Line-1 Couples Epithelial-Mesenchymal Transition Programming With the Acquisition of Oncogenic Phenotypes in Human Bronchial Epithelial Cells

Abstract

Lung cancer has the highest cancer-related mortality in the United States; there is an imminent need to identify novel molecular targets that can help improve the precision of current therapies. Recent studies have implicated the long interspersed nuclear element-1 (L1) in the onset and progression of lung cancers, but the molecular bases of this response remain largely unknown. L1 is an abundant and highly polymorphic genetic element that mobilizes via retrotransposition using L1-encoded ORF1p and ORF2p proteins. L1 expression is tightly regulated in somatic tissues and reactivated by tobacco carcinogens, such as benzo(a)pyrene (BaP). Its mobilization to new sites throughout the genome serves to globally regulate genetic structure and function and has been associated with epithelial-to-mesenchymal transition (EMT), a key event in malignant transformation which has been associated with chemoresistance and invasive phenotypes leading to metastasis. Therefore, to assess whether L1 induces EMT, chemoresistance, and invasive phenotypes involved in lung cancer progression and metastatic conversion, non-malignant, human bronchial epithelial BEAS-2B cells were stably transfected with vectors that constitutively expressed wildtype L1; a mutant counterpart that lacked reverse transcriptase activity, and was therefore unable to retrotranspose (mutant L1); or empty vector as a control. Immunoblotting showed that cells expressing L1 and mutant L1 proteins exhibited increased expression of mesenchymal markers coupled with decreased expression of epithelial markers compared to control cells. These changes in markers were seen in the cell lines transfected with both L1 and mutant L1, which is incapable of retrotransposition. Remarkably, blockade of L1 production with siRNA in cells constitutively expressing L1 and mutant L1 resulted in regression to a more epithelial phenotype. Furthermore, our data showed that cells transfected with L1 have increased survival rates when exposed to tyrosine kinase inhibitor sunitinib-malate. Finally, in-vivo experiments in which nu/nu mice were injected with cell lines constitutively expressing either L1 or mutant L1 resulted in tumor formation in both populations of mice, not seen in control mice. Based on these findings, we conclude that: 1) L1 alters non-malignant bronchial epithelial cell plasticity to induce EMT phenotypes via retrotransposition-independent mechanisms, 2) L1-mediated changes are partially reversible, 3) L1-mediated EMT may contribute to lung cancer progression by promotion of chemoresistance, and 4) tumorigenesis induced by L1 is mediated by both retrotransposition-competent and retrotransposition-deficient L1. These findings suggest that the retrotransposition-independent pathway of L1 is sufficient for cellular transformation, and that retrotransposition events are not necessary for L1-induced cellular transformation in the BEAS-2B cell line.