary acidic protein-positive cells, a subpopulation of Sox2-positive satellites, only gave birth to IB4-positive neurons. Local persistent delivery of tetrodotoxin to the sciatic nerve trunk significantly reduced the pain-induced neurogenesis. Furthermore, patch-clamp studies demonstrated that these glia-derived new neurons could fire action potentials and respond to capsaicin. Taken together, our data demonstrated a chronic pain-induced nociceptive neurogenesis in DRG from Sox2-positive satellite cells, indicating a possible contribution of DRG neurogenesis to the pathology of chronic pain., (© 2019 Wiley Periodicals, Inc.)

Aims: The acquired drug resistance has been regarded as a main barrier for the effective treatment of temozolomide (TMZ) in glioblastoma (GBM). MiR-126-3p is commonly down-regulated and exerts tumor-suppressive roles in kinds of human cancers, including GBM. This study was designed to investigate the functions and mechanisms of miR-126-3p in regulating TMZ resistance in GBM., Materials and Methods: qRT-PCR analysis was used to measure the expressions of miR-126-3p and SOX2 mRNA in GBM tissues and cells. Cell viability, colony forming ability and apoptosis were detected to evaluate the effect of miR-126-3p or SOX2 on TMZ resistance. Luciferase reporter experiments were applied to identify the target genes of miR-126-3p. Western blot analysis was performed to determine the protein levels associated with Wnt/β-catenin signaling. TOP/FOP Flash assays were conducted to determine the effects of miR-126-3p or SOX2 on Wnt/β-catenin signaling., Key Findings: miR-126-3p expression was decreased in TMZ-resistant GBM tissues and cells. High levels of miR-126-3p enhanced TMZ sensitivity by inhibiting cell viability, reducing colony forming potential and inducing apoptosis. Additionally, SOX2 was identified as a downstream target of miR-126-3p. On the contrary, SOX2 overexpression conferred TMZ resistance of GBM cells. Moreover, miR-126-3p-mediated TMZ sensitivity was reversed following increased expression of SOX2. Furthermore, miR-126-3p-induced inactivation of Wnt/β-catenin signaling was greatly abrogated by SOX2 up-regulation., Significance: MiR-126-3p sensitizes GBM cells to TMZ possibly by repressing SOX2 expression and blocking Wnt/β-catenin signaling. This study provides novel targets to overcome TMZ resistance in GBM chemotherapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Enhancers are important regulatory elements that can control gene activity across vast genetic distances. However, the underlying nature of this regulation remains obscured because it has been difficult to observe in living cells. Here, we visualize the spatial organization and transcriptional output of the key pluripotency regulator Sox2 and its essential enhancer Sox2 Control Region (SCR) in living embryonic stem cells (ESCs). We find that Sox2 and SCR show no evidence of enhanced spatial proximity and that spatial dynamics of this pair is limited over tens of minutes. Sox2 transcription occurs in short, intermittent bursts in ESCs and, intriguingly, we find this activity demonstrates no association with enhancer proximity, suggesting that direct enhancer-promoter contacts do not drive contemporaneous Sox2 transcription. Our study establishes a framework for interrogation of enhancer function in living cells and supports an unexpected mechanism for enhancer control of Sox2 expression that uncouples transcription from enhancer proximity., Competing Interests: JA, JG, BL, LM, MS, YS, BH, SL, OW No competing interests declared, (© 2019, Alexander et al.)

