Stem Cell Signaling Pathways Research Articles

Stem cell-derived paracrine factors by modulated reactive oxygen species to enhance cancer immunotherapy

Herein, we present an approach for manipulating paracrine factors and signaling pathways in adipose-derived stem cells (ADSCs) to achieve highly effective tumor immunotherapy. Our method involves precise control of reactive oxygen species concentration using the CD90-maleimide-pluronic F68-chlorin e6 conjugate (CPFC) to create ACPFC, which is then attached to ADSCs through the CD90 receptor-specific interaction. By regulating the irradiated laser power, ACPFC promotes signaling pathways such as cascade-3, VEGFR2, α2β1, C3AR1, CR1–4, and C5AR1, leading to the secretion of various inflammatory cytokines such as IFN-γ, TGF-β, and IL-6, while inhibiting AKT, ERK, NFkB, PAR1, and PAR3/4 signaling pathways to reduce the secretion of cell growth factors like TIMP-1, TIMP-2, VEGF, Ang-2, FGF-2, and HGF. When ACPFC is injected intravenously into a tumor animal model, it autonomously targets and accumulates at the tumor site, and upon laser irradiation, it generates various anti-inflammatory factors while reducing angiogenesis growth factors. The resulting antitumor response recruits CD3+CD8+ cytotoxic T cells and CD3+CD4+ helper T cells into the tumor and spleen, leading to highly effective melanoma and pancreatic tumor treatment in mice. Our technology for regulating stem cell paracrine factors holds significant promise for the treatment of various diseases.

Inhibition of Notch Signaling Enhances Antitumor Activity of Histone Deacetylase Inhibitor LAQ824.

As a novel histone deacetylase inhibitor (HDACi), LAQ824 (LAQ) effectively inhibits the proliferation of hematological malignancies and solid tumors. However, phase II trials of LAQ in solid tumors were terminated due to dose-dependent toxicity. Furthermore, LAQ has been shown to induce the activation of the Notch signaling pathway in hematopoietic stem cells, which is associated with tumor progression and drug resistance in colon and breast cancers. Therefore, in this study, we investigated the strategy of LAQ combined with a Notch signaling pathway inhibitor to treat solid tumors. We used RT-PCR and Western blot methods to demonstrate that LAQ upregulated the Notch signaling pathway in solid tumor cell lines at the molecular level. The combination of LAQ and a Notch signaling pathway inhibitor was shown by a Chou-Talalay assay to have a synergistic effect in inhibiting solid tumor cell line proliferation in vitro. We also demonstrated that the combination of LAQ and a Notch signaling pathway inhibitor significantly inhibited the growth of tumor cells in vivo using an allograft tumor model. This study indicates that inhibition of the Notch signaling pathway provides a valuable strategy for enhancing solid tumor sensitivity to LAQ.

Mixed-Lineage Leukemia 1 Inhibition Enhances the Differentiation Potential of Bovine Embryonic Stem Cells by Increasing H3K4 Mono-Methylation at Active Promoters.

Mixed-lineage leukemia 1 (MLL1) introduces 1-, 2- and 3-methylation into histone H3K4 through the evolutionarily conserved set domain. In this study, bovine embryonic stem cells (bESCs, known as bESCs-F7) were established from in vitro-fertilized (IVF) embryos via Wnt signaling inhibition; however, their contribution to the endoderm in vivo is limited. To improve the quality of bESCs, MM-102, an inhibitor of MLL1, was applied to the culture. The results showed that MLL1 inhibition along with GSK3 and MAP2K inhibition (3i) at the embryonic stage did not affect bESCs' establishment and pluripotency. MLL1 inhibition improved the pluripotency and differentiation potential of bESCs via the up-regulation of stem cell signaling pathways such as PI3K-Akt and WNT. MLL1 inhibition decreased H3K4me1 modification at the promoters and altered the distribution of DNA methylation in bESCs. In summary, MLL1 inhibition gives bESCs better pluripotency, and its application may provide high-quality pluripotent stem cells for domestic animals.

Potential Natural Products Regulation of Molecular Signaling Pathway in Dermal Papilla Stem Cells.

