Stem Cell Phenotype Research Articles

Use of proton pump inhibitors (PPI) in glioblastoma (GBM) and relationship to overall survival in a national real-world evidence (RWE) database.

e14027 Background: Aldehyde dehydrogenase-1A1 ( ALDH1A1) mediates therapy resistance and poor prognosis in multiple cancers. In GBM, ALDH1A1 expression above vs. below the mean is associated with a significant reduction in survival.1 ALDH enzymes activate transcriptional programs that induce the cancer stem cell phenotype, detoxify anticancer drugs, and function as antioxidants by reversing lipid peroxidation and etheno-DNA adducts, thus conferring chemotherapy and radiation resistance. PPI have been shown to increase enzyme activity of ALDH1A1 up to 6-fold, suggesting a potentially hazardous effect on tumor biology and survival. However, no prospective randomized data are available to address these concerns. Thus, we turned to XCELSIOR, an observational database that prospectively collects medical records including medication usage. Methods: Patients with GBM were e-consented to XCELSIOR ( NCT03793088 ), a nationwide observational research protocol that leverages patient consent to permit gathering of medical records from all sites of care. Clinical data were abstracted from medical documents into a central EDC system, source-verified, and standardized to generate comprehensive longitudinal histories for aggregate analysis. Identified patient records permitted the definitive identification of dates of diagnosis and death. Overall survival (OS) was calculated and stratified based for PPI exposure, extent of surgical resection (EOR), and MGMT methylation status. Results: XCELSIOR contained 320 GBM patients from 222 treatment sites. We stratified patients based on exposure to PPI and assessed median OS (mOS). For the entire population, mOS was substantially better for patients not receiving PPI. (TABLE) The 5-yr survival was 29.9% [21.0 – 42] and 6.6% [1.4 – 32.0] for patients not receiving vs. receiving PPI. In a multivariate analysis considering EOR and MGMT methylation, PPI exposure yielded a hazard ratio (HR) of 1.57 (p=0.009). For the interaction of m-MGMT and PPI exposure together, the HR is 2.63 (p=0.043), significant as the HR of m-MGMT alone is 0.40 (p=0.0028). Conclusions: In this RWE dataset, PPI use in GBM appeared detrimental to survival. This is strongly consistent with the hypothesis that PPI antagonizes chemotherapy. Additionally, use of PPI appeared to completely abrogate the survival benefit of chemotherapy associated with methylated-MGMT. While we recognize the utility of PPI in managing acute UGI bleeding, these data suggest avoiding PPIs in GBM patients. 1) Schmidt-Graf F, et al. Neuro Oncol. 2012;14(12):1452–1464. [Table: see text]

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.

Patchouli alcohol induces G0 /G1 cell cycle arrest and apoptosis in vincristine-resistant non-small cell lung cancer through ROS-mediated DNA damage.

Lung cancer, especially non-small cell lung cancer (NSCLC), is one of the leading causes of cancer-related deaths worldwide. Vincristine (VCR) is a chemotherapeutic agent for lung cancers; however, its effectiveness is limited by side effects and the development of drug resistance. Patchouli alcohol (PA), from Pogostemon cablin extract, is known to possess anti-inflammatory and anticancer properties. In this study, we investigated the role of PA in inducing reactive oxygen species (ROS)-mediated DNA damage in A549 and VCR-resistant A549/V16 NSCLC cells. The anticancer potential of PA was studied using the MTT assay, colony formation, flow cytometry analysis, western blotting, DCFDA staining, immunofluorescence staining, and TUNEL assay techniques. The intracellular ROS levels were enhanced in PA-treated cells, activating the CHK1 and CHK2 signaling pathways. PA further inhibited proliferation and colony-forming abilities and induced cell cycle arrest at the G0 /G1 phase by regulating p53/p21 and CDK2/cyclin E1 expression. Moreover, PA increased the percentage of cells in the subG1 phase and induced apoptosis by activating the Bax/caspase-9/caspase-3 intrinsic pathway. In addition, drug resistance (p-glycoprotein) and cancer stem cell (CD44 and CD133) markers were downregulated after PA treatment. Furthermore, combining PA and cisplatin exhibited synergistic inhibitory activity in A549 and A549/V16 cells. PA induced ROS-mediated DNA damage, triggered cell cycle arrest and apoptosis, attenuated drug resistance and cancer stem cell phenotypes, and synergistically inhibited proliferation in combination with cisplatin. These findings suggest that PA has the potential to be used for the treatment of NSCLC with or without VCR resistance.