Transplantation of dopaminergic precursors (DPs) is a promising therapeutic strategy of Parkinson's disease (PD). However, limited cell source for dopaminergic precursors has become a major obstacle for transplantation therapy. Our group demonstrated previously that mouse fibroblasts can be reprogrammed into induced dopaminergic precursors (iDPs) with high differentiation efficiency. In the current study, we hypothesized that a similar strategy can be applied to generate human iDPs for future cell therapy of PD. We overexpressed transcription factors Brn2, Sox2, and Foxa2 in human fibroblasts and observed formation of neurospheres. Subsequent characterization of the precursor colonies confirmed the generation of human induced dopaminergic precursors (hiDPs). These hiDPs were capable of self-renewal, proliferation, and differentiation. The hiDPs demonstrated high immunoreactivity for neural progenitor markers and high levels of gene expression for ventral mesencephalon-related neural progenitor markers such as Lmx1a, NIKX6.1, Corin, Otx2 and Mash1. Furthermore, the hiDPs could be differentiated into dopaminergic neurons with ˜80% efficiency, which significantly increased major functionally relevant proteins such as TH, DAT, AADC, Lmx1B, and VMAT2 compared to hiDPs. Additionally, hiDPs are more dopaminergic progenitor-restricted compare to those hiDP-like cells reprogrammed only by Brn2 and Sox2. Together, these results suggest that hiDPs with high differentiation efficiency can be generated by direct lineage reprogramming of fibroblasts with transcription factors Brn2, Sox2, and Foxa2. These hiDPs may serve as a safe and effective cell source for transplantation treatment of PD., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Individuals with sleep apnea often exhibit changes in cognitive behaviors consistent with alterations in the hippocampus. It is hypothesized that adult neurogenesis in the dentate gyrus is an ongoing process that maintains normal hippocampal function in many mammalian species, including humans. However, the impact of chronic intermittent hypoxia (IH), a principal consequence of sleep apnea, on hippocampal adult neurogenesis remains unclear. Using a murine model, we examined the impact of 30 d of IH (IH 30 ) on adult neurogenesis and synaptic plasticity in the dentate gyrus. Although IH 30 did not affect paired-pulse facilitation, IH 30 suppressed long-term potentiation (LTP). Immunohistochemical experiments also indicate that IH perturbs multiple aspects of adult neurogenesis. IH 30 increased the number of proliferating Sox2 + neural progenitor cells in the subgranular zone yet reduced the number of doublecortin-positive neurons. Consistent with these findings, cell lineage tracing revealed that IH 30 increased the proportion of radial glial cells in the subgranular zone, yet decreased the proportion of adult-born neurons in the dentate gyrus. While administration of a superoxide anion scavenger during IH did not prevent neural progenitor cell proliferation, it mitigated the IH-dependent suppression of LTP and prevented adult-born neuron loss. These data demonstrate that IH causes both reactive oxygen species-dependent and reactive oxygen species-independent effects on adult neurogenesis and synaptic plasticity in the dentate gyrus. Our findings identify cellular and neurophysiological changes in the hippocampus that may contribute to cognitive and behavioral deficits occurring in sleep apnea. SIGNIFICANCE STATEMENT Individuals with sleep apnea experience periods of intermittent hypoxia (IH) that can negatively impact many aspects of brain function. Neurons are continually generated throughout adulthood to support hippocampal physiology and behavior. This study demonstrates that IH exposure attenuates hippocampal long-term potentiation and reduces adult neurogenesis. Antioxidant treatment mitigates these effects indicating that oxidative signaling caused by IH is a significant factor that impairs synaptic plasticity and reduces adult neurogenesis in the hippocampus., (Copyright © 2019 the authors 0270-6474/19/391320-12$15.00/0.)

B7-H3 is a tumor-promoting glycoprotein that is expressed at low levels in most normal tissues, but is overexpressed in various human cancers which is associated with disease progression and poor patient outcome. Although numerous publications have reported the correlation between B7-H3 and cancer progression in many types of cancers, mechanistic studies on how B7-H3 regulates cancer malignancy are rare, and the mechanisms underlying the role of B7-H3 in drug resistance are almost unknown. Here we report a novel finding that upregulation of B7-H3 increases the breast cancer stem cell population and promotes cancer development. Depletion of B7-H3 in breast cancer significantly inhibits the cancer stem cells. By immunoprecipitation and mass spectrometry, we found that B7-H3 is associated with the major vault protein (MVP) and activates MEK through MVP-enhancing B-RAF and MEK interaction. B7-H3 expression increases stem cell population by binding to MVP which regulates the activation of the MAPK kinase pathway. Depletion of MVP blocks the activation of MEK induced by B7-H3 and dramatically inhibits B7-H3 induced stem cells. This study reports novel functions of B7-H3 in regulating breast cancer stem cell enrichment. The novel mechanism for B7-H3-induced stem cell propagation by regulating MVP/MEK signaling axis independent of the classic Ras pathway may have important implications in the development of strategies for overcoming cancer cell resistance to chemotherapy.

SOX2 (Sex-determining region Y box 2) is a transcription factor expressed in several foetal and adult tissues and its deregulated activity has been linked to chronic diseases associated with ageing. Nevertheless, the level of SOX2 expression in aged individuals at the tissue level has not previously been examined. In this work, we show that SOX2 expression decreases significantly in the brain with ageing, in both humans and rodents. The administration of resveratrol for 6 months in mice partly attenuated this reduction. We also identified an age-related decline in SOX2 mRNA and protein expression in several other organs, namely, the lung, heart, kidney, spleen and liver. Moreover, peripheral blood mononuclear cells (PBMCs) from elderly expressed lower levels of SOX2 than those from young individuals. Mechanistically, SOX2 expression inversely correlates with p16 Ink4a levels. Together, these data show a widespread decrease in SOX2 with age, suggesting that the decline in SOX2 expression might be used as a biomarker of ageing., (Copyright © 2018. Published by Elsevier B.V.)

