Expression Of Stem Cell Factors Research Articles

Developmental Genetic and Molecular Analysis of Drosophila Central Complex Lineages.

Complex behaviors are mediated by a diverse class of neurons and glia produced during development. Both neural stem cell-intrinsic and -extrinsic temporal cues regulate the appropriate number, molecular identity, and circuit assembly of neurons. The Drosophila central complex (CX) is a higher-order brain structure regulating various behaviors, including sensory-motor integration, celestial navigation, and sleep. Most neurons and glia in the adult CX are formed during larval development by 16 Type II neural stem cells (NSCs). Unlike Type I NSCs, which directly give rise to the ganglion mother cells (GMCs), Type II NSCs give rise to multiple intermediate neural progenitors (INPs), and each INP in turn generates multiple GMCs, hence fostering the generation of longer and more diverse lineages. This makes Type II NSCs a suitable model to unravel the molecular mechanisms regulating neural diversity in more complex lineages. In this review, we elaborate on the classification and identification of NSCs based on the types of division adopted and the molecular markers expressed in each type. In the end, we discuss genetic methods for lineage analysis and birthdating. We also explain the temporal expression of stem cell factors and genetic techniques to study how stem cell factors may regulate neural fate specification.

SRC-3/TRAF4 facilitates ovarian cancer development by activating the PI3K/AKT signaling pathway

ObjectiveOvarian cancer is the seventh most common cancer in women, and it causes many deaths in women worldwide. Patients with ovarian cancer have a poor prognosis and low survival rate. This study aimed to explore the role of the SRC-3/TRAF4/PI3K/AKT pathway in ovarian cancer development.MethodsSRC-3 and TRAF4 expression in ovarian cancer cell lines were assessed using qRT-PCR and western-blotting. The expression of SRC-3 and TRAF4 in ovarian cancer cells was downregulated by transient transfection with sh-RNAs. An MTT assay was performed to evaluate cell proliferation. Cell migration and invasion were measured using a Transwell assay. Cell stemness was detected using a cell spheroidization assay and western blotting. The expression levels of stem cell factors and PI3K/AKT pathway proteins were determined by qRT-PCR and western blot analysis.ResultsSRC-3 and TRAF4 were upregulated in ovarian cancer cell lines. TRAF4 is a downstream factor of SRC-3, and the protein level of TRAF4 was regulated by SRC-3. SRC-3 knockdown reduced TRAF4 expression. Silencing SRC-3 or TRAF4 inhibited cell proliferation, migration, and invasion, as well as the expression of stem cell factors. Furthermore, sh-TRAF4 as well as treatment with LY294002, the PI3K/Akt inhibitor, inhibited the phosphorylation of Akt and PI3K, thus repressing the activation of PI3K/AKT signaling pathway in ovarian cancer cell lines. However, TRAF4 overexpression reversed the effect of SRC-3 silencing on cell proliferation, migration, invasion, and stemness.ConclusionOur study demonstrated that SRC-3/TRAF4 promotes ovarian cancer cell growth, migration, invasion, and stemness by activating the PI3K/AKT pathway.

The Application of the SORE6 Reporter in the Studies of Acute Myeloid Leukemia - a Novel Model for Disease Relapses