Grade scoring system reveals distinct molecular subtypes and identifies KIF20A as a novel biomarker for predicting temozolomide treatment efficiency in gliomas.

The importance of molecular diagnostics is increasingly emphasized in the 2021 WHO guidelines for gliomas. There is considerable variability in molecular features and prognosis among glioma patients with the same pathological WHO grade. mRNA data and clinical information of human glioma patients were obtained from TCGA and CGGA databases, while expression profiles and TMZ resistance phenotypes of human glioma stem cells were acquired from the GEO database. Differentially expressed genes were identified across distinct WHO grades. Unsupervised clustering was performed on glioma patients based on DEG expression profiles. The Boruta algorithm was employed to identify feature genes for distinct molecular subtypes, and PCA was used to reduce the dimensionality of the feature gene expression data. Grade scores for each sample were calculated and correlated with patients' clinical molecular pathological features and immune microenvironment. Gene set enrichment analysis identified grade score-related functional pathways. Weighted gene co-expression network analysis identified grade score-associated biomarkers. The impact of the hub gene on malignant glioma behavior was validated through in vitro experiments, including CCK-8, EdU, colony formation, Transwell, wound healing, and immunofluorescence assays. A total of 672 and 687 samples were screened from TCGA and CGGA databases, respectively, along with 6 control, 24 low-grade, and 40 glioblastoma samples from our hospital. Two robust gene clusters were identified based on the expression profiles of 4,476 DEGs among grades 2, 3, and 4 tissues, revealing distinct prognoses. The grade scores exhibited significant heterogeneity across different WHO grade samples, representing diverse immune microenvironments. Grade scores served as independent risk factors for predicting patient prognosis, with higher sensitivity than traditional biomarkers. KIF20A, identified as a grade score-related biomarker, was independently associated with glioma prognosis. Exclusively expressed in tumor cells, KIF20A knockdown significantly inhibited tumor growth, invasion, and EMT biological behavior in glioma cells. Furthermore, KIF20A could serve as a biological marker for predicting patient response to TMZ treatment. The grade scoring system enhances our understanding of the glioma tumor microenvironment. KIF20A, a novel biomarker for predicting TMZ treatment efficiency, influences malignant tumor behavior by affecting the EMT biological behavior of glioma cells.

Phosphorylation of IWS1 by AKT maintains liposarcoma tumor heterogeneity through preservation of cancer stem cell phenotypes and mesenchymal-epithelial plasticity

Chemotherapy remains the mainstay of treatment for patients with advanced liposarcoma (LPS), but response rates are only 25% and the overall survival at 5 years is dismal at 20–34%. Translation of other therapies have not been successful and there has been no significant improvement in prognosis for nearly 20 years. The aberrant activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway has been implicated in the aggressive clinical behavior LPS and in resistance to chemotherapy, but the precise mechanism remains elusive and efforts to target AKT clinically have failed. Here we show that the AKT-mediated phosphorylation of the transcription elongation factor IWS1, promotes the maintenance of cancer stem cells in both cell and xenograft models of LPS. In addition, phosphorylation of IWS1 by AKT contributes to a “metastable” cell phenotype, characterized by mesenchymal/epithelial plasticity. The expression of phosphorylated IWS1 also promotes anchorage-dependent and independent growth, cell migration, invasion, and tumor metastasis. In patients with LPS, IWS1 expression is associated with reduced overall survival, increased frequency of recurrence, and shorter time to relapse after resection. These findings indicate that IWS1-mediated transcription elongation is an important regulator of human LPS pathobiology in an AKT-dependent manner and implicate IWS1 as an important molecular target to treat LPS.

Cementum protein 1 gene-modified adipose-derived mesenchymal stem cell sheets enhance periodontal regeneration in osteoporosis rat.