Objectives: Soluble IL-33 (interleukin (IL)-1-like cytokine) acts as endogenous alarm signal (alarmin). Since alarmins, besides activating immune system, act to restore tissue homeostasis, we investigated whether IL-33 exerts beneficial effects on oral stem cell pull., Materials and Methods: Clonogenicity, proliferation, differentiation and senescence of stem cells derived from human periodontal ligament (PDLSCs) and dental pulp (DPSCs) were determined after in vitro exposure to IL-33. Cellular changes were detected by flow cytometry, Western blot, immunocytochemistry and semiquantitative RT-PCR., Results: IL-33 stimulated proliferation, clonogenicity and expression of pluripotency markers, OCT-4, SOX-2 and NANOG, but it inhibited ALP activity and mineralization in both PDLSCs and DPSCs. Higher Ki67 expression and reduced β-galactosidase activity in IL-33-treated cells were demonstrated, whereas these trends were more conspicuous in osteogenic medium. However, after 7-day IL-33 pretreatment, differentiation capacity of IL-33-pretreated cells was retained, and increased ALP activity was observed in both cell types. Results showed that IL-33 regulates NF-κB and β-catenin signalling, indicating the association of these molecules with changes observed in IL-33-treated PDLSCs and DPSCs, particularly their proliferation, pluripotency-associated marker expression and osteogenesis., Conclusions: IL-33 treatment impairs osteogenesis of PDLSCs and DPSCs, while increases their clonogenicity, proliferation and pluripotency marker expression. After exposure to IL-33, osteogenic capacity of cells stayed intact. NF-κB and β-catenin are implicated in the effects achieved by IL-33 in PDLSCs and DPSCs., (© 2018 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

Endometrial carcinoma is a cancer derived from oncogenesis of the regenerating uterine cavity, in which cytokine stimulation shapes cell differentiation and tissue remodeling. Expression of the stem cell factors SOX2, OCT4, NANOG, and MYC has been linked to tumor malignancy in several cancers. However, how these stem cell factors crosstalk with cytokine signaling to promote malignancy in endometrial carcinoma is still elusive. Here we report that the expression of SOX2 and MYC, but not that of OCT4 and NANOG, correlate with poor histological differentiation and prognosis, while SOX2 expression is negatively associated with MYC level. We found that SOX2-high endometrial carcinoma cells possessed a higher colony-forming ability than their SOX2-low counterparts, and knockdown of SOX2 attenuated the colony-forming ability. We observed that SOX2 regulated EGFR expression in a SOX2-EGFR positive feedback loop. EGF stimulation induced SOX2 expression and promoted migration of endometrial carcinoma cells, whereas TGF-β stimulation inhibited SOX2 expression and attenuated the colony-forming ability. Immunohistochemistry analysis revealed that SOX2 expression correlated with lymph node infiltration of endometrial carcinoma. Our findings support that cytokine-induced stem cell factor SOX2 possesses oncogenic properties, with the potential to serve as a prognostic biomarker in endometrial carcinoma.

There are no effective treatments for corneal endothelial diseases, except for corneal transplantation, as human corneal endothelial cells (hCECs) do not regenerate. The regeneration of hCECs could be induced through regulation of the expression of specific genes. In this study, we investigated whether the overexpression of sex-determining region Y-box 2 (SOX2) can regenerate hCECs in vivo and in vitro. SOX2 was activated using the clustered regularly interspaced short palindromic repeats (CRISPR)/deactivated CRISPR-associated protein 9 (dCas9) activation system. Genes were transfected into the corneal endothelium of Sprague-Dawley rats. Central corneal thickness and opacity were measured, and alizarin red S staining was performed. Corneal opacity and central corneal thickness were reduced in the SOX2 group compared with the control group. The density of CECs was higher in the SOX2 group compared with the control group. Additionally, hCECs were cultured and analyzed after overexpressing SOX2. Cell viability, proliferation rate, and the number of cells in S-phase were increased after SOX2 overexpression (p < .05). Cyclin-dependent kinase 1 and cyclin D1 were found to be overexpressed (p < .05). WNT signaling was repressed, and the AKT pathway was activated by SOX2 overexpression. Mitochondrial oxidative stress and energy production were increased by SOX2 overexpression (p < .05). In conclusion, SOX2 activation promotes wound healing and regeneration in CECs. SOX2 activation using the CRISPR/dCas9 system may thus be useful for the treatment of hCEC diseases. Stem Cells 2018;36:1851-12., (© 2018 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Accumulation of amyloid-β peptide (Aβ) and massive neuronal death due to apoptosis were the essential steps in the pathogenesis of Alzheimer's disease (AD). MiR-429 was reported to play an important role in the pathogenesis of AD. However, the detailed function and underlying molecular mechanism of miR-429 in the pathogenesis of AD remain elusive. Cortical neurons were stimulated with 20 µM of Aβ 25-35 for 24 h to construct AD model in vitro. qRT-PCR assay was used to detect the expression of miR-429, and qRT-PCR or western blot analysis were performed to assess the levels of Sex-determining region Y-box 2 (SOX2) and B cell lymphoma-2 protein (BCL2) at mRNA or proteins levels in the AD mouse model and Aβ-induced treated cortical neurons. Luciferase reporter assay and western blot analysis were used to confirm the potential targets of miR-429. CCK-8 assay, flow cytometry analysis, and caspase3 activity assay were used to measure cell viability, cell apoptosis capacity and caspase3 activity, respectively. MiR-429 was upregulated and SOX2 and BCL2 were downregulated in the AD mouse model and Aβ-induced mouse cortical neurons. MiR-429 knockdown attenuated Aβ-induced cytotoxicity in mouse cortical neurons. SOX2 and BCL2 were direct targets of miR-429. Moreover, anti-miR-429-mediated neuroprotective effect was abated by the restoration of SOX2 or BCL2 expression. Knockdown of miR-429 might attenuate Aβ-induced cytotoxicity by targeting SOX2 and BCL2 in mouse cortical neurons, providing a novel prospect in AD therapy.