Stem cells have demonstrated significant potential for tissue engineering and repair, anti-aging, and rejuvenation. Hair follicle stem cells can be found in the dermal papilla at the base of the follicle and the bulge region, and they have garnered increased attention because of their potential to regenerate hair as well as their application for tissue repair. In recent years, these cells have been shown to affect hair restoration and prevent hair loss. These stem cells are endowed with mesenchymal characteristics and exhibit self-renewal and can differentiate into diverse cell types. As research in this field continues, it is probable that insights regarding stem cell maintenance, as well as their self-renewal and differentiation abilities, will benefit the application of these cells. In addition, an in-depth discussion is required regarding the molecular basis of cellular signaling and the influence of nature-derived compounds in stimulating the stemness properties of dermal papilla stem cells. This review summarizes (i) the potential of the mesenchymal cells component of the hair follicle as a target for drug action; (ii) the molecular mechanism of dermal papilla stem cells for maintenance of their stem cell function; and (iii) the positive effects of the natural product compounds in stimulating stemness in dermal papilla stem cells. Together, these insights may help facilitate the development of novel effective hair loss prevention and treatment.

Stem cell properties of cancer cells in ascitic fluid of patients with ovarian cancer: a key to control over cancer progression

Ovarian cancer is considered to be the most malignant and aggressive tumor of the female reproductive system, which is largely associated with early development of malignant ascites and peritoneal carcinomatosis. Cancer cells representing the primary focus, as well as those contained in the ascitic fluid, are extremely heterogeneous in terms of morphological, immunohistochemical, and molecular genetic aspects. Cancer stem cells play a significant role in tumor self-renewal, differentiation, metastasis, and development of chemoresistance.This literature review is aimed at summarizing the available data on cancer stem cells in ovarian cancer and their role in tumor progression. A bioinformatic search was carried out in the PubMed, NCBI, Google Scholar, and eLibrary databases using the keywords “cancer stem cells”, “ovarian cancer”, “malignant ascites”, “chemoresistance”, etc.The data presented in the review make it possible to comprehensively characterize the role of stem cell properties of ovarian cancer cells. The review presents up-to-date information on the molecular and biological parameters of cancer stem cells in ovarian cancer, which are the cellular component of malignant ascites, as well as data from the authors’ studies. Along with this, the article describes modern ideas about the mechanisms of formation of cellular spheroids and their contribution to cancer progression.Cancer stem cells are an extremely promising target in the development of future therapeutic strategies based on the study of signaling pathways in ovarian cancer stem cells, the mechanisms of spheroid formation, and the contribution of immune cells to the acquisition of cancer stem cell properties.

Extracellular matrix-derived mechanical force governs breast cancer cell stemness and quiescence transition through integrin-DDR signaling

The extracellular matrix (ECM) serves as signals that regulate specific cell states in tumor tissues. Increasing evidence suggests that extracellular biomechanical force signals are critical in tumor progression. In this study, we aimed to explore the influence of ECM-derived biomechanical force on breast cancer cell status. Experiments were conducted using 3D collagen, fibrinogen, and Matrigel matrices to investigate the role of mechanical force in tumor development. Integrin-cytoskeleton-AIRE and DDR-STAT signals were examined using RNA sequencing and western blotting. Data from 1358 patients and 86 clinical specimens were used for ECM signature-prognosis analysis. Our findings revealed that ECM-derived mechanical force regulated tumor stemness and cell quiescence in breast cancer cells. A mechanical force of ~45 Pa derived from the extracellular substrate activated integrin β1/3 receptors, stimulating stem cell signaling pathways through the cytoskeleton/AIRE axis and promoting tumorigenic potential and stem-like phenotypes. However, excessive mechanical force (450 Pa) could drive stem-like cancer cells into a quiescent state, with the removal of mechanical forces leading to vigorous proliferation in quiescent cancer stem cells. Mechanical force facilitated cell cycle arrest to induce quiescence, dependent on DDR2/STAT1/P27 signaling. Therefore, ECM-derived mechanical force governs breast cancer cell status and proliferative characteristics through stiffness alterations. We further established an ECM signature based on the fibrinogen/fibronectin/vitronectin/elastin axis, which efficiently predicts patient prognosis in breast cancer. Our findings highlight the vital role of ECM-derived mechanical force in governing breast cancer cell stemness/quiescence transition and suggest the novel use of ECM signature in predicting the clinical prognosis of breast cancer.

Fatty Acid Metabolites and the Tumor Microenvironment as Potent Regulators of Cancer Stem Cell Signaling.