Osteoporosis (OP) and periodontitis are both diseases with excessive bone resorption, and the number of patients who suffer from these diseases is expected to increase. OP has been identified as a risk factor that accelerates the pathological process of periodontitis. Achieving effective and safe periodontal regeneration in OP patients is a meaningful challenge. This study aimed to assess the efficacy and biosecurity of human cementum protein 1 (hCEMP1) gene-modified cell sheets for periodontal fenestration defect regeneration in an OP rat model. Rat adipose-derived mesenchymal stem cells (rADSCs) were isolated from Sprague-Dawley rats. After primary culture, rADSCs were subjected to cell surface analysis and multi-differentiation assay. And rADSCs were transduced with hCEMP1 by lentiviral vector, and hCEMP1 gene-modified cell sheets were generated. The expression of hCEMP1 was evaluated by reverse transcription polymerase chain reaction and immunocytochemistry staining, and transduced cell proliferation was evaluated by Cell Counting Kit-8. The hCEMP1 gene-modified cell sheet structure was detected by histological analysis and scanning electron microscopy. Osteogenic and cementogenic-associated gene expression was evaluated by real-time quantitative polymerase chain reaction. In addition, an OP rat periodontal fenestration defect model was used to evaluate the regeneration effect of hCEMP1 gene-modified rADSC sheets. The efficacy was assessed with microcomputed tomography and histology, and the biosecurity of gene-modified cell sheets was evaluated by histological analysis of the spleen, liver, kidney and lung. The rADSCs showed a phenotype of mesenchymal stem cells and possessed multi-differentiation capacity. The gene and protein expression of hCEMP1 through lentiviral transduction was confirmed, and there was no significant effect on rADSC proliferation. Overexpression of hCEMP1 upregulated osteogenic and cementogenic-related genes such as runt-related transcription factor 2, bone morphogenetic protein 2, secreted phosphoprotein 1 and cementum attachment protein in the gene-modified cell sheets. The fenestration lesions in OP rats treated with hCEMP1 gene-modified cell sheets exhibited complete bone bridging, cementum and periodontal ligament formation. Furthermore, histological sections of the spleen, liver, kidney and lung showed no evident pathological damage. This pilot study demonstrates that hCEMP1 gene-modified rADSC sheets have a marked ability to enhance periodontal regeneration in OP rats. Thus, this approach may represent an effective and safe strategy for periodontal disease patients with OP.

Zinc Finger E-Box Binding Homeobox Family: Non-Coding RNA and Epigenetic Regulation in Gliomas.

Gliomas are the most common malignant brain tumours. Among them, glioblastoma (GBM) is a grade four tumour with a median survival of approximately 15 months and still limited treatment options. Although a classical epithelial to mesenchymal transition (EMT) is not the case in glioma due to its non-epithelial origin, the EMT-like processes may contribute largely to the aggressive and highly infiltrative nature of these tumours, thus promoting invasive phenotype and intracranial metastasis. To date, many well-known EMT transcription factors (EMT-TFs) have been described with clear, biological functions in glioma progression. Among them, EMT-related families of molecules such as SNAI, TWIST and ZEB are widely cited, well-established oncogenes considering both epithelial and non-epithelial tumours. In this review, we aimed to summarise the current knowledge with a regard to functional experiments considering the impact of miRNA and lncRNA as well as other epigenetic modifications, with a main focus on ZEB1 and ZEB2 in gliomas. Although we explored various molecular interactions and pathophysiological processes, such as cancer stem cell phenotype, hypoxia-induced EMT, tumour microenvironment and TMZ-resistant tumour cells, there is still a pressing need to elucidate the molecular mechanisms by which EMT-TFs are regulated in gliomas, which will enable researchers to uncover novel therapeutic targets as well as improve patients' diagnosis and prognostication.

INFLUENCE OF THE CULTIVATION CONDITIONS OF THE GLIOBLASTOMA NEUROSPHERE ON THE EXPRESSION OF MALAT1 AND LINCROR LONG NON-CODING RNA GENES

ABSTRACT Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumor. One of the reasons for the resistance of MGB to treatment is the extreme heterogeneity of the tumor and, in particular, the presence of cancer stem cells (CSCs) in the population of glioblastoma cells. In this work, we investigated the effect of conditions that reduce the proportion of CSCs in the GBM cell population on the levels of long noncoding RNAs (lincROR and MALAT1) involved in the formation of the phenotype of glioblastoma cancer stem cells. We have shown that culturing under conditions that cause a decrease in cell stemness (when fetal calf serum is added to the culture medium) affected the content of these transcripts: in the cells of most of the analyzed lines, a decrease in the level of the positive stemness regulator lincROR and an increase in the content of MALAT1 were noted.