Severe uterine damage and infection lead to intrauterine adhesions, which result in hypomenorrhea, amenorrhea and infertility. Cell sheet engineering has shown great promise in clinical applications. Adipose-derived stem cells (ADSCs) are emerging as an alternative source of stem cells for cell-based therapies. In the present study, we investigated the feasibility of applying ADSCs as seed cells to form scaffold-free cell sheet. Data showed that ADSC sheets expressed higher levels of FGF, Col I, TGFβ, and VEGF than ADSCs in suspension, while increased expression of this gene set was associated with stemness, including Nanog, Oct4, and Sox2. We then investigated the therapeutic effects of 3D ADSCs sheet on regeneration in a rat model. We found that ADSCs were mainly detected in the basal layer of the regenerating endometrium in the cell sheet group at 21 days after transplantation. Additionally, some ADSCs differentiated into stromal-like cells. Moreover, ADSC sheets transplanted into partially excised uteri promoted regeneration of the endometrium cells, muscle cells and stimulated angiogenesis, and also resulted in better pregnancy outcomes. Therefore, ADSC sheet therapy shows considerable promise as a new treatment for severe uterine damage.

Inner ear formation requires that a series of cell fate decisions and morphogenetic events occur in a precise temporal and spatial pattern. Previous studies have shown that transcription factors, including Pax2, Sox2, and Prox1, play important roles during the inner ear development. However, the temporospatial expression patterns among these transcription factors are poorly understood. In the current study, we present a comprehensive description of the temporal and spatial expression profiles of Pax2, Sox2, and Prox1 during auditory and vestibular sensory organ development in mice. Using immunohistochemical analyses, we show that Sox2 and Pax2 are both expressed in the prosensory cells (the developing hair cells), but Sox2 is later restricted to only the supporting cells of the organ of Corti. In the vestibular sensory organ, however, the Pax2 expression is localized in hair cells at postnatal day 7, while Sox2 is still expressed in both the hair cells and supporting cells at that time. Prox1 was transiently expressed in the presumptive hair cells and developing supporting cells, and lower Prox1 expression was observed in the vestibular sensory organ compared to the organ of Corti. The different expression patterns of these transcription factors in the developing auditory and vestibular sensory organs suggest that they play different roles in the development of the sensory epithelia and might help to shape the respective sensory structures.

Stem cell surface markers may facilitate a better understanding of stem cell biology through molecular function studies or serve as tools to monitor the differentiation status and behavior of stem cells in culture or tissue. Thus, it is important to identify additional novel stem cell markers. We used glycoproteomics to discover surface glycoproteins on human embryonic stem cells (hESCs) that may be useful stem cell markers. We found that a surface glycoprotein, leucine-rich repeat neuronal protein 1 (LRRN1), is expressed abundantly on the surface of hESCs before differentiation into embryoid bodies (EBs). Silencing of LRRN1 with short hairpin RNA (shLRRN1) in hESCs resulted in decreased capacity of self-renewal, and skewed differentiation toward endoderm/mesoderm lineages in vitro and in vivo. Meanwhile, the protein expression levels of the pluripotency factors OCT4, NANOG, and SOX2 were reduced. Interestingly, the mRNA levels of these pluripotency factors were not affected in LRRN1 silenced cells, but protein half-lives were substantially shortened. Furthermore, we found LRRN1 silencing led to nuclear export and proteasomal degradation of all three pluripotency factors. In addition, the effects on nuclear export were mediated by AKT phosphorylation. These results suggest that LRRN1 plays an important role in maintaining the protein stability of pluripotency factors through AKT phosphorylation, thus maintaining hESC self-renewal capacity and pluripotency. Overall, we found that LRRN1 contributes to pluripotency of hESC by preventing translocation of OCT4, NANOG, and SOX2 from nucleus to cytoplasm, thereby lessening their post-translational modification and degradation. Stem Cells 2018;36:1514-1524., (© AlphaMed Press 2018.)