Individual cancer cells are not equal but are organized into a cellular hierarchy in which only a rare few leukemia cells can self-renew in a manner reminiscent of the characteristic stem cell properties. The PI3K/AKT pathway functions in a variety of cancers and plays a critical role in the survival and proliferation of healthy cells under physiologic conditions. In addition, cancer stem cells might exhibit a variety of metabolic reprogramming phenotypes that cannot be completely attributed to the intrinsic heterogeneity of cancer. Given the heterogeneity of cancer stem cells, new strategies with single-cell resolution will become a powerful tool to eradicate the aggressive cell population harboring cancer stem cell phenotypes. Here, this article will provide an overview of the most important signaling pathways of cancer stem cells regarding their relevance to the tumor microenvironment and fatty acid metabolism, suggesting valuable strategies among cancer immunotherapies to inhibit the recurrence of tumors.

Functionalized Exosomes Promote Neural Differentiation of P19 Cells by Activating the Wnt Signaling Pathway.

Global population growth and population aging continue to accelerate, and lead to a further increase in the risk of neurological diseases worldwide. Extracellular vesicles secreted by mesenchymal stem cells carry many proteins, lipids, and genetic material that mediate cell-to-cell communication and improve therapeutic outcomes for neurological disorders. Stem cells from human exfoliated deciduous teeth are considered a suitable cell source for tissue regeneration, which exerts therapeutic effects via the secretion of exosomes. This study was performed to assess the effect of functionalized exosomes on neural differentiation of embryonic carcinoma cell line P19. We stimulated stem cells from human exfoliated deciduous teeth with the glycogen synthase kinase-3β inhibitor TWS119 and then extracted its exosomes. P19 cells were induced to differentiate using functionalized exosomes, and the biological functions and involved signaling pathways of differentially expressed genes were analyzed by RNA-sequencing. Immunofluorescence techniques detected neuronal specific markers. It was found that TWS119 activated the Wnt signaling pathway in stem cells from human exfoliated deciduous teeth. RNA-sequencing showed that upregulated differentially expressed genes in the functionalized exosome-treated group were responsible for cell differentiation, neurofilament, and structural constituent of the synapse. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that the functionalized exosome-treated group activated the Wnt signaling pathway. Immunofluorescence showed that functionalized exosomes induced neurite outgrowth in P19 cells. Our results demonstrated that functionalized exosomes promoted neural differentiation of P19 cells by activating the Wnt signaling pathway.

Profiling and targeting cancer stem cell signaling pathways for cancer therapeutics.

Tumorigenic cancer stem cells (CSCs) represent a subpopulation of cells within the tumor that express genetic and phenotypic profiles and signaling pathways distinct from the other tumor cells. CSCs have eluded many conventional anti-oncogenic treatments, resulting in metastases and relapses of cancers. Effectively targeting CSCs' unique self-renewal and differentiation properties would be a breakthrough in cancer therapy. A better characterization of the CSCs' unique signaling mechanisms will improve our understanding of the pathology and treatment of cancer. In this paper, we will discuss CSC origin, followed by an in-depth review of CSC-associated signaling pathways. Particular emphasis is given on CSC signaling pathways' ligand-receptor engagement, upstream and downstream mechanisms, and associated genes, and molecules. Signaling pathways associated with regulation of CSC development stand as potential targets of CSC therapy, which include Wnt, TGFβ (transforming growth factor-β)/SMAD, Notch, JAK-STAT (Janus kinase-signal transducers and activators of transcription), Hedgehog (Hh), and vascular endothelial growth factor (VEGF). Lastly, we will also discuss milestone discoveries in CSC-based therapies, including pre-clinical and clinical studies featuring novel CSC signaling pathway cancer therapeutics. This review aims at generating innovative views on CSCs toward a better understanding of cancer pathology and treatment.

Understanding the Wnt Signaling Pathway in Acute Myeloid Leukemia Stem Cells: A Feasible Key against Relapses.

Wnt signaling is a highly conserved pathway in evolution which controls important processes such as cell proliferation, differentiation and migration, both in the embryo and in the adult. Dysregulation of this pathway can favor the development of different types of cancer, such as acute myeloid leukemia and other hematological malignancies. Overactivation of this pathway may promote the transformation of pre-leukemic stem cells into acute myeloid leukemia stem cells, as well as the maintenance of their quiescent state, which confers them with self-renewal and chemoresistance capacity, favoring relapse of the disease. Although this pathway participates in the regulation of normal hematopoiesis, its requirements seem to be greater in the leukemic stem cell population. In this review, we explore the possible therapeutic targeting of Wnt to eradicate the LSCs of AML.