Abstract A05: Multiplexed CyTOF analysis of FLT3-ITD AML landscape identifies proteomic profiles associated with resistance to targeted therapies

Abstract Targeted therapies(TT) combining FLT3 inhibitors with hypomethylating agents (HMA) and Venetoclax (Ven) are highly effective against FLT3-ITD acute myeloid leukemia (AML) with high response rates. Signaling mutations along with emergence of rarer mutations are the key contributors to primary and secondary resistance to TT. We hypothesize that resistant leukemia clones have unique proteomic profiles that facilitate survival under therapy pressure(TP) and deciphering proteomic profiles at the single-cell level will delineate resistance mechanisms and adaptive responses driven by TP. We performed multiplexed single-cell proteomic analysis of serial PB and BM samples (n:162) from patients treated with FLT3i+HMA+/-VEN using CyTOF. Unsupervised analysis identified leukemia cells and non-malignant cellular elements of the leukemia compartment in an unbiased manner. Next, we interrogated the leukemia proteomic landscape to identify leukemia associated proteomic features. Since Ven targets BCL2 and FLT3i modulates the expression of anti-apoptotic molecules we assessed the expression of apoptosis regulators and found that leukemia cells with immature phenotype, including those with leukemia stem cell(LSC) phenotype, almost always expressed moderate-high levels of BCL2. Monocytic cells(MC) lacked BCL2 expression and had the highest levels of MCL1. Despite expressing moderate levels of MCL1 and BCL-XL, TT was effective in substantially eliminating CD34+ immature leukemia cells. We observed a relative enrichment of MCs, either benign or malignant, after TT. Since cells with MC phenotype were inherently resistant to TT we hypothesized that leukemia cell subsets having similar proteomic profiles to MCs will persist after TT. Phenotypic interrogation of leukemia cells (UMAP) revealed that LSCs generally clustered on the opposite pole distant from MCs and leukemia cells on poles facing MCs were more differentiated. Remarkably, FLT3-ITD mutation partners differentially altered the proteomic landscape. NPM1 mutant FLT3-ITD AML cells displayed a less diverse leukemia architectural organization with differentiation block. Contrarily, signaling mutations diversified the leukemia landscape into a diverse continuum of differentiation states. We also observed that RAS/MAPK mutations could overcome differentiation block and gave rise to a differentiation continuum encompassing less-differentiated, transitional and differentiated leukemia cells. The transitional CD34+ leukemia cells, mapped in close vicinity to MCs, and differentiated MCs that preferentially persisted at D28, had active signaling pathways and expressed CD36. The presence of CD34+ leukemia cells with persistent signaling at D28 was indicative of poor clinical outcomes. Conclusion: Multiplexed single-cell proteomic analysis identified a unique mode of resistance and proteomic features of surviving cells in with FLT3-ITD AML patients treated with TT, and elucidated how different mutation partners in FLT3-ITD AML differentially altered proteomic landscape to drive resistance to TT. Citation Format: Muharrem Muftuoglu, Musa Yilmaz, Mahesh Basyal, Li Li, Naval Daver, Michael Andreeff. Multiplexed CyTOF analysis of FLT3-ITD AML landscape identifies proteomic profiles associated with resistance to targeted therapies [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 A05.

Prediction of Stem Cell State Using Cell Image‐Based Deep Learning

Stem cells represent an ideal source for regenerative medicine; however, longitudinal assessment of stem cell phenotype and function is challenging. Contrastingly, a convolutional neural network (CNN) algorithm can automatically extract the image features and produce highly accurate image recognition. Thus, this study implements CNN to establish stable and reproducible cell culture experiments by predicting a unique morphology of pluripotent stem cell (PSC) lines. Interestingly, the algorithm distinguishes the PSC lines cultured in the different cell culture conditions, such as the presence or absence of small molecules and/or the long‐ or short‐term culture in our induced PSC (iPSC) models, which include iPSC lines with abnormal gene expression patterns and genomic abnormalities. Our deep learning technology accurately classifies the various cell lines with or without genetic defects using only the cell images, without any labeling process. This suggests that the CNN system may simplify the various tasks involving stable cell cultures and their differentiation.

RAB27B Drives a Cancer Stem Cell Phenotype in NSCLC Cells Through Enhanced Extracellular Vesicle Secretion.

Expression of RAB27B in CSCs leads to elevated levels of EVs that mediate communication between CSCs and BCCs that maintains a stem-like phenotype in NSCLC cells.

Self-Assembled Copolymeric Nanowires as a New Class of 3D Scaffold for Stem Cells Growth and Proliferation

Stem cell therapy has emerged as the most vibrant area of research, due to the capacity of stem cells for self-renewal and differentiation into different types of cell lines upon their culture. But lately, scientists become increasingly aware of the limitations of conventional 2D culture and stem cell culture media, due to several key drawbacks associated with this model, such as immune response upon transplantation, animal pathogen contamination, and complication, during developmental studies due to undefined factors in the cultural media. In this study, an attempt has been made to develop a new type of polymeric 3D scaffold based on the self-assembly of a star-like amphiphilic copolymer of poly(caprolactone)–poly(ethylene oxide) unit into nanowires (nanofibers), that have a scale similar to the native extracellular matrix and are capable of mimicking the extracellular microenvironment where the functional properties of stem cells can be observed and manipulated. The obtained data showed that polymeric-based nanofibers can be used as a 3D scaffold for mouse embryonic stem cells (mESCs) growth without losing their stem cell phenotype. The results obtained suggest that the polymeric 3D scaffolds (nanofibers) not only support stem cells’ growth and proliferation but also preserve the mESC pluripotency.