Previous studies have suggested that cancer stem cells (CSCs) resisted radiotherapy and chemotherapy. P16 INK4A is a biomarker for cervical carcinogenesis and reduces proliferation of stem cells. We aimed to investigate the expression and clinical significance of cyclin-dependent kinase inhibitor 2A (P16 INK4A ), sex determining region Y-box 2 (SOX2), and Aldehyde dehydrogenase 1 family, member A1 (ALDH1A1) in cervical cancer treated with radiotherapy and cervical cell line models. The expressions of P16 INK4A , SOX2, and ALDH1A1 were performed by immunohistochemical staining of tumor samples from 139 cervical cancer patients with International Federation of Gynecology and Obstetrics stages Ib to IV. The staining showed high expression in 100, 107, and 13 patients with P16 INK4A (>80%), SOX2 (≥10%), and ALDH1A1 (50%), respectively. The high-P16 INK4A group had a higher five-year overall survival (OS) rate and disease-free survival (DFS) than the low-P16 INK4A group (OS: 62.0% and 35.2%, p = 0.016; DFS: 60.0% and 31.2%, p = 0.002). The low-P16 INK4A /high-SOX2 and low-P16 INK4A /high-ALDH1A1 groups had a worse five-year OS and DFS rate than the high-P16 INK4A /low-SOX2 and high-P16 INK4A /low-ALDH1A1 groups, respectively. Depletion of P16 INK4A promoted chemoresistance and radioresistance of cervical cancer cells increased the expression of SOX2 and ALDH1A1 and exhibited higher self-renewal ability. These results suggest that lower P16 INK4A expression associated with higher CSC markers predicts poor prognostic outcomes and is a promising target in patients with cervical cancer.

Upregulation of Cdx2 expression in outer cells is a key event responsible for cell lineage segregation between the inner cell mass and the trophoderm (TE) in mouse morula-stage embryos. In TE cells, polarization can regulate Hippo and Rho-associated kinase (Rho-ROCK) signaling to induce the nuclear location of YAP, which has been demonstrated to further induce the expression of Cdx2. However, we found that CDX2 expression could not be detected in the outer cells of porcine morula-stage embryos but only in some TE cells at the early blastocyst stage. The biological significance and the regulation mechanism of this species-specific CDX2 expression pattern have still not been determined. We show here that an asynchronous CDX2 expression pattern exists in porcine TE cells during the development of the blastocyst. We demonstrate that CDX2 expression in porcine TE cells depends on the nuclear localization of YAP and polarization of the embryo through Y27632 treatment. We found that the polarization process in the morula to the late blastocyst stage porcine embryos was asynchronous, which was revealed by the apical localization of phosphorylated EZRIN staining. Artificially enhancing the number of polarized blastomeres by culturing the separated blastomeres of four-cell stage porcine embryos resulted in increased CDX2-positive cell numbers. These results indicate that the mechanism of CDX2 expression regulation is conserved, but the polarization progress is not conserved between the pig and the mouse, and results in a species-specific trophoblast determination progress model., (© 2018 Wiley Periodicals, Inc., A Wiley Company.)

Sex determining region Y-box protein 3 (SOX3) is involved in embryonic development and tumorigenesis. However, the expression and precise role of SOX3 in osteosarcoma remain unclear. In this study, we reported that SOX3 expression was upregulated in osteosarcoma tissues compared with non-cancerous bone cyst tissues. To elucidate the cellular and molecular function of SOX3, we examined the consequences of SOX3 knockdown in osteosarcoma cells. We found that the downregulation of SOX3 inhibited the proliferation, migration and invasion of osteosarcoma cells. SOX3 downregulation also increased the cell population in the G1 phase and induced cell apoptosis. SOX3 knockdown-mediated cell cycle arrest and cell apoptosis were associated with decreased levels of Cdc25A, cyclin D1, proliferating cell nuclear antigen (PCNA) and Bcl-2, as well as an increased Bax expression. We also found that the downregulation of SOX3 decreased the expression of Snail, Twist and matrix metalloproteinase-9 (MMP-9), and increased E-cadherin expression, resulting in the inhibition of cell migration and invasion. Taken together, our data indicate that SOX3 may serve as an oncogene in osteosarcoma, and SOX3 downregulation may prove to be a novel approach for the inhibition of osteosarcoma progression.