Most patients diagnosed with acute myeloid leukemia (AML) suffer from disease relapses within two years after the initial remission. Treatment options for relapsed AML are limited and the clinical outcome associated with this stage of disease is dismal. Partly due to the lack of experimental models, the biology underlying AML relapses is not well understood. We hypothesized that the cancer stem-like (CSL) cells, which represent a minority of the neoplastic cell population, are the major contributors of AML relapses. SORE6, a reporter designed to detect the expression and activity of inducible pluripotent stem cell factors, was used to identify/purify CSL cells in two FLT3-mutated AML cell lines, MOLM-13 and MV4-11. Both cell lines contained ~10% of SORE6+ cells at the steady state, as revealed by their expression of the reporter green fluorescence protein. Compared to SORE6- cells, SORE6+ cells had significantly more spheroid formation in the hanging drop assay (75 vs 35%, p=.006), and were significantly more resistant to Ara-C (inhibitory concentration at 50%, 60 vs 25 nM, p=.0005) and Venetoclax (2.6 vs 8.2 nM, p=.002) in the presence of low-dose Ara-C. By western blots, SORE6+ cells were found to express substantially higher levels of Myc/FLT3/pSTAT5 than SORE6- cells. Myc appeared to be a key driver of SORE6 activity, and the reporter activity was effectively suppressed by Myc pharmacologic inhibitors or shRNA. By subjecting a mixture of bar-coded SORE6- and SORE6+ MOLM-13 cells (in 9:1 ratio to mimic that at the steady state) to the lowest concentration of Ara-C that resulted in no detectable viability in the cell culture, we consistently found evidence of cell regeneration after 8-12 days of undetectable cell viability. Regeneration initially occurred slowly, and the cell growth went into a log phase after 1-2 weeks. Using PCR to detect the relative proportion of bar-coded SORE6- and SORE6+ cells, we found that the regenerated cell population contained a substantially higher proportion of SORE6+ cells, with 64% SORE6+ cells in the initial slow-growth phase. In comparison, the proportion of cells originating from SORE6+ cells increased to an average of 86% during the log-growth phase. Importantly, cells in the log-growth phase were found to have a significantly higher resistance to Ara-C and had significantly more spheroid formation in the hanging drop assay compared to untreated cells. To further validate the concept that CSL cells are the key contributors to the AML relapse, we collected pairs of initially diagnostic and relapsed bone marrow specimens derived from the same patients. By immunocytochemistry, we found that relapsed AML blasts were more frequently positive for Myc, a surrogate marker of SORE6 activity, compared to those in the pre-treatment samples. To conclude, using SORE6, we have developed a technically simple experimental model that capitulates some of the key features of AML relapses. Our model has provided direct evidence that CSL cells are key contributors to the disease relapse in AML. We believe that this in-vitro model is valuable in testing the efficacy of new therapeutic agents against AML, especially those designed to target the CSL cell population.

Interstitial Cells of Cajal: Potential Targets for Functional Dyspepsia Treatment Using Medicinal Natural Products.

Introduction The pathophysiology of functional dyspepsia (FD) remains uncertain, but the interstitial cells of Cajal (ICCs), pacemakers that regulate gastrointestinal motility, are garnering attention as key modulators and therapeutic targets in FD. This review comprehensively discusses the involvement of ICCs in the pharmacologic actions of FD and as therapeutic targets for herbal products for FD. Methods A search of the literature was performed using PubMed by pairing “interstitial cells of Cajal” with “medicinal plant, herbal medicine, phytotherapy, flavonoids, or traditional Chinese medicine (TCM).” Results From the 55 articles screened in the initial survey, 34 articles met our study criteria. The search results showed that herbal products can directly depolarize ICCs to generate pacemaker potentials and increase the expression of c-kit and stem cell factors, helping to repair ICCs. Under certain pathological conditions, medicinal plants also protect ICCs from oxidative stress and/or inflammation-induced impairment. Two representative herbal decoctions (Banhasasim-tang, 半夏泻心汤, and Yukgunja-tang, 六君子汤) have been shown to modulate ICC functions by both clinical and preclinical data. Conclusion This review strongly indicates the potential of herbal products to target ICCs and suggests that further ICC-based studies would be promising for the development of FD treatment agents.

ANO1 regulates the maintenance of stemness in glioblastoma stem cells by stabilizing EGFRvIII

Glioblastoma multiforme (GBM) or glioblastoma is the most deadly malignant brain tumor in adults. GBM is difficult to treat mainly due to the presence of glioblastoma stem cells (GSCs). Epidermal growth factor receptor variant III (EGFRvIII) has been linked to stemness and malignancy of GSCs; however, the regulatory mechanism of EGFRvIII is largely unknown. Here, we demonstrated that Anoctamin-1 (ANO1), a Ca2+-activated Cl- channel, interacts with EGFRvIII, increases its protein stability, and supports the maintenance of stemness and tumor progression in GSCs. Specifically, shRNA-mediated knockdown and pharmacological inhibition of ANO1 suppressed the self-renewal, invasion activities, and expression of EGFRvIII and related stem cell factors, including NOTCH1, nestin, and SOX2 in GSCs. Conversely, ANO1 overexpression enhanced the above phenomena. Mechanistically, ANO1 protected EGFRvIII from proteasomal degradation by directly binding to it. ANO1 knockdown significantly increased survival in mice and strongly suppressed local invasion of GSCs in an in vivo intracranial mouse model. Collectively, these results suggest that ANO1 plays a crucial role in the maintenance of stemness and invasiveness of GSCs by regulating the expression of EGFRvIII and related signaling molecules, and can be considered a promising therapeutic target for GBM treatment.