Abstract 2444: RAB27B drives a cancer stem cell phenotype in non-small cell lung cancer cells through enhanced extracellular vesicle secretion

Abstract Non-small cell lung cancer (NSCLC), a major subtype of lung cancer, accounts for ~85% of all lung cancer diagnoses. A sub-population of cells within human NSCLC tumors called cancer stem cells (CSCs) have been found to exhibit distinct properties that contribute to factors associated with poor outcomes in NSCLC patients including metastasis, relapse, and therapeutic resistance. We previously performed RNAseq analysis on bulk cancer cells (BCCs) and CSCs from a panel of primary NSCLC tumors and cell lines to identify signaling mechanisms involved in the tumorigenic behavior of NSCLC CSCs. Our analysis revealed that expression of RAB27B, a small GTPase that plays a critical role in extracellular vesicle (EV) formation and secretion, was significantly upregulated in CSCs when compared to BCCs. Interestingly, RAB27B is overexpressed and predicts patient survival in NSCLC. The goal of this study was to assess the role of RAB27B in the tumorigenic behavior of NSCLC CSCs and determine the mechanism by which RAB27B promotes an aggressive stem-cell like phenotype in NSCLC cells an aspect of RAB27B function that has not been investigated in NSCLC. QPCR and immunoblot analysis was used to compare RAB27B mRNA and protein expression in a panel of NSCLC BCCs and CSCs. Two independent shRNA constructs were used to knockdown (KD) RAB27B in NSCLC CSCs. The effects of RAB27B KD on CSC expansion, transformed growth and invasion were analyzed by clonal expansion, soft agar colony formation and Boyden chamber assays, respectively. Xenograft and tail vein mouse models were used to assess the effects of RAB27B KD on CSC tumor growth and lung colonization. EVs were isolated from the conditioned media of BCCs, CSCs and RAB27B KD CSCs using differential ultracentrifugation, and characterized by nanoparticle tracking analysis, transmission electron microscopy and immunoblotting according to MISEV guidelines. Our analysis revealed that RAB27B is significantly upregulated in NSCLC CSCs compared to BCCs. RAB27B KD cells exhibited a significant decrease in clonal expansion, transformed growth and invasion when compared to non-target shRNA control CSCs. In vivo studies demonstrated that RAB27B is important for the tumorigenicity of CSCs and promotes tumor cell proliferation, and angiogenesis. EVs were positive for EV-specific markers and negative for an intracellular vesicle marker. CSCs secrete significantly more EVs when compared to BCCs and KD of RAB27B in CSCs led to a decrease in the levels of EVs secreted. Furthermore, delivery of CSC-derived, but not BCC-derived EVs to BCCs induced spheroid growth, clonal expansion, and invasion in BCCs. Taken together, our results indicate that RAB27B is required for maintenance of a highly tumorigenic, cancer-initiating and invasive stem-like cell population in NSCLC and RAB27B is involved in propagating EV-mediated communication from NSCLC CSCs to BCCs. Citation Format: Kayleah M. Meneses, Jennifer A. Lindemann, Prita Pandya, Dania S. Al-Qasrawi, Ryan Argo, Celeste Weems, Danielle J. Beetler, Irene K. Yan, Joy Wolfram, Tushar C. Patel, Verline Justilien. RAB27B drives a cancer stem cell phenotype in non-small cell lung cancer cells through enhanced extracellular vesicle secretion [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 2444.

Abstract 2456: Transcriptional signatures of stem cell phenotypes in normal and clonal hematopoiesis