In mice, the incisors grow throughout the animal's life, and this continuous renewal is driven by dental epithelial and mesenchymal stem cells. Sox2 is a principal marker of the epithelial stem cells that reside in the mouse incisor stem cell niche, called the labial cervical loop, but relatively little is known about the role of the Sox2 + stem cell population. In this study, we show that conditional deletion of Sox2 in the embryonic incisor epithelium leads to growth defects and impairment of ameloblast lineage commitment. Deletion of Sox2 specifically in Sox2 + cells during incisor renewal revealed cellular plasticity that leads to the relatively rapid restoration of a Sox2 -expressing cell population. Furthermore, we show that Lgr5 -expressing cells are a subpopulation of dental Sox2 + cells that also arise from Sox2 + cells during tooth formation. Finally, we show that the embryonic and adult Sox2 + populations are regulated by distinct signalling pathways, which is reflected in their distinct transcriptomic signatures. Together, our findings demonstrate that a Sox2 + stem cell population can be regenerated from Sox2 - cells, reinforcing its importance for incisor homeostasis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Aim: To investigate the expression of cancer stem cell markers in meningiomas., Material and Methods: CD133, Nestin and Sox2 expression levels in 35 paraffin-embedded meningioma tissue samples were assessed using immunohistochemistry., Results: In this study, five cases were atypical (WHO Grade II), two were anaplastic (WHO Grade III), and 28 were benign (WHO Grade I). Among atypical and anaplastic meningiomas, all were positive for Nestin and CD133, and 4 were positive for Sox2. Of the 28 benign meningiomas, 23 were positive for Nestin, 11 were positive for CD133, and none were positive for Sox2. In addition, Nestin and CD133 were expressed at significantly higher levels in the non-benign group than in the benign group., Conclusion: Nestin, CD133 and Sox2 expression levels may be correlated with the WHO pathological grade. Specifically, more aggressive meningiomas are characterized by higher positivity rates and higher levels of Nestin, CD133 and Sox2 expression in positive cells.

Elongation of the body axis is a key aspect of body plan development. Bipotential neuromesoderm progenitors (NMPs) ensure axial growth of embryos by contributing both to the spinal cord and mesoderm. The current model for the mechanism controlling NMP deployment invokes Tbx6, a T-box factor, to drive mesoderm differentiation of NMPs. Here, we identify a new population of Tbx6 + cells in a subdomain of the NMP niche in mouse embryos. Based on co-expression of a progenitor marker, Sox2, we identify this population as representing a transient cell state in the mesoderm-fated NMP lineage. Genetic lineage tracing confirms the presence of the Tbx6 + NMP cell state. Furthermore, we report a novel aspect of the documented Tbx6 mutant phenotype, namely an increase from two to four ectopic neural tubes, corresponding to the switch in NMP niche, thus highlighting the importance of Tbx6 function in NMP fate decision. This study emphasizes the function of Tbx6 as a bistable switch that turns mesoderm fate 'on' and progenitor state 'off', and thus has implications for the molecular mechanism driving NMP fate choice., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

SOX2 is a regulatory factor of embryonic stem cells that has been implicated in carcinogenesis and cancer progression. We aimed to investigate the potential role of SOX2 in the stepwise progression from pleomorphic adenoma (PA) to invasive carcinoma ex pleomorphic adenoma (CXPA), evaluating its prognostic significance as well. Thirty PAs without malignant transformation and 25 CXPAs presenting both luminal or myoepithelial differentiation (7 intracapsular and 18 extracapsular) were evaluated immunohistochemically for SOX2 expression. Of these, 24 CXPAs (96%) were positive to SOX2, being 6 intracapsular carcinomas (85.7%) and all the 18 extracapsular carcinomas (100%). Residual PA areas and PA without malignant transformation were negative. High SOX2 expression levels (> 50% of positive cells) were correlated with high histological grade (p = 0.02), brisk mitotic activity (p = 0.01), advanced pT stage (p = 0.01), tumor recurrence (p = 0.01), and development of distant metastasis (p = 0.004). Still, overall survival rates were shorter in patients with extracapsular CXPA exhibiting diffuse SOX2 expression. These results suggest that SOX2 may play an important role in carcinogenesis and progression of CXPA and is also related with prognostic indicators in CXPAs with extracapsular invasion. Although direct therapeutic intervention in SOX2 may result in unwanted complications due to its constitutive functions, strategic approach to SOX2-related pathways may provide new therapeutic opportunities for patients with invasive CXPA.