Neural Progenitor Cells Alter Chromatin Organization and Neurotrophin Expression in Response to 3D Matrix Degradability.

Neural progenitor cells (NPCs) are promising therapeutic candidates for nervous system regeneration. Significant efforts focus on developing hydrogel-based approaches to facilitate the clinical translation of NPCs, from scalable platforms for stem cell production to injectable carriers for cell transplantation. However, fundamental questions surrounding NPC-hydrogel interactions remain unanswered. While matrix degradability is known to regulate the stemness and differentiation capacity of NPCs, how degradability impacts NPC epigenetic regulation and secretory phenotype remains unknown. To address this question, NPCs encapsulated in recombinant protein hydrogels with tunable degradability are assayed for changes in chromatin organization and neurotrophin expression. In high degradability gels, NPCs maintain expression of stem cell factors, proliferate, and have large nuclei with elevated levels of the stemness-associated activating histone mark H3K4me3. In contrast, NPCs in low degradability gels exhibit more compact, rounded nuclei with peripherally localized heterochromatin, are non-proliferative yet non-senescent, and maintain expression of neurotrophic factors with potential therapeutic relevance. This work suggests that tuning matrix degradability may be useful to direct NPCs toward either a more-proliferative, stem-like phenotype for cell replacement therapies, or a more quiescent-like, pro-secretory phenotype for soluble factor-mediated therapies.

Molecular Mechanisms of Stem Cell Reprogramming by CD47 Antagonists in Primary Human Cells

CD47 is a ubiquitously expressed transmembrane receptor whose interaction with signal regulatory protein alpha on macrophages initiates anti‐phagocytic signaling. Additionally, thrombospondin‐1 (THBS1) binding to CD47 induces cell‐autonomous signaling that regulates cell proliferation, differentiation, and recovery from genotoxic stress. Previous studies showed that CD47‐ or THBS1‐null primary lung endothelial cells exposed to ionizing radiation have higher viability than primary wildtype cells. The mechanisms that enhance the viability of CD47‐null primary cells include enhanced autophagy, anabolic metabolism, and increased expression of c‐Myc and other stem cell transcription factors. The latter enable null cells to spontaneously form embryoid bodies. These results suggest that loss of CD47 may generally facilitate cell reprogramming and self‐renewal. Induced pluripotent stem cells (iPS) can be induced by introducing Yamanaka stem cell factors (MYC, KLF4, SOX2, and OCT4) into primary somatic cells, but iPS cells can form teratomas and thus pose risk of cancer when used for regenerative medicine. Therefore, alternative approaches are needed to overcome these limitations. We propose that CD47 gene knock out (KO) via CRISPR or treating with CD47 antagonists from primary human cells could be a new, simpler, and more effective way to develop multipotent stem cells. To investigate this, we have studied the effects of CD47 knockout and antagonists on embryoid body formation and mRNA expression of stem cell factors using fibroblasts, adipocytes, and human umbilical vein cells (HUVEC). Reprogramming was measured by quantifying embryoid body formation, cell viability, and mRNA expression using real‐time PCR. Preliminary results with HUVEC, fibroblast, and adipocyte primary cells treated with CD47 knockout or antagonists have shown enhanced cell proliferation and expression of stem cell factors similar to murine cd47‐null lung endothelial cells. These results suggest that CD47 plays a key regulatory role in stem cell reprogramming of human primary cells.Support or Funding InformationThis work was supported by the Intramural Research Program of the NIH/National Cancer Institute.