Abstract Clonal hematopoiesis (CH) is an age-related clonal expansion of hematopoietic stem cells (HSCs). Factors that shape the progression of CH are only beginning to be characterized. HSCs are a heterogeneous population of cells with distinct clonal outputs over time, which are hypothesized to be largely scripted in epigenetic and transcriptional cell state. Molecular properties of HSC states that mediate clonal behaviors of two major driver mutations in CH - DNMT3A and TET2 - represent potential therapeutical targets. Recent studies have identified the landscape of human CH mutant cells compared to non-mutated ones. However, the notion of a specific molecular target identified in CH cells associated with selective advantage or predisposition for subsequent transformation requires a formal validation by longitudinal studies. To uncover molecular underpinnings of CH-driven clonal phenotypes in human HSCs in vivo, we combined clonal analysis using heritable expressed DNA barcodes with single-cell transcriptomics using transplant in immunodeficient mice. We developed a statistical approach to associate transcriptional differences of HSCs with the clone phenotypes, such as size and HSC density, across more than a hundred of detected clones. It revealed robust transcriptional signatures underlying clone size and HSC density, which are largely complementary to transcriptional programs activated in TET2 and DNMT3A mutant HSCs. Of note, the clone size and density of HSCs in the clone tend to be uncoupled and associated with distinct transcriptional processes. Citation Format: Ruslan Soldatov, Giulia Schiroli, Peter Kharchenko, David Scadden. Transcriptional signatures of stem cell phenotypes in normal and clonal hematopoiesis [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 2456.

PD25-05 CHRONIC EXPOSURE OF PHYSIOLOGICAL CONCENTRATION OF ARSENIC INDUCED BLADDER MALIGNANCY IN PRECLINICAL MODELS

You have accessJournal of UrologyCME1 Apr 2023PD25-05 CHRONIC EXPOSURE OF PHYSIOLOGICAL CONCENTRATION OF ARSENIC INDUCED BLADDER MALIGNANCY IN PRECLINICAL MODELS Hangcheng Fu, Chendil Damodaran, and Ankem Murali Hangcheng FuHangcheng Fu More articles by this author , Chendil DamodaranChendil Damodaran More articles by this author , and Ankem MuraliAnkem Murali More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003303.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Arsenic is a naturally occurring metalloid classified as a group 1 carcinogen in humans. Chronic environmental exposure to inorganic arsenic has been frequently associated with different types of cancer, including squamous cell carcinoma of the skin and lung and bladder cancers. The bladder is one of the identified target tissues for arsenic toxicity as some of the toxic metabolites of arsenic excreted through urine have been detected in organs, particularly in the bladder. Hence, the study aims to understand how arsenic causes bladder carcinogenesis and dissect the molecular signaling responsible for transforming normal bladder epithelial cells. METHODS: To develop an in vitro model of arsenic (250 nM: 33 mg/L))-induced malignant transformation, we chronically exposed healthy bladder epithelial cells (HU-hTERT1) for over 12 months to a median physiological concentration of arsenic and the transformed (AST) cells like that observed in BCa patients. Cell proliferation, qRT-PCR, and Western blot analyses were used to assess the carcinogenicity effect of arsenic in TRT-HU1 cells. In vivo, xenotransplanted mice models were used to confirm the tumorigenicity potential of the transformed bladder epithelial cells. Computational Data and statistical analysis were performed. RESULTS: Our results demonstrate that arsenic levels in the urine of our bladder cancer patient's cohort are significantly higher (4–6 fold) than that observed in healthy controls. Arsenic-exposed cells began to form anchorage-dependent colonies and -independent spheroids after four months, and the number of colonies increased with exposure time. Our transcriptomic and RPPA analysis suggested stem cell activation may be responsible for arsenic-induced transformation in bladder epithelial cells. In addition, we found a robust induction of ALDH1A1 and CD44 transcript and protein expression correlating with stem cell enrichment in arsenic-mediated transformation. Silencing the stem cell activators (ALDH1A1 and CD44) abrogates arsenic-induced malignant transformation. Finally, the tumorgenicity potential of the transformed bladder epithelial cells was confirmed in xenografted mice models. CONCLUSIONS: Our results suggested that stem cell activators may significantly facilitate the arsenic-exposed cells' survival advantage, enabling the healthy epithelial cells to reprogram into a cancer stem cell phenotype, leading to malignant transformation. Source of Funding: N/A © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e730 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hangcheng Fu More articles by this author Chendil Damodaran More articles by this author Ankem Murali More articles by this author Expand All Advertisement PDF downloadLoading ...

Effect of miR-18a-5p, miR-19a-3p, and miR-20a-5p on In Vitro Cardiomyocyte Differentiation of Human Endometrium Tissue-Derived Stem Cells Through Regulation of Smad4 Expression.