Acute myeloid leukemia is driven by leukemic stem cells which can be identified by cross lineage expression or arrest of differentiation compared to normal hematopoietic stem cells. Self-renewal and lack of differentiation are also features of stem cells and have been associated with the expression of embryonic genes. The aim of our study was to evaluate the expression of embryonic antigens (OCT4, NANOG, SOX2, SSEA1, SSEA3) in hematopoietic stem cell subsets (CD34 + CD38 - and CD34 + CD38 + ) from normal bone marrows and in samples from acute myeloid leukemia patients. We observed an upregulation of the transcription factors OCT4 and SOX2 in leukemic cells as compared to normal cells. Conversely, SSEA1 protein was downregulated in leukemic cells. The expression of OCT4, SOX2, and SSEA3 was higher in CD34 + CD38 - than in CD34 + CD38 + subsets in leukemic cells. There was no correlation with biological characteristics of the leukemia. We evaluated the prognostic value of marker expression in 69 patients who received an intensive treatment. The rate of complete remission was not influenced by the level of expression of markers. Overall survival was significantly better for patients with high SOX2 levels, which was unexpected because of the inverse correlation with favorable genetic subtypes. These results prompt us to evaluate the potential role of these markers in leukemogenesis and to test their relevance for better leukemic stem cell identification.

SOX2 is an important transcription factor involved in pluripotency maintenance, pluripotent reprogramming and differentiation towards neural lineages. Here we engineered the previously described HEL24.3 hiPSC to generate a SOX2 reporter by knocking-in a T2A fused nuclear tdTomato reporter cassette before the STOP codon of the SOX2 gene coding sequence. CRISPR/SaCas9-mediated stimulation of homologous recombination was utilized to facilitate faithful targeted insertion. This line accurately reports the expression of endogenous SOX2 and therefore constitutes a useful tool to study the SOX2 expression dynamics upon hiPSC culture, differentiation and somatic cell reprogramming., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Accumulating evidence has shown that miR-429 plays an important role in the development and progression of tumour. However, the role of miR-429 in glioblastoma multiforme (GBM) remains largely unknown. The present study is designed to investigate the function of miR-429 in GBM and to explore the molecular mechanism underlying its function. The expression level of miR-429 was detected in GBM tissues and cell lines by quantitative real-time polymerase chain reaction. The effect of overexpression of miR-429 on in vitro cell proliferation, apoptosis and invasion was examined. Western blot analysis was used to detect the influence of miR-429 on the expression of target gene, and Pearson analysis was used to calculate the correlation between the expression of targets gene and the miR-429 in GBM tissues. Our study shows that miR-429 is downregulated in GBM tissues compared with noncancerous tissues (P < .01). In addition, the expression of miR-429 in GBM cell lines is also significantly lower (P < .01). Enforced expression of miR-429 inhibits GBM cells proliferation, induces apoptosis and suppresses invasion and leads to the downregulation of the SOX2 protein. Moreover, the expression level of miR-429 in GBM tissues shows inverse relationship with the expression level of SOX2 protein. Our findings suggest that miR-429 represents a potential tumour-suppressive miRNA and plays an important role in GBM progression by directly targeting SOX2., (Copyright © 2017 John Wiley & Sons, Ltd.)

The mammalian post-implantation embryo has been extensively investigated at the tissue level. However, to unravel the molecular basis for the cell-fate plasticity and determination, it is essential to study the characteristics of individual cells. In particular, the individual definitive endoderm (DE) cells have not been characterized in vivo Here, we report gene expression patterns in single cells freshly isolated from mouse embryos on days 5.5 and 6.5. Initial transcriptome data from 124 single cells yielded signature genes for the epiblast, visceral endoderm, and extra-embryonic ectoderm and revealed a unique distribution pattern of fibroblast growth factor (FGF) ligands and receptors. Further analysis indicated that early-stage epiblast cells do not segregate into lineages of the major germ layers. Instead, some cells began to diverge from epiblast cells, displaying molecular features of the premesendoderm by expressing higher levels of mesendoderm markers and lower levels of Sox3 transcripts. Analysis of single-cell high-throughput quantitative RT-PCR data from 441 cells identified a late stage of the day 6.5 embryo in which mesoderm and DE cells emerge, with many of them coexpressing Oct4 and Gata6 Analysis of single-cell RNA-sequence data from 112 cells of the late-stage day 6.5 embryos revealed differentially expressed signaling genes and networks of transcription factors that might underlie the segregation of the mesoderm and DE lineages. Moreover, we discovered a subpopulation of mesoderm cells that possess molecular features of the extraembryonic mesoderm. This study provides fundamental insight into the molecular basis for lineage segregation in post-implantation mouse embryos., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Specific activation of endogenous genes can be achieved by programmable artificial transcription factors (ATFs). In this study, we compared two artificial, programmable, clustered regularly interspaced short palindromic repeats (CRISPR)-based, ubiquitous transcription factors: deficient CRISPR-associated protein 9 (dCas9)-VP64 (CRISPRa) alone, or a combination of dCas9-VP64 and MS2-P65-HSF1 [synergistic activation mediator (SAM) system] mediated activation of five pluripotency genes: KLF4 (K), LIN28 (L), MYC (M), OCT4 (O), and SOX2 (S) in human cells (HEK293T, HeLa, HepG2, and primary fibroblasts). Activation potential was monitored using a luciferase reporter system and we found that both CRISPRa and SAM can efficiently activate the proximal promoter of all five genes. We also observed that the guide RNA (gRNA) target sites and number of gRNAs have a major effect on gRNA-guided activation efficiency. Furthermore, increased activation efficiency (>3-folds) could be achieved by the SAM system compared to CRISPRa. In addition, we discovered that only the SAM system could efficiently activate LIN28, OCT4, and SOX2 expression (up to 100-folds compared to coexpression with a scrambled gRNA) in primary human fibroblasts. This SAM-mediated activation of LOS can be stably maintained for over 20 days in fibroblasts cultured in either fibroblasts or stem cell medium. However, when attempting to use the SAM-LOS activation as an approach for induced pluripotent stem cells-reprogramming, no embryonic stem-like colonies could be obtained from these SAM fibroblasts. In conclusion, our study showed that CRISPR/Cas9-based ATFs are potent to activate and maintain transcription of endogenous human pluripotent genes. However, future improvements of the system are still required to improve activation efficiency and cellular reprogramming using ATFs.