Discovery of a new pyrimidine synthesis inhibitor eradicating glioblastoma-initiating cells.

Glioblastoma-initiating cells (GICs) comprise a tumorigenic subpopulation of cells that are resistant to radio- and chemotherapies and are responsible for cancer recurrence. The aim of this study was to identify novel compounds that specifically eradicate GICs using a high throughput drug screening approach. We performed a cell proliferation/death-based drug screening using 10 560 independent compounds. We identified dihydroorotate dehydrogenase (DHODH) as a target protein of hit compound 10580 using ligand-fishing and mass spectrometry analysis. The medical efficacy of 10580 was investigated by in vitro cell proliferation/death and differentiation and in vivo tumorigenic assays. Among the effective compounds, we identified 10580, which induced cell cycle arrest, decreased the expression of stem cell factors in GICs, and prevented tumorigenesis upon oral administration without any visible side effects. Mechanistic studies revealed that 10580 decreased pyrimidine nucleotide levels and enhanced sex determining region Y-box 2 nuclear export by antagonizing the enzyme activity of DHODH, an essential enzyme for the de novo pyrimidine synthesis. In this study, we identified 10580 as a promising new drug against GICs. Given that normal tissue cells, in particular brain cells, tend to use the alternative salvage pathway for pyrimidine synthesis, our findings suggest that 10580 can be used for glioblastoma therapy without side effects.Key Points1. Chemical screening identified 10580 as a novel GIC-eliminating drug that targets DHODH, an essential enzyme for the de novo pyrimidine synthesis pathway. 2. Compound 10580 induced cell cycle arrest, apoptosis, and differentiation in GICs.

Effects of salinomycin and niclosamide on small cell lung cancer and small cell lung cancer circulating tumor cell lines

SummaryTumor dissemination and recurrence is attributed to highly resistant cancer stem cells (CSCs) which may constitute a fraction of circulating tumor cells (CTCs). Small cell lung cancer (SCLC) constitutes a suitable model to investigate the relation of CTCs and CSCs due to rapid tumor spread and a high number of CTCs. Expansion of five SCLC CTC lines (BHGc7, 10, 16, 26 and UHGc5) in vitro at our institution allowed for the analysis of CSC markers and cytotoxicity of the CSC-selective drugs salinomycin and niclosamide against CTC single cell suspensions or CTC spheroids/ tumorospheres (TOS). Salinomycin exerted dose-dependent cytotoxicity against the SCLC lines but, with exception of BHGc7 TOS, there was no markedly enhanced activity against TOS. Similarly, niclosamide exhibits high activity against BHGc7 TOS and UHGc5 TOS but not against the other CTC spheroids. High expression of the CSC marker CD133 was restricted to three SCLC tumor lines and the BHGc10 CTC line. All SCLC CTCs are CD24-positive but lack expression of CD44 and ABCG2 in contrast to the SCLC tumor lines which show a phenotype more similar to that of CSCs. The stem cell marker SOX2 was found in all CTC lines and SCLC GLC14/16, whereas elevated expression of Oct-3/4 and Nanog was restricted to BHGc26 and UHGc5. In conclusion, the SCLC CTCs established from patients with relapsed disease lack a typical CSC phenotype in respect to chemosensitivity to CSC-selective drugs, surface markers, expression of pluripotent stem cell and transcription factors.

Abstract 1153: Hypoxic microenvironments promote stemness and enhance the directed migration of glioma stem cells