Smad4 regulates the expression of the genes required for heart homeostasis. Regarding the central role of microRNAs in cardiac biology, we investigated the expression of the three Smad4-targeting miRNAs, namely miR-18a-5p, miR-19a-3p, and miR-20a-5p, as well as Smad4 during differentiation of human endometrium-derived mesenchymal stem cells (hEMSCs) into cardiomyocytes (CMs). To evaluate mesenchymal phenotype and multi-lineage differentiation ability of hEMSCs, immunophenotyping by flow cytometry and differentiation into osteoblasts and adipocytes were performed, respectively. For transdifferentiation into CMs, hEMSCs were exposed to a cardiomyogenic medium composed of 5-aza and bFGF for 30 days. The comparison between transcriptional expression levels of Nkx2-5, GATA4, Smad4, TNNT2, TBX5, miR-18a-5p, miR-19a-3p, and miR-20a-5p by qRT-PCR, as well as protein levels of Nkx2-5, Smad4, and cTnT by immunofluorescence staining, was conducted in every 6 days. In vitro, the mesenchymal stem cell phenotype of hEMSCs and their potency for differentiation into other MSCs were confirmed. Differentiated hEMSCs had morphological characteristics of CMs. The percentage of positive cells for Nkx2-5, Smad4, and cTnT proteins was increased following induction and culminated on the 24th day. Also, mRNA levels of Nkx2-5, GATA4, Smad4, TNNT2, and TBX5 exhibited the same trend. The expression of investigated miRNAs was significantly decreased sequentially. A significant negative correlation between expressions of Smad4 and investigated miRNAs was observed. Our results indicate that miR-18a-5p, miR-19a-3p, and miR-20a-5p are involved in the cardiac differentiation propensity of hEMSCs potentially by regulation of Smad levels. Although, more mechanistic experiments are required to confirm this idea.

Dual inhibition of EZH2 and G9A/GLP histone methyltransferases by HKMTI-1-005 promotes differentiation of acute myeloid leukemia cells.

All-trans-retinoic acid (ATRA)-based differentiation therapy of acute promyelocytic leukemia (APL) represents one of the most clinically effective examples of precision medicine and the first example of targeted oncoprotein degradation. The success of ATRA in APL, however, remains to be translated to non-APL acute myeloid leukemia (AML). We previously showed that aberrant histone modifications, including histone H3 lysine 4 (H3K4) and lysine 27 (H3K27) methylation, were associated with this lack of response and that epigenetic therapy with small molecule inhibitors of the H3K4 demethylase LSD1/KDM1A could reprogram AML cells to respond to ATRA. Serving as the enzymatic component of Polycomb Repressive Complex 2, EZH2/KMT6A methyltransferase plays a critical role in normal hematopoiesis by affecting the balance between self-renewal and differentiation. The canonical function of EZH2 is methylation of H3K27, although important non-canonical roles have recently been described. EZH2 mutation or deregulated expression has been conclusively demonstrated in the pathogenesis of AML and response to treatment, thus making it an attractive therapeutic target. In this study, we therefore investigated whether inhibition of EZH2 might also improve the response of non-APL AML cells to ATRA-based therapy. We focused on GSK-343, a pyridone-containing S-adenosyl-L-methionine cofactor-competitive EZH2 inhibitor that is representative of its class, and HKMTI-1-005, a substrate-competitive dual inhibitor targeting EZH2 and the closely related G9A/GLP H3K9 methyltransferases. We found that treatment with HKMTI-1-005 phenocopied EZH2 knockdown and was more effective in inducing differentiation than GSK-343, despite the efficacy of GSK-343 in terms of abolishing H3K27 trimethylation. Furthermore, transcriptomic analysis revealed that in contrast to treatment with GSK-343, HKMTI-1-005 upregulated the expression of differentiation pathway genes with and without ATRA, while downregulating genes associated with a hematopoietic stem cell phenotype. These results pointed to a non-canonical role for EZH2, which was supported by the finding that EZH2 associates with the master regulator of myeloid differentiation, RARα, in an ATRA-dependent manner that was enhanced by HKMTI-1-005, possibly playing a role in co-regulator complex exchange during transcriptional activation. In summary, our results strongly suggest that addition of HKMTI-1-005 to ATRA is a new therapeutic approach against AML that warrants further investigation.