Abstract PR01: Novel anti-leukemia activity of the IRE1-XBP1 signaling pathway in hematopoietic stem and progenitor cells

Abstract Most patients with acute myeloid leukemia (AML) initially achieve clinical remission after chemotherapy but eventually die from disease relapse as they succumb to chemotherapy-resistant AML cells, including the rare fraction of leukemia stem cells (LSC) that persist and often reinitiate a more aggressive disease. While eliminating LSCs has become a desirable strategy to achieve durable AML remission, much remains unknown about LSC vulnerabilities and the specific cellular programs that enable their disease activity. Spurred by increasing evidence that disruptions in proteome homeostasis or “proteostasis” impair stem cell integrity, we investigated how acute myeloid leukemogenesis is impacted by the inositol-requiring enzyme-1 alpha (IRE1a), the most conserved enzyme of the endoplasmic reticulum unfolded protein response that resolves proteotoxic stress in eukaryotic cells. Activated IRE1a catalyzes an unconventional splicing of the XBP1U mRNA to generate an XBP1S transcript that encodes the transcription factor XBP1 (X-box binding protein-1), the main effector of IRE1a-transduced signals. Using transcriptomics and genomics to profile the activation and function of IRE1a in primary human AML samples and murine AML models, we identified the IRE1a-XBP1 axis as a cell-intrinsic brake against pro-myeloid leukemogenic programs in hematopoietic stem and progenitor cells (HSPC). Notably, compared to blast cells from the same AML patient, LSCs expressed significantly reduced levels of the genes encoding IRE1a (ERN1) and its spliced product XBP1S suggesting that downregulation of IRE1a-XBP1 activity promotes LSC activity and acute myeloid leukemogenesis. Consistent with this hypothesis, genetic induction of XBP1S-encoded XBP1 in patient-derived AML cells suppressed their LSC transcriptional program, impaired their clonogenic capacity, and limited their ability to cause AML in immunodeficient animals. By contrast, genetically engineered IRE1a or XBP1 insufficiency enriched the LSC signature in HSPCs and cooperated with the myeloproliferative oncogene FLT3-ITD to initiate a lethal AML disease in mice. Integrated RNA-Seq and ChIP-Seq of XBP1 target genes in HSPCs revealed a mechanism in which IRE1a-induced XBP1 functions as a potent transcriptional repressor of signature pro-LSC gene programs, exemplified by the Wnt/beta-catenin pathway. Accordingly, we defined an independently prognostic “18-gene” signature of XBP1-repressed beta-catenin target genes that when highly expressed correlated with poor survival outcomes in multiple cohorts of AML patients. Taken together, our findings uncover a novel anti-leukemia function for IRE1a-XBP1 signaling that restrains acute myeloid leukemogenesis in HSPCs and suggest that this pathway is likely subverted in LSCs to facilitate AML initiation and progression. Our results further suggest that IRE1a activation is a vulnerability in AML stem cells that can be harnessed therapeutically to achieve durable disease remission. Citation Format: Brendan M Barton, Francheska Son, Akanksha Verma, Laurie Glimcher, Stanley Adoro. Novel anti-leukemia activity of the IRE1-XBP1 signaling pathway in hematopoietic stem and progenitor cells [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr PR01.

Abstract 3805: Revealing the role of microRNAs in the emergence of cancer stem cell heterogeneity during colorectal cancer progression