Abstract Glioblastoma (GBM) is a highly malignant primary brain tumor and has a poor prognosis and is resistant to the most advanced therapies. The development of highly hypoxic centrally located necrotic regions is strongly associated with accelerated GBM progression, yet mechanistic explanations are not complete. Hypoxic regions within GBM are highly enriched for glioma stem cells (GSC), which are critical for tumor initiation, recurrence and therapeutic resistance due to their self-renewing capabilites. While most GBM cells migrate outward, away from hypoxia, GSCs accumulate there, forming a hypercellular border called pseudopalisades that promote their survival. Mechanisms that mediate GSC accumulation within pseudopalisades are unknown. We hypothesize that GSCs are enriched in the pseudopalisades due to the homing of a subpopulation towards the supportive hypoxic microenvironment. This in turn activates critical hypoxia-induced drivers to promote stemness, survival and migratory behavior encouraging the recruitment of more GSCs. Using two genetically distinct patient-derived GBM cell lines, we found that exposure to hypoxia in vitro led to enrichment of GSCs among a heterogeneous population of GSCs and non-GSCs. We found that hypoxia enhanced the expression of stem cell, pro-survival and pro-migratory factors in GSCs, such as the pro-migratory chemokine SDF-1α, and its cognate receptor CXCR4. Importantly, we found hypoxia also induced upregulation of OLIG2, one of four core transcription factors necessary and sufficient to induce stemness, in GSCs and non-GSCs exposed to hypoxia. We determined that OLIG2 was necessary for the expression of stem cell markers, and that its knockdown reduced cellular migration in hypoxia vs normoxia in GSCs. This suggests a critical role for OLIG2 in both hypoxia-induced stemness and homing behavior. We also generated a unique model of localized hypoxia in one brain hemisphere through vaso-cclusion and observed the homing of xenografted GBM cells towards hypoxia, regulated by OLIG2 in vivo. These data shed light on how hypoxic tumor microenvironments facilitate mechanisms vital to GBM progression and therapeutic resistance. Citation Format: Nitya V. Sharma, Monica Chau, Subhas Mukherjee, James Ross, Brandon Miller, Changming Zhang, Jun Kong, Carol Tucker-Burden, Emily Kaissi, John W. Reitnauer, Bilge Dundar, Cheryl L. Olson, Gregory Bix, Daniel J. Brat. Hypoxic microenvironments promote stemness and enhance the directed migration of glioma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1153.

Induced differentiation and molecular characterization of monocyte-derived multipotential cells (MOMCs) generated from commonly discarded leukapheresis filters

Background & Aim Μonocyte-derived multipotential cells (MOMCs), easily isolated from the peripheral blood, include progenitors capable of differentiating into endothelial lineages in a specific permissive environment and thus represent an autologous transplantable cell source for therapeutic neovasculogenesis (Kuwana M et al, 2012). Methods, Results & Conclusion discarded leukapheresis filters (T.P.H. Meyer et al, 2005)and estimate the expression of several stem cell factors that play a key role in their pluripotency and differentiation capacity. A leukocyte elution medium composed of Citrate phosphate dextrose (CPD), hypertonic SAGM and Phosphate Buffered Saline was prepared and used for back-flushing of the filters, opposite to the primary blood flow during the leukocyte depletion step. Upon centrifugation in density gradient, mononuclear cells were collected and subsequently incubated in 37οC 5% CO2conditions for 10 days in poly-L-lysine+gelatin coated plates in the presence of 50 ng/ml SDF-1a chemotactic factor enabling the MOMCs generation. Aiming the characterization of the harvested cells, flow cytometry was performed for the detection of typical surface marker expression (CD45, CD14, CD34, Collagen type I and CD184). The induced differentiation capacity of MOMCs towards bone and endothelium in specific culture mediums was estimated via alizarin red staining and flow cytometry, respectively. Finally, the expression levels of genes related with self-renewal and differentiation were estimated with Real-time PCR. The generated MOMCs were characterized by high expression of the CD45/CD14/CD34/CD184/Col I markers while progressively lost their native markers and gained endothelial or bone features upon induced differentiation. The MOMCs expressed several stem cell factors like Nanog, Oct4, c-Myc, Sox2, Klf4, Sall4, ESRRB and ZNF214. All these factors are responsible to activate the networks related with the pluripotency, differentiation and reprogramming of cells while are highly expressed in embryonic stem cells. To conclude, we successfully optimized conditions to elute leukocytes adsorbed to leukapheresis filters resulting in leukocyte suspensions collection for multiple uses. The easy and quick generation of MOMCs from monocytes derived from leukapheresis filters, makes them potential candidates for future therapeutic application in tissue regeneration.

Constant Degradation of the Androgen Receptor by MDM2 Conserves Prostate Cancer Stem Cell Integrity.

