Long non-coding RNA NORAD promotes pancreatic cancer stem cell proliferation and self-renewal by blocking microRNA-202-5p-mediated ANP32E inhibition

Abstract Background Cancer stem cells (CSCs) are key regulators in the processes of tumor initiation, progression, and recurrence. The mechanism that maintains their stemness remains enigmatic, although the role of several long noncoding RNAs (lncRNAs) has been highlighted in the pancreatic cancer stem cells (PCSCs). In this study, we first established that PCSCs overexpressing lncRNA NORAD, and then investigated the effects of NORAD on the maintenance of PCSC stemness. Methods Expression of lncRNA NORAD, miR-202-5p and ANP32E in PC tissues and cell lines was quantified after RNA isolation. Dual-luciferase reporter assay, RNA pull-down and RIP assays were performed to verify the interactions among NORAD, miR-202-5p and ANP32E. We then carried out gain- and loss-of function of miR-202-5p, ANP32E and NORAD in PANC-1 cell line, followed by measurement of the aldehyde dehydrogenase activity, cell viability, apoptosis, cell cycle distribution, colony formation, self-renewal ability and tumorigenicity of PC cells. Results LncRNA NORAD and ANP32E were upregulated in PC tissues and cells, whereas the miR-202-5p level was down-regulated. LncRNA NORAD competitively bound to miR-202-5p, and promoted the expression of the miR-202-5p target gene ANP32E thereby promoting PC cell viability, proliferation, and self-renewal ability in vitro, as well as facilitating tumorigenesis of PCSCs in vivo. Conclusion Overall, lncRNA NORAD upregulates ANP32E expression by competitively binding to miR-202-5, which accelerates the proliferation and self-renewal of PCSCs.

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A novel lncRNA ROPM-mediated lipid metabolism governs breast cancer stem cell properties

Journal of Hematology & Oncology ◽

10.1186/s13045-021-01194-z ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Shuiqing Liu ◽

Yan Sun ◽

Yixuan Hou ◽

Liping Yang ◽

Xueying Wan ◽

...

Keyword(s):

Breast Cancer ◽

Stem Cells ◽

Cancer Stem Cells ◽

Phospholipid Metabolism ◽

Luciferase Reporter ◽

Breast Cancer Patients ◽

Loss Of Function ◽

Gain And Loss

Abstract Background Cancer stem cells (CSCs) are considered as the major cause to tumor initiation, recurrence, metastasis, and drug resistance, driving poor clinical outcomes in patients. Long noncoding RNAs (lncRNAs) have emerged as crucial regulators in cancer development and progression. However, limited lncRNAs involved in CSCs have been reported. Methods The novel lncROPM (a regulator of phospholipid metabolism) in breast CSCs (BCSCs) was identified by microarray and validated by qRT-PCR in BCSCs from breast cancer cells and tissues. The clinical significance of lncROPM was evaluated in two breast cancer cohorts and TANRIC database (TCGA-BRCA, RNAseq data). Gain- and loss-of-function assays were performed to examine the role of lncROPM on BCSCs both in vitro and in vivo. The regulatory mechanism of lncROPM was investigated by bioinformatics, RNA FISH, RNA pull-down, luciferase reporter assay, and actinomycin D treatment. PLA2G16-mediated phospholipid metabolism was determined by UHPLC-QTOFMS system. Cells’ chemosensitivity was assessed by CCK8 assay. Results LncROPM is highly expressed in BCSCs. The enhanced lncROPM exists in clinic breast tumors and other solid tumors and positively correlates with malignant grade/stage and poor prognosis in breast cancer patients. Gain- and loss-of-function studies show that lncROPM is required for the maintenance of BCSCs properties both in vitro and in vivo. Mechanistically, lncROPM regulates PLA2G16 expression by directly binding to 3'-UTR of PLA2G16 to increase the mRNA stability. The increased PLA2G16 significantly promotes phospholipid metabolism and the production of free fatty acid, especially arachidonic acid in BCSCs, thereby activating PI3K/AKT, Wnt/β-catenin, and Hippo/YAP signaling, thus eventually involving in the maintenance of BCSCs stemness. Importantly, lncROPM and PLA2G16 notably contribute to BCSCs chemo-resistance. Administration of BCSCs using clinic therapeutic drugs such as doxorubicin, cisplatin, or tamoxifen combined with Giripladib (an inhibitor of cytoplasmic phospholipase A2) can efficiently eliminate BCSCs and tumorigenesis. Conclusions Our study highlights that lncROPM and its target PLA2G16 play crucial roles in sustaining BCSC properties and may serve as a biomarker for BCSCs or other cancer stem cells. Targeting lncROPM-PLA2G16 signaling axis may be a novel therapeutic strategy for patients with breast cancer.