Abstract Our goal is to identify microRNAs (miRNAs) that target mRNAs involved in stem cell (SC) signaling pathways involved in colorectal cancer (CRC) tumorigenesis. SC overpopulation drives CRC progression but exact mechanism is unclear. Hypothesis: specific miRNAs target SC genes, which leads to the emergence of multiple cancer SC (CSC) sub-populations and tumor heterogeneity. We used flow cytometry to identify CSC sub-populations based on expression of SC markers including ALDH1, LGR5 and others in the HT-29 CRC cell line. CSC sub-populations were isolated and measured using FACS and expression of miRNAs in each CSC sub-population was quantified using NanoString profiling. The level of miRNA upregulation in each CSC sub-population was ranked based on differential expression of the miRNAs between different CSC sub-populations. We chose to further analyze the LGR5 and ALDH SC sub-populations because they showed: i) the largest difference between CSC sub-population sizes, ii) the smallest size (<1%) of the co-positive (LGR5+/ALDH+) cell sub-population, iii) the highest numbers of differentially expressed miRNAs. Note that WNT signaling mainly occurs via LGR5+ CSC and retinoic acid (RA) signaling mainly occurs via ALDH+ CSCs. We report herein miRNAs that are differentially expressed between ALDH+/LGR5- compared to ALDH-/LGR5- subpopulation and LGR5+/ALDH- versus ALDH-/LGR5-. The mRNAs expressed in CSC subpopulations identified from Nanostring profiling were analyzed using bioinformatics for their predicted binding by upregulated miRNAs. Our findings show that: 1) multiple CSC subpopulations exist in the HT-29 cell population; 2) each CSC subpopulation has a unique miRNA signature. Notably, RXRA mRNA is significantly upregulated in the ALDH+/LGR5- CSC sub-population but not in the LGR5+/ALDH- CSC subpopulation. We identified 5 miRs upregulated in the LGR5+/ALDH- CSC subpopulation that are predicted to target the 3’UTR of RXRA mRNA. One of these miRs, miR-660-3p, is specifically upregulated in the LGR5+/ALDH- CSC subpopulation and binding of miR-660-3p to RXRA mRNA was validated using luciferase assay in HT-29 cells. We previously reported that RXRA is selectively expressed in ALDH + CSC sub-populations. Also, RXRA is known to bind with β-catenin leading to its degradation and suppression of WNT-based transcriptional activity. Taken together, these results indicate that expression of miR-660-3p, by inhibiting RXRA expression, increases WNT signaling and decreases RA signaling in LGR5+/ALDH- CSCs. Thus, upregulated miR-660-3p targets a key gene in the RA signaling pathway (RXRA) which could contribute to the emergence of SC sub-populations during CRC development. Citation Format: Molly Lausten, Victoria Stark, Caroline Facey, Lynn Opdenaker, Bruce M. Boman. Revealing the role of microRNAs in the emergence of cancer stem cell heterogeneity during colorectal cancer progression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3805.

Abstract 4811: Oxygen tension - dependent differences in cancer cell kinome

Abstract Current approaches to preclinical cancer research often fail to consider the impact of ambient oxygen (O2; ~21%) on cancer cells. This is also true for hypoxia studies that typically involves cancer cells previously grown in ambient O2 before subsequent transfer to hypoxia. However, the tumor microenvironment is characterized by significantly lower O2 levels. We have previously demonstrated the impact of ambient O2 on stem cell populations, signaling pathways and resistance to therapy. We developed an experimental approach that allows us to collect and process tumor tissues from transgenic mammary tumor mouse models and ovarian cancer patients under physioxia (3% O2), such that they are never exposed to ambient O2. In this study, our goal was to explore oxygen-dependent signaling pathway alterations and to determine how these pathways are influenced by targeted drugs in the context of physioxia or ambient air (AA). Our studies revealed increased basal phosphorylation levels of EGFR (Y1068) in the tumor cells in AA, relative to physioxia. However, downstream signaling effectors AKT and ERK showed higher phosphorylation levels under physioxia, compared to AA, suggesting that their activation is independent of EGFR signaling. These findings correlate with the decreased sensitivity of the tumor cells under physioxia to target drugs lapatinib and alpelisib. We then sought to examine basal and target drug induced kinome changes in tumor cells under physioxia and AA via Multiplexed Inhibitor Beads (MIBs) kinome assay. This assay revealed significant differences in the kinome of the tumor cells under physioxia compared to AA. Although direct comparison between vehicle and lapatinib treated cells in physioxia and ambient air showed very minimal changes, pairwise comparison between lapatinib treated physioxia cells and vehicle treated AA cells revealed an increase in the activity of PDGFRB in lapatinib treated physioxia cells. Similarly, a receptor tyrosine kinase (RTK) array and western blotting showed increased basal and lapatinib induced phosphorylation of PDGFRB (Y751) under physioxia. Next, we determined the potential role of PDGFRB in downstream signaling pathway activation of AKT and ERK and resistance to lapatinib. We found that sunitinib, a multitarget RTK inhibitor with high affinity for PDGFR effectively decreased PGDFRB activity under physioxia, with a concurrent decrease in the phosphorylation of AKT. Moreover, tumor cells under physioxia were more sensitive to sunitinib treatment, relative to ambient air. Furthermore, a combination of lapatinib and sunitinib rendered tumor cells under physioxia more sensitive to treatment than with lapatinib alone. These findings suggest that ambient and physioxic oxygen tensions differentially impact cancer relevant signaling pathways. Therefore, it may be necessary to carry out preclinical cancer studies in the context of physiologically relevant oxygen tensions to aid translatability. Citation Format: Adedeji K. Adebayo, Brijesh Kumar, Christopher Davis, Steven P. Angus, Harikrishna Nakshatri. Oxygen tension - dependent differences in cancer cell kinome. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4811.