Prostate cancer stem cells (CSC) are implicated in tumor initiation, cancer progression, metastasis, and the development of therapeutic-resistant disease. It is well known that the bulk of prostate cancer cells express androgen receptor (AR) and that androgens are required for prostate cancer growth, progression, and emergence of castration-resistant disease. In contrast, the small subpopulation of self-renewing CSCs exhibits an AR-negative (AR-) signature. The mechanisms underlying the absence of AR are unknown. Using CSC-like cell models isolated from clinical biopsy tissues, we identify the E3 ligase MDM2 as a key regulator of prostate CSC integrity. First, unlike what has been reported for the bulk of AR+ tumor cells where MDM2 regulates the temporal expression of AR during transcriptional activity, MDM2 in CSCs promoted the constant ubiquitination and degradation of AR, resulting in sustained loss of total AR protein. Second, MDM2 promoted CSC self-renewal, the expression of stem cell factors, and CSC proliferation. Loss of MDM2 reversed these processes and induced expression of full-length AR (and not AR variants), terminal differentiation into luminal cells, and cell death. Selectively blocking MDM2-mediated activity in combination with androgen/AR-targeted therapy may offer a novel strategy for eliminating AR- CSCs in addition to the bulk of AR+ prostate cancer cells, decreasing metastatic tumor burden and inhibiting the emergence of therapeutic resistance.Significance: These findings provide a novel mechanistic aspect of prostate cancer cell stemness that advances our understanding of the diverse transcriptional activity that bypasses AR in contributing to therapeutic resistance, tumor progression, and metastasis.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/6/1124/F1.large.jpg.

Hmga2 regulation of tooth formation and association with Sox2 and Nanog expression

Tooth formation is accomplished under strict genetic programs. Although patients with chromosome 12q14 aberration shows tooth phenotype including the size and eruption timing with bone growth anomaly, its etiology is uncertain. Here, we examined expression of Hmga2, which is encoded at chromosome 12q14, in mouse tooth germs and analyzed the involvement in lower first molar (M1) and mandibular bone development. Hmga2 expression was immunohistochemically detected at enamel organ and the surrounding mesenchyme of the M1 germs. The expression was dynamically changed with gestation and rapidly decreased in postnatal mice. In Hmga2−/− mice, the M1 germs and crowns were diminished in size, and formation and eruption of molars were delayed with mandibular bone growth retardation. Hmga2 cDNA or siRNA transfection showed that Hmga2 transcriptionally up-regulates expression of stem cell factors, Sox2 and Nanog. They were co-localized with Hmga2 in the germs, but differentially distributed at enamel organ and mesenchyme in Hmga2−/− mice. These results demonstrate that Hmga2 expressed in tooth germs regulates the growth, sizing and eruption and stem cell factor expression in different compartment of the germ and associates with mandibular bone growth. Although future studies are needed, the present study demonstrates HMGA2 regulation of tooth genesis with skeletal development.

Epigenetically jump starting de novo shoot regeneration.

The ability to regenerate lost organs or tissues is a central requirement for animals and plants in order to cope with injury. Regeneration of a whole body is rare in animals but is more commonly found in plants where in vitro regeneration has become a widely used tool in plant research. In a new study, Kim et al find that epigenetic changes via the histone acetyltransferase HAG1 establish the competency for shoot regeneration from callus by promoting the expression of root stem cell factors.

C/EBPδ links IL-6 and HIF-1 signaling to promote breast cancer stem cell-associated phenotypes.