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Long noncoding RNA LINC00261 upregulates ITIH5 to impair tumorigenic ability of pancreatic cancer stem cells

Cell Death Discovery ◽

10.1038/s41420-021-00575-0 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Lijuan Zou ◽

Hengpeng He ◽

Zhiguo Li ◽

Ou Chen ◽

Xiukun Jia ◽

...

Keyword(s):

Pancreatic Cancer ◽

Stem Cells ◽

Cell Proliferation ◽

Drug Resistance ◽

Cancer Stem Cells ◽

Noncoding Rna ◽

Cell Subset ◽

Luciferase Reporter ◽

Stem Cell Markers ◽

Self Renewal

AbstractLong noncoding RNAs (lncRNAs) are implicated tumor development in a range of different cancers, including pancreatic cancer (PC). Cancer stem cells (CSCs), a drug-resistant cancer cell subset, drive tumor progression in PC. In this work, we aimed to investigate the mechanism by which lncRNA LINC00261 affects the biological functions of CSCs during the progression of PC. Microarray analysis of differentially expressed genes and lncRNAs suggested that LINC00261 is downregulated in PC. Both LINC00261 and ITIH5 were confirmed to be downregulated in PC cells and PC stem cells. Gain-of-function and loss-of-function investigations were performed to analyze their effects on cell proliferation, drug resistance, cell cycle distribution, self-renewal, invasion, and ultimately overall tumorigenicity. These experiments revealed that the expression of stem cell markers was reduced, and cell proliferation, self-renewal ability, cell invasion, drug resistance, and tumorigenicity were all suppressed by upregulation of LINC00261 or ITIH5. The results of dual-luciferase reporter gene, ChIP, and RIP assays indicated that LINC00261 binds directly to GATA6, increasing its activity at the ITIH5 promoter. The presence of LINC00261 and GATA6 inhibited the self-renewal and tumorigenesis of PC stem cells, while silence of ITIH5 rescued those functions. Collectively, this study identifies the tumor suppressive activity of LINC00261 in PC, showing that this lncRNA limits the functions of PC stem through an ITIH5/GATA6 regulatory pathway.

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Transcriptome Profiling of Panc-1 Spheroid Cells with Pancreatic Cancer Stem Cells Properties Cultured by a Novel 3D Semi-Solid System

Cellular Physiology and Biochemistry ◽

10.1159/000491479 ◽

2018 ◽

Vol 47 (5) ◽

pp. 2109-2125 ◽

Cited By ~ 6

Author(s):

Zhaocong Yang ◽

Yanfeng Zhang ◽

Tingting Tang ◽

Qinhua Zhu ◽

Wanyue Shi ◽

...

Keyword(s):

