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Tinzaparin inhibits VL30 retrotransposition induced by oxidative stress and/or VEGF in HC11 mouse progenitor mammary cells: Association between inhibition of cancer stem cell proliferation and mammosphere disaggregation.

ABSTRACT: Tinzaparin is an anticoagulant and antiangiogenic drug with inhibitory properties against tumor growth. VEGF stimulates angiogenesis, while an association between reactive oxygen species (ROS) and angiogenesis is involved in tumor progression. The present study aimed to investigate the effect of tinzaparin on VL30 retrotransposition‑positive mouse HC11 mammary stem‑like epithelial cells, previously reported to be associated with induced mammosphere/cancer stem cell (CSC) generation and tumorigenesis. Under 24 h serum starvation, 15.2% nominal retrotransposition frequency was increased to 29%. Additionally, while treatment with 3‑12 ng/ml VEGF further induced retrotransposition frequency in a dose‑dependent manner (up to 40.3%), pre‑incubation with tinzaparin (2 IU/ml) for 0.5‑4 h reduced this frequency to 18.3% in a time‑dependent manner, confirmed by analogous results in NIH3T3 fibroblasts. Treatment with 10‑40 pg/ml glucose oxidase (GO) for 24 h induced HC11 cell retrotransposition in a dose‑dependent manner (up to 82.5%), while a 3 h pre‑incubation with tinzaparin (1 or 2 IU/ml) elicited a 13.5 or 25.5% reduction in retrotransposition, respectively. Regarding tumorigenic VL30 retrotransposition‑positive HC11 cells, treatment with 2 IU/ml tinzaparin for 5 days reduced proliferation rate in a time‑dependent manner (up to ~55%), and after 3 weeks, disaggregated soft agar‑formed foci, as well as low‑adherent mammospheres, producing single mesenchymal‑like cells with a ~50% reduced retrotransposition. With respect to the VL30 retrotransposition mechanism: While 12 ng/ml VEGF increased the level of VL30 and endogenous reverse transcriptase (enRT) transcripts ~1.41‑ and ~1.16‑fold, respectively, subsequent tinzaparin treatment reduced both endogenous/ROS‑ and VEGF‑induced levels 1.15‑ and 0.40‑fold (VL30) and 0.60‑ and 0.52‑fold (enRT), respectively. To the best of our knowledge, these data demonstrate for the first time, the novel inhibition activity of tinzaparin against ROS‑ and VEGF‑induced VL30 retrotransposition, and the proliferation and/or aggregation of mouse HC11 mammosphere/tumor‑initiating CSCs, thus contributing to the inhibition of VL30 retrotransposition‑induced primary tumor growth.

SUBMITTER: Mantziou S

PROVIDER: S-EPMC8485018 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Retrotransposons copy their sequences via an RNA intermediate, followed by reverse transcription into cDNA and random insertion, into a new genomic locus. New retrotransposon copies may lead to cell transformation and/or tumorigenesis through insertional mutagenesis. Methylation is a major defense mechanism against retrotransposon RNA expression and retrotransposition in differentiated cells, whereas stem cells are relatively hypo?methylated. Epithelial?to?mesenchymal transition (EMT), which transforms normal epithelial cells into mesenchymal?like cells, also contributes to tumor progression and tumor metastasis. Cancer stem cells (CSCs), a fraction of undifferentiated tumor?initiating cancer cells, are reciprocally related to EMT. In the present study, the outcome of long terminal repeat (LTR)?Viral?Like 30 (VL30) retrotransposition was examined in mouse mammary stem?like/progenitor HC11 epithelial cells. The transfection of HC11 cells with a VL30 retrotransposon, engineered with an EGFP?based retrotransposition cassette, elicited a higher retrotransposition frequency in comparison to differentiated J3B1A and C127 mouse mammary cells. Fluorescence microscopy and PCR analysis confirmed the specificity of retrotransposition events. The differentiated retrotransposition?positive cells retained their epithelial morphology, while the respective HC11 cells acquired mesenchymal features associated with the loss of E?cadherin, the induction of N?cadherin, and fibronectin and vimentin protein expression, as well as an increased transforming growth factor (TGF)??1, Slug, Snail?1 and Twist mRNA expression. In addition, they were characterized by cell proliferation in low serum, and the acquisition of CSC?like properties indicated by mammosphere formation under anchorage?independent conditions. Mammospheres exhibited an increased Nanog and Oct4 mRNA expression and a CD44+/CD24?/low antigenic phenotype, as well as self?renewal and differentiation capacity, forming mammary acini?like structures. DNA sequencing analysis of retrotransposition?positive HC11 cells revealed retrotransposed VL30 copies integrated at the vicinity of EMT?, cancer type? and breast cancer?related genes. The inoculation of these cells into Balb/c mice produced cytokeratin?positive tumors containing pancytokeratin?positive cells, indicative of cell invasion features. On the whole, the findings of the present study demonstrate, for the first time, to the best of our knowledge, that stem?like epithelial HC11 cells are amenable to VL30 retrotransposition associated with the induction of EMT and CSC generation, leading to tumorigenesis.

Project description:The development of mammary gland as a lactogenic tissue is a highly coordinated multistep process. The epithelial cells of lactiferous tubules undergo profound changes during the developmental window of puberty, pregnancy, and lactation. Several hormones including estrogen, progesterone, glucocorticoids and prolactin act in concert, and orchestrate the development of mammary gland. Understanding the gene regulatory networks that coordinate proliferation and differentiation of HC11 Mammary Epithelial stem-like Cells (MEC) under the influence of lactogenic hormones is critical for elucidating the mechanism of lactogenesis in detail. In this study, we analyzed transcriptome profiles of undifferentiated MEC (normal) and compared them with Murine Embryonic Stem Cells (ESC) using next-generation mRNA sequencing. Further, we analyzed the transcriptome output during lactogenic differentiation of MEC following treatment with glucocorticoids (primed state) and both glucocorticoids and prolactin together (prolactin state). We established stage-specific gene regulatory networks in ESC and MEC (normal, priming and prolactin states). We validated the top up-and downregulated genes in each stage of differentiation of MEC by RT-PCR and found that they are comparable with that of RNA-seq data. HC11 MEC display decreased expression of Pou5f1 and Sox2, which is crucial for the differentiation of MEC, which otherwise ensure pluripotency to ESC. Cited4 is induced during priming and is involved in milk secretion. MEC upon exposure to both glucocorticoids and prolactin undergo terminal differentiation, which is associated with the expression of several genes, including Xbp1 and Cbp that are required for cell growth and differentiation. Our study also identified differential expression of transcription factors and epigenetic regulators in each stage of lactogenic differentiation. We also analyzed the transcriptome data for the pathways that are selectively activated during lactogenic differentiation. Further, we found that selective expression of chromatin modulators (Dnmt3l, Chd9) in response to glucocorticoids suggests a highly coordinated stage-specific lactogenic differentiation of MEC.

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Tinzaparin inhibits VL30 retrotransposition induced by oxidative stress and/or VEGF in HC11 mouse progenitor mammary cells: Association between inhibition of cancer stem cell proliferation and mammosphere disaggregation.

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