To improve cancer patient outcome significantly, we must understand the mechanisms regulating stem-like cancer cells, which have been implicated as a cause of metastasis and treatment resistance. The transcription factor C/EBPδ can exhibit pro- and anti-tumorigenic activities, but the mechanisms underlying the complexity of its functions are poorly understood. Here, we identify a role for breast cancer cell intrinsic C/EBPδ in promoting phenotypes that have been associated with cancer stem cells (CSC). While C/EBPδ expression is not abundant in most metastatic breast cancers, our data support a pro-tumorigenic role of C/EBPδ when expressed in subsets of tumor cells and/or through transient activation by the tumor microenvironment or loss of substrate adhesion. Using genetic mouse models and human breast cancer cell lines, we show that deletion or depletion of C/EBPδ reduced expression of stem cell factors and stemnness markers, sphere formation and self-renewal, along with growth of tumors and established experimental metastases in vivo. C/EBPδ is also known as a mediator of the innate immune response, which is enhanced by hypoxia and interleukin-6 (IL-6) signaling, two conditions that also play important roles in cancer progression. Our mechanistic data reveal C/EBPδ as a link that engages two positive feed-back loops, in part by directly targeting the IL-6 receptor (IL6RA) gene, and, thus, amplifying IL-6 and HIF-1 signaling. This study provides a molecular mechanism for the synergism of tumor micro-environmental conditions in cancer progression with potential implications for the targeting of cancer stem cells.

Contribution of KCTD12 to esophageal squamous cell carcinoma

BackgroundIt has been shown that the expression of potassium channel tetramerization domain containing 12 (KCTD12) as a regulator of GABAB receptor signaling is reversely associated with gastrointestinal stromal tumors. In present study we examined the probable role of KCTD12 in regulation of several signaling pathways and chromatin remodelers in esophageal squamous cell carcinoma (ESCC).MethodsKCTD12 ectopic expression was done in KYSE30 cell line. Comparative quantitative real time PCR was used to assess the expression of stem cell factors and several factors belonging to the WNT/NOTCH and chromatin remodeling in transfected cells in comparison with non-transfected cells.ResultsWe observed that the KCTD12 significantly down regulated expression of NANOG, SOX2, SALL4, KLF4, MAML1, PYGO2, BMI1, BRG1, MSI1, MEIS1, EGFR, DIDO1, ABCC4, ABCG2, and CRIPTO1 in transfected cells in comparison with non-transfected cells. Migration assay showed a significant decrease in cell movement in ectopic expressed cells in comparison with non-transfected cells (p = 0.02). Moreover, KCTD12 significantly decreased the 5FU resistance in transfected cells (p = 0.01).ConclusionsKCTD12 may exert its inhibitory role in ESCC through the suppression of WNT /NOTCH, stem cell factors, and chromatin remodelers and can be introduced as an efficient therapeutic marker.

Effects of oil extracts from Arachis hypogea on stem cell factors for follicular maturation and implantation in the superovulated rat

Oil extracts from Arachis hypogea is often used clinically for the administration of exogenous hormone during assisted reproduction. But its capacity to induce follicular growth after fertilisation to maintain the formation of progesterone has not yet been clarified. We assessed the effects of peanut oil on stem cell factors (SCFs) for follicular maturation and meiotic activities in the superovulated rat. Female rats were superovulated and treated with peanut oil (S + Pn), while the controls were either superovulated and treated with saline solution (S + N), or non-superovulated and treated with saline solution (N) or peanut oil (Pn). After mating, uterine horns and ovaries were obtained and a morphometry of granulosa cells and theca interna (TI) was carried out. The mitotic index (MI) of the granulosa cells and the protein expression of SCF on ovarian stromal cells were evaluated, as well as the ultramorphology of the endometrium. Results showed a successful implantation in S + Pn, N and Pn groups. The MI was high in the Pn group, moderate in the S + Pn and N groups, and poor in the S + N group. However, both follicular size and TI levels varied significantly (p < 0.05) across intervention groups. In multiple comparisons, both follicular size and TI values in the experimental group were the highest (p < 0.0001). Protein expressions of SFC were high on ovarian stromal cells in both the S + Pn and N groups, while decidualisation was observed in the experimental groups. We conclude that peanut oil may be implicated in mitotic activities of granolusa cells, induce the release of SCF and sustain implantation.