Pancreatic Cancer ◽

Stem Cells ◽

Cancer Stem Cells ◽

Culture System ◽

High Throughput Sequencing ◽

Semi Solid ◽

Pancreatic Cancer Stem Cells ◽

Solid System

Background/Aims: Pancreatic cancer remains one of the deadliest human malignancies, the lethality of which may be attributed to the presence of pancreatic cancer stem cells (PCSCs), a small subpopulation of cells existing within pancreatic tumor with high carcinogenesis. Therefore, it is crucial to establish an efficient enrichment and culture system of PCSCs and identify the key genes involved in the regulation of PCSCs. The three-dimensional (3D) liquid suspension mammosphere culture system has been established for enrichment and culture of PCSCs in vitro as the cell spheres are likely to originate from individual cell clone, but it has been challenged because the cell spheroids could be a result of cell aggregation. Methods: We optimized the existing culture system by adding methylcellulose to create a 3D semi-solid system which prevented the non-specific aggregation. Then we identified the CSC properties of Panc-1 spheroid cells cultured by this system by detecting the genes associated with stemness and by evaluation of the tumorigenicity in vitro and in vivo through invasion, migration and xenograft experiments methods. Subsequently, we performed high-throughput sequencing (HTS) of the Panc-1 spheroid cells. Results: We confirmed the PCSCs properties and high malignancy of the Panc-1 spheroid cells enriched by our novel 3D semi-solid system both in vitro and in vivo. Hundreds of mRNA, microRNA (miRNA) and dozens of long non-coding RNA (LncRNA) were identified to be differentially regulated in PCSCs-like Panc-1 spheroid cells compared with their parental cells by HTS. Conclusions: Our results demonstrate an efficient enrichment and culture system for Panc-1 spheroid cells with the PCSCs properties. The differentially expressed genes and their targets identified by the HTS of the Panc-1 spheroid cells can serve as new potential biomarkers for pancreatic cancer diagnosis and targeted therapy.

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MicroRNA-28-5p Regulates Liver Cancer Stem Cell Expansion via IGF-1 Pathway

Stem Cells International ◽

10.1155/2019/8734362 ◽

2019 ◽

Vol 2019 ◽

pp. 1-16 ◽

Cited By ~ 2

Author(s):

Qing Xia ◽

Tao Han ◽

Pinghua Yang ◽

Ruoyu Wang ◽

Hengyu Li ◽

...

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Liver Cancer ◽

Critical Role ◽

Self Renewal ◽

Sorafenib Treatment ◽

The Impact

Background. MicroRNAs (miRNAs) play a critical role in the regulation of cancer stem cells (CSCs). However, the role of miRNAs in liver CSCs has not been fully elucidated. Methods. Real-time PCR was used to detect the expression of miR-miR-28-5p in liver cancer stem cells (CSCs). The impact of miR-28-5p on liver CSC expansion was investigated both in vivo and in vitro. The correlation between miR-28-5p expression and sorafenib benefits in HCC was further evaluated in patient-derived xenografts (PDXs). Results. Our data showed that miR-28-5p was downregulated in sorted EpCAM- and CD24-positive liver CSCs. Biofunctional investigations revealed that knockdown miR-28-5p promoted liver CSC self-renewal and tumorigenesis. Consistently, miR-28-5p overexpression inhibited liver CSC’s self-renewal and tumorigenesis. Mechanistically, we found that insulin-like growth factor-1 (IGF-1) was a direct target of miR-28-5p in liver CSCs, and the effects of miR-28-5p on liver CSC’s self-renewal and tumorigenesis were dependent on IGF-1. The correlation between miR-28-5p and IGF-1 was confirmed in human HCC tissues. Furthermore, the miR-28-5p knockdown HCC cells were more sensitive to sorafenib treatment. Analysis of patient-derived xenografts (PDXs) further demonstrated that the miR-28-5p may predict sorafenib benefits in HCC patients. Conclusion. Our findings revealed the crucial role of the miR-28-5p in liver CSC expansion and sorafenib response, rendering miR-28-5p an optimal therapeutic target for HCC.