The composition and roles of gastric stem cells in epithelial homeostasis, regeneration, and tumorigenesis.

The epithelial lining of the stomach undergoes rapid turnover to preserve its structural and functional integrity, a process driven by long-lived stem cells residing in the antral and corpus glands. Several subpopulations of gastric stem cells have been identified and their phenotypic and functional diversities linked to spatiotemporal specification of stem cells niches. Here, we review the biological features of gastric stem cells at various locations of the stomach under homeostatic conditions, as demonstrated by reporter mice, lineage tracing, and single cell sequencing. We also review the role of gastric stem cells in epithelial regeneration in response to injury. Moreover, we discuss emerging evidence demonstrating that accumulation of oncogenic drivers or alteration of stemness signaling pathways in gastric stem cells promotes gastric cancer. Given a fundamental role of the microenvironment, this review highlights the role reprogramming of niche components and signaling pathways under pathological conditions in dictating stem cell fate. Several outstanding issues are raised, such as the relevance of stem cell heterogeneity and plasticity, and epigenetic regulatory mechanisms, to Helicobacter pylori infection-initiated metaplasia-carcinogenesis cascades. With the development of spatiotemporal genomics, transcriptomics, and proteomics, as well as multiplexed screening and tracing approaches, we anticipate that more precise definition and characterization of gastric stem cells, and the crosstalk with their niche will be delineated in the near future. Rational exploitation and proper translation of these findings may bring forward novel modalities for epithelial rejuvenation and cancer therapeutics.

Sorting nexin 10 sustains PDGF receptor signaling in glioblastoma stem cells via endosomal protein sorting.

Glioblastoma is the most malignant primary brain tumor, the prognosis of which remains dismal even with aggressive surgical, medical, and radiation therapies. Glioblastoma stem cells (GSCs) promote therapeutic resistance and cellular heterogeneity due to their self-renewal properties and capacity for plasticity. To understand the molecular processes essential for maintaining GSCs, we performed an integrative analysis comparing active enhancer landscapes, transcriptional profiles, and functional genomics profiles of GSCs and non-neoplastic neural stem cells (NSCs). We identified sorting nexin 10 (SNX10), an endosomal protein sorting factor, as selectively expressed in GSCs compared with NSCs and essential for GSC survival. Targeting SNX10 impaired GSC viability and proliferation, induced apoptosis, and reduced self-renewal capacity. Mechanistically, GSCs utilized endosomal protein sorting to promote platelet-derived growth factor receptor β (PDGFRβ) proliferative and stem cell signaling pathways through posttranscriptional regulation of the PDGFR tyrosine kinase. Targeting SNX10 expression extended survival of orthotopic xenograft-bearing mice, and high SNX10 expression correlated with poor glioblastoma patient prognosis, suggesting its potential clinical importance. Thus, our study reveals an essential connection between endosomal protein sorting and oncogenic receptor tyrosine kinase signaling and suggests that targeting endosomal sorting may represent a promising therapeutic approach for glioblastoma treatment.

Effect of 3D Spheroid Culturing on NF-κB Signaling Pathway and Neurogenic Potential in Human Amniotic Fluid Stem Cells.

Human amniotic fluid stem cells (hAFSCs) are known for their advantageous properties when compared to somatic stem cells from other sources. Recently hAFSCs have gained attention for their neurogenic potential and secretory profile. However, hAFSCs in three-dimensional (3D) cultures remain poorly investigated. Therefore, we aimed to evaluate cellular properties, neural differentiation, and gene and protein expression in 3D spheroid cultures of hAFSCs in comparison to traditional two-dimensional (2D) monolayer cultures. For this purpose, hAFSCs were obtained from amniotic fluid of healthy pregnancies and cultivated in vitro, either in 2D, or 3D under untreated or neuro-differentiated conditions. We observed upregulated expression of pluripotency genes OCT4, NANOG, and MSI1 as well as augmentation in gene expression of NF-κB-TNFα pathway genes (NFKB2, RELA and TNFR2), associated miRNAs (miR103a-5p, miR199a-3p and miR223-3p), and NF-κB p65 protein levels in untreated hAFSC 3D cultures. Additionally, MS analysis of the 3D hAFSCs secretome revealed protein upregulation of IGFs signaling the cascade and downregulation of extracellular matrix proteins, whereas neural differentiation of hAFSC spheroids increased the expression of SOX2, miR223-3p, and MSI1. Summarizing, our study provides novel insights into how 3D culture affects neurogenic potential and signaling pathways of hAFSCs, especially NF-κB, although further studies are needed to elucidate the benefits of 3D cultures more thoroughly.

