Murine hepatocellular carcinoma derived stem cells reveal epithelial-to-mesenchymal plasticity

Jayachandran, Aparna; Shrestha, Ritu; Dhungel, Bijay; Huang, I-Tao; Vasconcelos, Maria Helena Ferreira; Morrison, Brian J.; Ramlogan‐Steel, Charmaine A.; Steel, Jason C.

doi:10.4252/wjsc.v9.i9.159

Cited by 13 publications

(21 citation statements)

References 35 publications

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“…CSCs exhibit biological traits of the epithelial-to mesenchymal transition (EMT) [24, 25]. EMT is involved in various pathological processes including tumor development [26, 27].…”

Section: Introductionmentioning

confidence: 99%

Tobacco smoke induced hepatic cancer stem cell-like properties through IL-33/p38 pathway

Xie

Zhu

Wang

et al. 2019

J Exp Clin Cancer Res

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BackgroundTobacco smoke (TS) critically contributes to the development of hepatocellular carcinoma. Cancer stem cells (CSCs) induced by TS is an early event in the initiation of carcinogenesis. Tumor specific microenvironment including inflammatory factors is key mediator for maintaining the stemness of CSCs through various pathways such as p38 MAPK. However, the mechanisms of inflammatory factors in TS-induced acquisition of liver CSCs properties remain undefined. The aim of this study was to investigate the role of IL-33/p38 axis in long term TS-induced acquisition of hepatic CSCs properties in mouse liver tissues and human liver cells.MethodsBALB/c mice were exposed to TS for 12 weeks, along with or without 1 mg/kg SB203580 (p38 inhibitors) treatment. Histopathological analysis, alterations in the levels of IL-33, liver CSCs markers, EMT-like changes and p38 MAPK activation in liver tissues of mice were analyzed by immunohistochemical staining, immunofluorescence assay and Western blot analysis. Moreover, LO2 immortalized human liver cells were exposed to cigarette smoke extract (CSE) and the tumorsphere formation ability was determined. LO2 cells were further treated with IL-33 or CSE and the expression of phosphorylated p38, liver CSCs markers and EMT-related proteins was examined.ResultsLong term TS exposure increased the levels of CSCs markers, induced epithelial-to mesenchymal transition (EMT) and inflammatory factor IL-33 expression. Moreover, we showed that p38 MAPK modulated TS-stimulated hepatic CSC-like properties, as evidenced by the findings that long term TS exposure activated p38, and that TS-induced stemness was abolished by p38 inhibition. In addition, data from in vitro model showed that similar to cigarette smoke extract (CSE), IL-33 treatment promoted the activation of p38, increased the levels of liver CSCs markers expression and EMT-like changes.ConclusionsCollectively, these data suggested that IL-33/p38 axis plays an important role in long term TS exposure-induced acquisition of hepatic CSC-like properties.

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“…CSCs exhibit biological traits of the epithelial-to mesenchymal transition (EMT) [24, 25]. EMT is involved in various pathological processes including tumor development [26, 27].…”

Section: Introductionmentioning

confidence: 99%

Tobacco smoke induced hepatic cancer stem cell-like properties through IL-33/p38 pathway

Xie

Zhu

Wang

et al. 2019

J Exp Clin Cancer Res

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“…These heterogeneous 3D tumor-like cultures have also been shown to enrich for cancer stem cells. 16 Given the reported tumor-suppressing roles of miRNA199a and its downregulation in cancer stem cells for other cancer types, we first investigated whether maintaining Hepa1-6 cells as 3D tumorspheres could enrich for classic cancer stem cell markers. We quantified the levels of expression of HCC stemness markers CD44 ( Figure 6 B), CD133 ( Figure 6 C), and Oct4 ( Figure 6 D) in these tumorspheres and observed a significant upregulation of these genes when compared to 2D-grown Hepa1-6 cells (p < 0.05).…”

Section: Resultsmentioning

confidence: 99%

MicroRNA199a-Based Post-transcriptional Detargeting of Gene Vectors for Hepatocellular Carcinoma