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Chemosensitization of prostate cancer stem cells in mice by angiogenin and plexin-B2 inhibitors

Communications Biology ◽

10.1038/s42003-020-0750-6 ◽

2020 ◽

Vol 3 (1) ◽

Cited By ~ 3

Author(s):

Shuping Li ◽

Kevin A. Goncalves ◽

Baiqing Lyu ◽

Liang Yuan ◽

Guo-fu Hu

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Ribosomal Rna ◽

Protein Translation ◽

Chemotherapeutic Agents ◽

Self Renewal ◽

5S Ribosomal Rna ◽

Prostate Cancer Stem Cells

AbstractCancer stem cells (CSCs) are an obstacle in cancer therapy and are a major cause of drug resistance, cancer recurrence, and metastasis. Available treatments, targeting proliferating cancer cells, are not effective in eliminating quiescent CSCs. Identification of CSC regulators will help design therapeutic strategies to sensitize drug-resistant CSCs for chemo-eradication. Here, we show that angiogenin and plexin-B2 regulate the stemness of prostate CSCs, and that inhibitors of angiogenin/plexin-B2 sensitize prostate CSCs to chemotherapy. Prostate CSCs capable of self-renewal, differentiation, and tumor initiation with a single cell inoculation were identified and shown to be regulated by angiogenin/plexin-B2 that promotes quiescence and self-renewal through 5S ribosomal RNA processing and generation of the bioactive 3′-end fragments of 5S ribosomal RNA, which suppress protein translation and restrict cell cycling. Monoclonal antibodies of angiogenin and plexin-B2 decrease the stemness of prostate CSCs and sensitize them to chemotherapeutic agents in vitro and in vivo.

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IL-17B/IL-17RB signaling cascade contributes to self-renewal and tumorigenesis of cancer stem cells by regulating Beclin-1 ubiquitination

Oncogene ◽

10.1038/s41388-021-01699-4 ◽

2021 ◽

Author(s):

Qingli Bie ◽

Hui Song ◽

Xinke Chen ◽

Xiao Yang ◽

Shuo Shi ◽

...

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Molecular Mechanisms ◽

Tumor Necrosis Factor Receptor ◽

Beclin 1 ◽

Signaling Cascade ◽

Interleukin 17 ◽

Self Renewal

AbstractCancer stem cells (CSCs) are characterized by robust self-renewal and tumorigenesis and are responsible for metastasis, drug resistance, and angiogenesis. However, the molecular mechanisms for the regulation of CSC homeostasis are incompletely understood. This study demonstrated that the interleukin-17 (IL-17)B/IL-17RB signaling cascade promotes the self-renewal and tumorigenesis of CSCs by inducing Beclin-1 ubiquitination. We found that IL-17RB expression was significantly upregulated in spheroid cells and Lgr5-positive cells from the same tumor tissues of patients with gastric cancer (GC), which was closely correlated with the degree of cancer cell differentiation. Recombinant IL-17B (rIL-17B) promoted the sphere-formation ability of CSCs in vitro and enhanced tumor growth and metastasis in vivo. Interestingly, IL-17B induced autophagosome formation and cleavage-mediated transformation of LC3 in CSCs and 293T cells. Furthermore, inhibition of autophagy activation by ATG7 knockdown reversed rIL-17B-induced self-renewal of GC cells. In addition, we showed that IL-17B also promoted K63-mediated ubiquitination of Beclin-1 by mediating the binding of tumor necrosis factor receptor-associated factor 6 to Beclin-1. Silencing IL-17RB expression abrogated the effects of IL-17B on Beclin-1 ubiquitination and autophagy activation in GC cells. Finally, we showed that IL-17B level in the serum of GC patients was positively correlated with IL-17RB expression in GC tissues, and IL-17B could induce IL-17RB expression in GC cells. Overall, the results elucidate the novel functions of IL-17B for CSCs and suggest that the intervention of the IL-17B/IL-17RB signaling pathway may provide new therapeutic targets for the treatment of cancer.

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Lgr5+CD44+EpCAM+ Strictly Defines Cancer Stem Cells in Human Colorectal Cancer

Cellular Physiology and Biochemistry ◽

10.1159/000488743 ◽

2018 ◽

Vol 46 (2) ◽

pp. 860-872 ◽

Cited By ~ 16

Author(s):

Zhengwei Leng ◽

Qinghua Xia ◽

Jinhuang Chen ◽

Yong Li ◽

Jiqian Xu ◽

...

