ABSTRACT: 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.