MiR-197-3p Promotes Osteosarcoma Stemness and Chemoresistance by Inhibiting SPOPL.

First-line treatment for osteosarcoma includes chemotherapy and surgery. However, the five-year survival rate of refractory osteosarcoma remains unsatisfactory. Osteosarcoma cancer stem cells, possessing stemness and chemoresistance, are one of the critical causes of poor response to chemotherapy. Elucidating regulatory signaling pathways of osteosarcoma cancer stem cells may provide a rationale for improving regimens against chemoresistant osteosarcoma. Methotrexate (MTX)-resistant osteosarcoma cells were established. microRNA expression profiles were used for detecting differentially expressed microRNA in resistant clones and the parental cells. microRNA target databases were employed to predict potential microRNA and mRNA interactions. Flow cytometry was performed to measure stem cell marker Prominin-1 (CD133)-positive cells. Immunofluorescence staining was applied to detect CD133 expression. miR-197-3p mimic or anti-miR-197-3p stably transfected cells were used to generate xenograft models. In the study, we found that miR-197-3p was increased in MTX-resistant cell lines. Overexpression of miR-197-3p enhanced the expression of cancer stem cell markers CD133, Octamer-binding protein 4 (OCT4), Transcription factor SOX-2 (SOX2), and Homeobox protein NANOG (NANOG), as well as chemoresistance-associated genes ATP-dependent translocase ABCB1 (ABCB1) and Broad substrate specificity ATP-binding cassette transporter ABCG2 (ABCG2), whereas miR-197-3p knockdown inhibited stemness and recovered sensitivity to MTX. We also classified the tumor suppressor Speckle-type POZ protein-like (SPOPL) as a target of miR-197-3p. The miR-197-3p mutation that could not combine SPOPL promoter regions was unable to sustain stemness or chemoresistance. Collectively, we discovered miR-197-3p conferred osteosarcoma stemness and chemotherapy resistance by targeting SPOPL, prompting promising therapeutic candidates for refractory osteosarcoma treatment.

Targeting crosstalk of signaling pathways in cancer stem cells: a promising approach for development of novel anti-cancer therapeutics

Wnt, Hedgehog (Hh), and Notch signaling pathways are the evolutionarily conserved signaling pathways that regulate the embryonic development and also play crucial role in maintaining stemness properties of cancer stem cells (CSCs) and inducing epithelial-to-mesenchymal transition (EMT), metastasis, and angiogenesis. It has been highly challenging to inhibit the CSCs growth and proliferation as these are capable of evading chemotherapeutic drugs and cause cancer recurrence through multiple signaling pathways. Therefore, novel therapeutic strategies to target the key players involved in the crosstalk of these signaling pathways need to be developed. In this review, we have identified the interacting molecules of Wnt, Hh, and Notch pathways responsible for enhancing the malignant properties of CSCs. Analyzing the functions of these crosstalk molecules will help us to find an approach toward the development of new anti-cancer drugs for inhibition of CSCs growth and progression. Long non-coding RNAs (LncRNAs) play a significant role in various cellular processes, like chromatin remodeling, epigenetic modifications, transcriptional, and post-transcriptional regulations. Here, we have highlighted the research findings suggesting the involvement of LncRNAs in maintenance of the stemness properties of CSCs through modulation of the above-mentioned signaling pathways. We have also discussed about the different therapeutic approaches targeting those key players responsible for mediating the crosstalk between the pathways. Overall, this review article will surely help the cancer biologists to design novel anti-CSCs agents that will open up a new horizon in the field of anti-cancer therapeutics.

Correlation between stem cell molecular phenotype and atherosclerotic plaque neointima formation and analysis of stem cell signal pathways.