Keyword(s):

Colorectal Cancer ◽

Stem Cells ◽

Cancer Stem Cells ◽

Colony Formation ◽

Epithelial Mesenchymal Transition ◽

Tumor Xenograft ◽

Self Renewal ◽

Mesenchymal Transition

Background/Aims: Although EpCAM+CD44+ cells exhibit more stem-like properties than did EpCAM-CD44- cells, the specificity of EpCAM combined with CD44 in defining CSCs needs further improvement. Lgr5 is used as a biomarker to isolate cancer stem cells (CSCs) in colorectal cancer. However, it remains unclear whether Lgr5, along with EpCAM and CD44, can further identify and define CSCs in colorectal cancer. Methods: Lgr5+CD44+EpCAM+, Lgr5+CD44+EpCAM-, Lgr5+CD44-EpCAM+, Lgr5-CD44+EpCAM+, and Lgr5-CD44-EpCAM-cells were separately isolated using fluorescence-activated cell sorting (FACS). Colony formation, self-renewal, differentiation, and tumorigenic properties of these cells were investigated through in vitro experiments and in vivo tumor xenograft models. The expression of stemness genes and CSC- and epithelial-mesenchymal transition (EMT)-related genes, such as KLF4, Oct4, Sox2, Nanog, CD133, CD44, CD166, ALDH1, Lgr5, E-cadherin, ZO-1, Vimentin, Snail, Slug, and Twist, was examined using real-time PCR. Results: Lgr5-positive subpopulations exhibited higher capacities for colony formation, self-renewal, differentiation, and tumorigenicity as well as higher expression of stemness genes and mesenchymal genes and lower expression of epithelial genes than did Lgr5-negative subpopulations. Conclusion: Our data revealed that tumorigenic cells were highly restricted to Lgr5-positive subpopulations. Most importantly, Lgr5+CD44+EpCAM+ cells exhibited more pronounced CSC-like traits than did any other subpopulation, indicating that Lgr5 combined with CD44 and EpCAM can further improve the stem-like traits of CSCs in colorectal cancer.

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Targeting Lung Cancer Stem Cells with Antipsychological Drug Thioridazine

BioMed Research International ◽

10.1155/2016/6709828 ◽

2016 ◽

Vol 2016 ◽

pp. 1-7 ◽

Cited By ~ 14

Author(s):

Haiying Yue ◽

Dongning Huang ◽

Li Qin ◽

Zhiyong Zheng ◽

Li Hua ◽

...

Keyword(s):

Lung Cancer ◽

Cell Cycle ◽

Stem Cells ◽

Cancer Stem Cells ◽

Cancer Progression ◽

Cell Cycle Distribution ◽

Lung Cancer Stem Cells ◽

Sphere Formation

Lung cancer stem cells are a subpopulation of cells critical for lung cancer progression, metastasis, and drug resistance. Thioridazine, a classical neurological drug, has been reported with anticancer ability. However, whether thioridazine could inhibit lung cancer stem cells has never been studied. In our current work, we used different dosage of thioridazine to test its effect on lung cancer stem cells sphere formation. The response of lung cancer stem cells to chemotherapy drug with thioridazine treatment was measured. The cell cycle distribution of lung cancer stem cells after thioridazine treatment was detected. The in vivo inhibitory effect of thioridazine was also measured. We found that thioridazine could dramatically inhibit sphere formation of lung cancer stem cells. It sensitized the LCSCs to chemotherapeutic drugs 5-FU and cisplatin. Thioridazine altered the cell cycle distribution of LCSCs and decreased the proportion of G0 phase cells in lung cancer stem cells. Thioridazine inhibited lung cancer stem cells initiated tumors growth in vivo. This study showed that thioridazine could inhibit lung cancer stem cells in vitro and in vivo. It provides a potential drug for lung cancer therapy through targeting lung cancer stem cells.