TFAP2C promotes stemness and chemotherapeutic resistance in colorectal cancer via inactivating hippo signaling pathway

BackgroundAberrant expression of transcription Factor AP-2 Gamma (TFAP2C) has been reported to be implicated in malignant process of many cancers. The purpose of this study is to investigate the clinical significance and biological roles of TFAP2C in colorectal cancer (CRC).MethodsTFAP2C expression was evaluated by real-time PCR, Western blot and immunohistochemistry (IHC) respectively in clinical CRC tissues. Statistical analysis was performed to explore the correlation between TFAP2C expression and clinicopathological features, and overall and progression-free survival in CRC patients. In vitro and in vivo assays were performed to assess the biological roles of TFAP2C in CRC cells. Western blot, luciferase and Chromatin immunoprecipitation (ChIP) assays were used to identify the underlying pathway mediating the biological roles of TFAP2C in CRC.ResultsTFAP2C is robustly upregulated in CRC tissues and cells, and high expression of TFAP2C correlates with advanced clinicopathological features, poor prognosis and disease progression in CRC patients. Furthermore, upregulating TFAP2C enhances spheroids formation ability, the fraction of SP cells, expression of stem cell factors and the mitochondrial potential, and reduces the apoptosis induced by 5-fluorouracil in colorectal cancer cells in vitro, and promotes stemness and chemoresistance of CRC cells in vivo; while silencing TFAP2C yields an opposite effect. Importantly, downregulation of TFAP2C dramatically restores chemotherapeutic sensitivity of CRC cells to 5-FU in vivo. Our results further demonstrate that TFAP2C promotes stemness and chemoresistance of CRC cells to 5-FU by inhibiting Hippo signaling via transcriptionally upregulating ROCK1 and ROCK2 in CRC cells.ConclusionOur findings indicate that TFAP2C may serve as a novel prognostic factor in CRC patients, and a therapeutic target for the treatment of CRC, suggesting that silencing TFAP2C in combination with 5-FU may be an effective therapeutic strategy to improve survival in CRC patients.

Interleukin 7-expressing fibroblasts promote breast cancer growth through sustenance of tumor cell stemness

ABSTRACTThe tumor microenvironment harbors cancer-associated fibroblasts that function as major modulators of cancer progression. Here, we assessed to which extent distinct cancer-associated fibroblast subsets impact mammary carcinoma growth and cancer cell stemness in an orthotopic murine model. We found that fibroblasts expressing the Cre recombinase under the control of the interleukin 7 promoter occupied mainly the tumor margin where they physically interacted with tumor cells. Intratumoral ablation of interleukin 7-expressing fibroblasts impaired breast tumor growth and reduced the clonogenic potential of cancer cells. Moreover, cDNA expression profiling revealed a distinct oncogenic signature of interleukin 7-producing fibroblasts. In particular, Cxcl12 expression was strongly enhanced in interleukin 7-producing fibroblasts and cell type-specific genetic ablation and systemic pharmacological inhibition revealed that the CXCL12/CXCR4 axis impacts breast tumor cell stemness. Elevated expression of CXCL12 and other stem cell factors in primary human breast cancer-associated fibroblasts indicates that certain fibroblast populations support tumor cell stemness and thereby promote breast cancer growth.

Maintenance of stemness by miR-589-5p in hepatocellular carcinoma cells promotes chemoresistance via STAT3 signaling

The strength and duration of STAT3 signaling are tightly controlled by multiple negative feedback mechanisms under physical conditions. However, how these serial feedback loops are simultaneously disrupted in cancers, leading to constitutive activation of STAT3 signaling in hepatocellular carcinoma (HCC), remains obscure. Here we report that miR-589-5p is elevated in HCC tissues, which is caused by recurrent gains. Overexpression of miR-589-5p correlates with poor overall and relapse-free survival in HCC patients. Upregulating miR-589-5p enhances spheroid formation ability, fraction of CD133 positive and side population cells, expression of cancer stem cell factors and the mitochondrial potential, and represses the apoptosis induced by doxorubicin in vitro and tumorigenicity in vivo in HCC cells; conversely, silencing miR-589-5p yields an opposite effect. Our findings further demonstrate miR-589-5p promotes the cancer stem cell characteristics and chemoresistance via targeting multiple negative regulators of STAT3 signaling pathway, including SOCS2, SOCS5, PTPN1 and PTPN11, leading to constitutive activation of STAT3 signaling. Collectively, our results unravel a novel mechanism by which miR-589-5p promotes the maintenance of stemness and chemoresistance in HCC, providing a potential rational registry of anti-miR-589-5p combining with conventional chemotherapy against HCC.