Dhungel¹,

Ramlogan‐Steel²,

Layton³

et al. 2018

Molecular Therapy - Nucleic Acids

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A gene therapeutic platform needs to be both efficient and safe. The criterion of safety is particularly important for diseases like hepatocellular carcinoma (HCC), which develop in a background of an already compromised liver. Gene vectors can be constructed either by targeting HCC or by detargeting liver and/or other major organs. miRNA-based negative detargeting has gained considerable attention in recent times due to its effectiveness and the ease with which it can be adapted into current gene delivery vectors. In this study, we provide a proof-of-concept using miRNA199a as a negative targeting agent. We introduced vectors harboring reporters with miRNA199a binding sites in cells expressing high endogenous levels of miRNA199a and compared the reporter expression in HCC cells with low endogenous miRNA199a. We observed that the expression of reporters with miRNA199a binding sites is significantly inhibited in miRNA199a-positive cells, whereas minimal effect was observed in miRNA199a-negative HCC cells. In addition, we created a post-transcriptionally regulated suicide gene therapeutic system based on cytosine deaminase (CD)/5-fluorocytosine (5-FC) exploiting miRNA199a binding sites and observed significantly lower cell death for miRNA199a-positive cells. Furthermore, we observed a decrease in the levels of miRNA199 in 3D tumorspheres of miRNA199a-positive Hepa1-6 cells and a reduction in the inhibition of reporter expression after transfection in these 3D models when compared with 2D Hepa1-6 cells. In summary, we provide evidence of miRNA199a-based post-transcriptional detargeting with relevance to HCC gene therapy.

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“…After establishing the efficacy of miRNA122a binding site based targeting of HCC, we examined whether a similar targeting approach could be used to direct transgene expression to hepatocellular CSCs. To do this we utilized 3D tumorspheres cultures of HuH7 to enrich for CSCs [ 34 ] and showed increases in stemness markers and a concurrent downregulation of miRNA122a. The CSC enriched cultures were readily transfected with luciferase and eGFP vectors with or with the miRNA122a binding sites and showed enhanced transgene expression in the CSCs compared to the non-enriched, miRNA122a expressing cultures.…”

Section: Discussionmentioning

confidence: 99%

miRNA122a regulation of gene therapy vectors targeting hepatocellular cancer stem cells

et al. 2018

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In this study, we report a miRNA122a based targeted gene therapy for hepatocellular cancer stem cells (CSCs). First, we assessed the levels of miRNA122a in normal human hepatocytes, a panel of hepatocellular carcinoma (HCC) cell lines and hepatocellular CSCs observing its significant downregulation in HCC and CSCs. The miRNA122a binding site was then incorporated at the 3’-UTR of reporter genes gaussia luciferase (GLuc) and eGFP which resulted in significant hepatocyte detargeting. Using this strategy for the delivery of gene directed enzyme prodrug therapy (GDEPT) utilizing the cytosine deaminase/5-fluorocytosine (CD/5-FC) system, we showed significant killing in cells with low or no miRNA122a while those cells, such as hepatocytes with high miRNA122a were largely spared. Next, we showed that CSC enriched tumorspheres exhibit a significant downregulation of miRNA122a expression providing a rational to exploit its binding site for targeted gene delivery. Using plasmids harboring reporters GLuc and eGFP with or without miR122a binding sites, we showed high reporter expression in the CSCs and little reported expression in the non-enriched cultures. Finally, we demonstrate the efficacy of miRNA122a based post-transcriptionally targeted GDEPT for hepatocellular CSCs.

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Murine hepatocellular carcinoma derived stem cells reveal epithelial-to-mesenchymal plasticity

Cited by 13 publications

References 35 publications

Tobacco smoke induced hepatic cancer stem cell-like properties through IL-33/p38 pathway

Tobacco smoke induced hepatic cancer stem cell-like properties through IL-33/p38 pathway

MicroRNA199a-Based Post-transcriptional Detargeting of Gene Vectors for Hepatocellular Carcinoma

miRNA122a regulation of gene therapy vectors targeting hepatocellular cancer stem cells

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