The epithelial-mesenchymal transition is the key process driving cancer metastasis. MicroRNA-194 inhibits epithelial-mesenchymal transition in several cancers and its downregulation indicates a poor prognosis in human endometrial carcinoma. Self-renewal factor Sox3 induces epithelial-mesenchymal transition at gastrulation and is also involved epithelial-mesenchymal transition in several cancers. We intended to determine the roles of Sox3 in inducing epithelial-mesenchymal transition in endometrial cancer stem cells and the possible role of microRNA-194 in controlling Sox3 expression. Firstly, we found that Sox3 and microRNA-194 expressions were associated with the status of endometrial cancer stem cells in a panel of endometrial carcinoma tissue, the CD133+ cell was higher in tumorsphere than in differentiated cells, and overexpression of microRNA-194 would decrease CD133+ cell expression. Silencing of Sox3 in endometrial cancer stem cell upregulated the epithelial marker E-cadherin, downregulated the mesenchymal marker vimentin, and significantly reduced cell invasion in vitro; overexpression of Sox3 reversed these phenotypes. Furthermore, we discovered that the expression of Sox3 was suppressed by microRNA-194 through direct binding to the Sox3 3'-untranslated region. Ectopic expression of microRNA-194 in endometrial cancer stem cells induced a mesenchymal-epithelial transition by restoring E-cadherin expression, decreasing vimentin expression, and inhibiting cell invasion in vitro. Moreover, overexpression of microRNA-194 inhibited endometrial cancer stem cell invasion or metastasis in vivo by injection of adenovirus microRNA-194. These findings demonstrate the novel mechanism by which Sox3 contributes to endometrial cancer stem cell invasion and suggest that repression of Sox3 by microRNA-194 may have therapeutic potential to suppress endometrial carcinoma metastasis. The cancer stem cell marker, CD133, might be the surface marker of endometrial cancer stem cell.

Epstein-Barr virus (EBV) infection is closely related to carcinogenesis of various cancers, and is also associated with the development of drug resistance in cancer stem cells. However, in EBV-positive cancer cells, the mechanistic details of the downstream signaling and the connection of PI3K with the NF-κB pathway for development of drug resistance remain controversial. Diffuse large B-cell lymphoma (DLBCL) and multiple myeloma (MM) cells infected by EBV display drug resistance-related proteins (MDR1, MRP1 and MRP2) and stem cell markers (OCT4 and SOX2). EBV-infected HT (HT/EBV) and H929 (H929/EBV) cells activated p110δ expression, but downregulated the expression of p110α and p110β. A combination of CAL-101, a p110δ-specific inhibitor, with bortezomib treatment of HT/EBV cells synergistically suppressed proliferation, reduced levels of drug resistance-related proteins, activated caspase cleavage and recovered expression of p110α/p110β. Additionally, co-treatment with CAL-101 and bortezomib attenuated the expression of OCT4 and SOX2 via inhibition of activated NF-κB. Co-treatment with CAL-101 and bortezomib also attenuated drug resistance and NF-κB activity of EBV-infected H929 cells. Our results provide supportive evidence for the clinical application of CAL-101 and bortezomib to treat EBV-infected hematologic cancer.