Epigallocatechin-3-Gallate Prevents the Acquisition of a Cancer Stem Cell Phenotype in Ovarian Cancer Tumorspheres through the Inhibition of Src/JAK/STAT3 Signaling

Three-dimensional tumorsphere cultures recapitulate the expression of several cancer stem cell (CSC) biomarkers and represent an effective in vitro platform to screen the anti-CSC properties of drugs. Whereas ovarian carcinoma is among the leading causes of death for women, ovarian CSC (OvCSC), a highly malignant subpopulation of ovarian cancer cells, is thought to be responsible for therapy resistance, metastasis, and tumor relapse. Epigallocatechin-3-gallate (EGCG), a diet-derived active polyphenol found in green tea leaves, can suppress ovarian cancer cell proliferation and induce apoptosis. However, its capacity to prevent the acquisition of cancer stemness traits in ovarian malignancies remains unclear. Here, we exploited the in vitro three-dimensional tumorsphere culture model to explore the capacity of EGCG to alter CSC biomarkers expression, signal transducing events and cell chemotaxis. Total RNA and protein lysates were isolated from human ES-2 ovarian cancer cell tumorspheres for gene assessment by RT-qPCR and protein expression by immunoblot. Real-time cell chemotaxis was assessed with xCELLigence. Compared with their parental adherent cells, tumorspheres expressed increased levels of the CSC markers NANOG, SOX2, PROM1, and Fibronectin. EGCG treatment reduced dose-dependently tumorspheres size and inhibited the transcriptional regulation of those genes. Src and JAK/STAT3 signaling pathways appeared to be relevant for CSC phenotype and chemotactic response. In conclusion, these data highlight and support the chemopreventive benefits of the diet-derived EGCG and its capacity to target intracellular transducing events that regulate the acquisition of an invasive CSC phenotype.

Tumor Microenvironment-Responsive Nanoherb Delivery System for Synergistically Inhibition of Cancer Stem Cells.

Multidrug resistance in cancer stem cells (CSCs) is a major barrier to chemotherapy; hence, developing CSC-specific targeted nanocarriers for efficient drug delivery is critical. In this study, monodisperse hollow-structured MnO2 (H-MnO2) with a mesoporous shell was created for efficient targeted drug delivery. An effective therapeutic compound isoliquiritigenin (ISL) was confirmed to inhibit the lung cancer stem-cell phenotype by natural compound screening based on integrated microfluidic devices. The resultant H-MnO2 showed a high drug-loading content of the potent CSC-targeting compound ISL and near-infrared fluorescent dye indocyanine green (ICG). In addition, H-MnO2 was successively modified with hyaluronic acid (HA) to enhance targeting CSCs with high CD44 expression levels. The H-MnO2@(ICG + ISL)@HA nanocomposites displayed promising chemotherapeutic and photothermal treatment capabilities, as well as NIR-triggered drug release, which showed excellent CSC-killing effects and tumor inhibition efficacy. Meanwhile, the development of the tumor was effectively restrained by NIR-triggered phototherapy and prominent chemotherapy without obvious side effects after tail vein injection of the nanocomposites in vivo. In summary, the prepared nanocomposites accomplished synergistic cancer therapy that targets CSCs, offering a versatile platform for lung cancer diagnosis and treatment.

An unbiased proteomics approach to identify the senescence-associated secretory phenotype of human bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) derived from bone marrow can support skeletal tissue repair and regeneration owing to their self-renewing capacity, differentiation ability, and trophic functions. Bone marrow-derived MSCs undergo dramatic changes with aging, including the senescence-associated secretory phenotype (SASP) which may largely contribute to age-related changes in bone tissue leading to osteoporosis. A mass spectrometry-based proteomics approach was used to investigate the MSC SASP. Replicative senescence was achieved by exhaustive in vitro sub-cultivation and confirmed by standard proliferation criteria. Conditioned media from non-senescent and senescent MSCs underwent mass spectrometry. Proteomics and bioinformatics analyses enabled the identification of 95 proteins expressed uniquely in senescent MSCs. Protein ontology analysis revealed the enrichment of proteins linked to the extracellular matrix, exosomes, cell adhesion, and calcium ion binding. The proteomic analysis was independently validated by taking ten identified proteins with relevance to bone aging and confirming their increased abundance in conditioned media from replicatively senescent versus non-senescent MSCs (ACTα2, LTF, SOD1, IL-6, LTBP2, PXDN, SERPINE 1, COL1α1, THBS1, OPG). These target proteins were used to further investigate changes in the MSC SASP profile in response to other inducers of senescence, ionizing radiation (IR) and H2O2. Similar secreted protein expression profiles with replicatively senescent cells were seen with H2O2 treatment except for LTF and PXDN, which were increased by IR treatment. With both IR and H2O2 treatment there was a decrease in THBS1. In vivo investigation of these secreted proteins with aging was shown by significant changes in the abundance of OPG, COL1α1, IL-6, ACTα2, SERPINE 1, and THBS1 in the plasma of aged rats. This unbiased, comprehensive analysis of the changes in the MSC secretome with senescence defines the unique protein signature of the SASP in these cells and provides a better understanding of the aging bone microenvironment.