Vascular stem cells exist in the three-layer structure of blood vessel walls and play an indispensable role in angiogenesis under physiological conditions and vascular remodeling under pathological conditions. Vascular stem cells are mostly quiescent, but can be activated in response to injury and participate in endothelial repair and neointima formation. Extensive studies have demonstrated the differentiation potential of stem/progenitor cells to repair endothelium and participate in neointima formation during vascular remodeling. The stem cell population has markers on the surface of the cells that can be used to identify this cell population. The main positive markers include Stem cell antigen-1 (Sca1), Sry-box transcription factor 10 (SOX10). Stromal cell antigen 1 (Stro-1) and Stem cell growth factor receptor kit (c-kit) are still controversial. Different parts of the vessel have different stem cell populations and multiple markers. In this review, we trace the role of vascular stem/progenitor cells in the progression of atherosclerosis and neointima formation, focusing on the expression of stem cell molecular markers that occur during neointima formation and vascular repair, as well as the molecular phenotypic changes that occur during differentiation of different stem cell types. To explore the correlation between stem cell molecular markers and atherosclerotic diseases and neointima formation, summarize the differential changes of molecular phenotype during the differentiation of stem cells into smooth muscle cells and endothelial cells, and further analyze the signaling pathways and molecular mechanisms of stem cells expressing different positive markers participating in intima formation and vascular repair. Summarizing the limitations of stem cells in the prevention and treatment of atherosclerotic diseases and the pressing issues that need to be addressed, we provide a feasible scheme for studying the signaling pathways of vascular stem cells involved in vascular diseases.

The NLRC4 Inflammasome Drives Myelodysplastic Syndrome By Linking Epigenetic Reprogramming and Innate Immune Signaling

Introduction Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic stem cell (HSC) malignancies characterized by dysplastic and ineffective hematopoiesis. Despite emerging evidence that chronic inflammation contributes to the development of MDS, the mechanisms underlying the dysregulation of innate immune and inflammatory signaling pathways in hematopoietic stem and progenitor cells (HSPCs) remain poorly characterized. We have used multiple MDS mouse models to identify a tumor suppressive role for the EP300 epigenetic histone acetyltransferase enzyme in the progression of MDS to AML. Based on the gene expression changes we observed, we have used one specific mouse model, that lacks Tet2 expression, to define the role that chronic inflammation plays in promoting MDS development and the mechanisms involved. Methods We have conducted a series of in vitro, in vivo, and in silico experiments using Tet2-deficient mice, and chronic, low-dose LPS treatment to model chronic inflammation. Cytokine array immunoassays were performed to evaluate the inflammatory response of WT or Tet2-deficient mice to LPS treatment. Chimeric mice transplanted with bone marrow (BM) cells isolated from LPS or vehicle (PBS) treated WT or Tet2-deficient mice were monitored for the development of MDS and survival. Serial competitive bone marrow transplantation assays and flow cytometry profiling were used to determine the competitive advantage of LPS or PBS-treated BM cells, and their differentiation. Bioinformatic analyses of RNA sequencing (RNA-seq) results from our mouse model were performed to characterize the transcriptional profiling and identify novel innate immune pathways. Colony-forming unit (CFU) and qPCR assays were used to evaluate HSPC proliferative capacity, self-renewal, and gene expression changes. Results We find that mice lacking Tet2 develop a hyper-inflammatory status, marked by the expression of a number of pro-inflammatory cytokines, such as Il-1b and Il-6, which are further fueled by LPS-induced chronic inflammation. Chimeric mice transplanted with BM cells isolated from LPS-treated Tet2-deficient mice exhibit a significantly shorter life span and early onset of MDS phenotypes, including dysplastic hematopoiesis and thrombocytopenia, compared to age-matched chimeric mice transplanted with BM cells isolated from PBS-treated Tet2-deficient mice or LPS-treated WT mice. Chronic inflammation promotes the self-renewal and myeloid differentiation of Tet2-deficient HSPCs in the short term; however, it accelerates HSC exhaustion in the long run. Transcription profiling and subsequent bioinformatic analyses identify functional clusters in LPS-treated Tet2-deficient HSPCs, including genes involved in the regulation of epigenetics, immunity, metabolism, and cell cycle. Detailed inspection of the most highly differentially expressed genes reveals that many genes in the Nlrc4 inflammasome pathway are upregulated in LPS-treated Tet2-deficient HSPCs. We validate changes in the expression of these genes by qPCR, and then show that deleting Nlrc4 suppresses the colony formation of Tet2-deficient HSPCs in vitro. Ongoing work focuses on evaluating the effects of deleting Nlrc4 or blocking Nlrc4 function using a small molecule inhibitor on MDS development in vivo. Conclusions The combinatory role of intrinsic and extrinsic factors drives the development of MDS in this Tet2-deficient MDS mouse model. It is likely that different genetic mutations generate unique sensitivity to certain signaling pathways, and we demonstrate the epigenetic rewiring and activation of the Nlrc4 inflammasome pathway as a mechanistic basis for the inflammatory component of MDS [or clonal hematopoiesis of indeterminate potential (CHIP)] pathogenesis in Tet2-deficient cells. Our work has the potential to provide novel targeting opportunities in MDS management and confirm the importance of different components of inflammasome signaling in MDS.