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A Novel Therapeutic Approach for Colorectal Cancer Stem Cells: Blocking the PI3K/Akt Signaling Axis With Caffeic Acid

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.585987 ◽

2020 ◽

Vol 8 ◽

Author(s):

Se-Ra Park ◽

Soo-Rim Kim ◽

In-Sun Hong ◽

Hwa-Yong Lee

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Caffeic Acid ◽

Akt Signaling ◽

Chemotherapeutic Agents ◽

Cancer Therapies ◽

Self Renewal ◽

Colorectal Cancer Stem Cells

Cancer stem cells (CSCs) have been identified in a multiple of cancer types and resistant to traditional cancer therapies such as chemotherapeutic agents and radiotherapy, which may destroy bulk tumor cells but not all CSCs, contributing to reformation tumor masses and subsequent relapse. Moreover, it is very difficult to effectively identify and eliminate CSCs because they share some common phenotypic and functional characteristics of normal stem cells. Therefore, finding better therapeutic strategies to selectively target CSCs might be helpful to reduce subsequent malignancies. In the present study, we found that caffeic acid effectively suppresses self-renewal capacity, stem-like characteristics, and migratory capacity of CD44+ and CD133+ colorectal CSCs in vitro and in vivo. In addition, we also revealed that PI3K/Akt signaling may be linked to multiple colorectal CSC-associated characteristics, such as radio-resistance, stem-like property, and tumorigenic potential. To the best of our knowledge, this is the first study demonstrating that caffeic acid effectively targets colorectal CSC populations by inhibiting the growth and/or self-renewal capacity of colorectal CSCs through PI3K/Akt signaling in vitro and in vivo.

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LncRNA ZNFTR functions as an inhibitor in pancreatic cancer by modulating ATF3/ZNF24/VEGFA pathway

Cell Death and Disease ◽

10.1038/s41419-021-04119-3 ◽

2021 ◽

Vol 12 (9) ◽

Author(s):

Wei Li ◽

Shengbo Han ◽

Ping Hu ◽

Ding Chen ◽

Zhu Zeng ◽

...

Keyword(s):

Pancreatic Cancer ◽

Zinc Finger Protein ◽

Clinical Results ◽

Clinicopathological Characteristics ◽

Luciferase Reporter ◽

Activating Transcription Factor 3 ◽

Loss Of Function ◽

Poor Overall Survival

AbstractThe majority of long non-coding RNAs (lncRNAs) have been discovered to be overexpressed in pancreatic cancer (PC) and served as promoters in the tumorigenesis of PC, while the inhibitory functions of lncRNAs in the development of PC have not been fully elucidated yet. LncRNA microarray was adopted to analyze the differential expression of lncRNAs in PC tissues and that in normal peritumoral (NP) tissues. Functional role of lncRNA BM466146.1 on PC was evaluated by gain- and loss-of-function experiments in vivo and in vitro. RNA pull-down, RNA immunoprecipitation, luciferase reporter, and Chromatin-immunoprecipitation assays were performed to assess the mechanism of ZNFTR, respectively. The correlation between the expression of ZNFTR and various clinicopathological characteristics was accessed in PC specimens. This study displayed lncRNA BM466146.1 was downregulated in PC tissues and functioned as a suppressor through regulating the expression of adjacent gene Zinc finger protein 24 (ZNF24), which was assigned as ZNFTR. Mechanistically, ZNFTR interacted with activating transcription factor 3 (ATF3) and sequestered ATF3 away from the ZNF24 promoter, which consequently increased the expression of ZNF24. Further, ZNF24 inhibited the proliferative, metastatic, and pro-angiogenic abilities of PC cells by suppressing transcription of vascular endothelial growth factor A (VEGFA). Therefore, the downregulation of ZNFTR in PC led to the decreased expression of ZNF24, which further resulted in the upregulation of VEGFA to facilitate the development of PC. Meanwhile, ZNFTR was transcriptionally inhibited by the HIF-1α/HDAC1 complex-mediated deacetylation. Clinical results further demonstrated that the low expression of ZNFTR was associated with poor overall survival time. Taken together, our results implicated that ZNFTR was a hypoxia-responsive lncRNA, and functioned as an inhibitor by modulating ATF3/ZNF24/VEGFA pathway in PC.

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