Abstract 261: L1-MET transcription silencing modulates MET and EGFR gene and their protein expression and induces apoptosis and cell-death in different types of cancer cells

Abstract

The activation of the LINE-1 sequence located within the second intron of MET leads to the onset of L1-MET transcript. We recently characterized the full L1-MET structure in breast cancer and showed that high levels of this transcript recognize a subset of more aggressive breast carcinomas, mainly of triple negative phenotype. However, at present, the relationship between L1-MET and MET is still poorly understood. In order to elucidate this function, we silenced L1-MET transcription using cells expressing different levels of L1-MET/MET, including lung cancer (A549 and EBC1), gastric cancer (GTL16), and breast cancer (MDA-MB231), that were transiently transfected with Gapmers-LNA (Exiqon), specifically targeting L1-MET sequence. Cell viability and apoptosis were evaluated after 24 h by cell count, Cell Titer Glow (Promega) and propidium iodide/annexin based-cytofluorimeter assays. RNA was purified from sample and control cells to assess the L1-METsilencing by qRT-PCR and to evaluate the gene-expression of a subset of cancer-related genes using Nanostring Technology, whereas western blot analyses were carried out to measure the protein expressions. A significant decrease of cell viability was detected in A549, EBC1 and GTL16, but not in MDA-MB231 cells, characterized by the lowest level of L1-MET overall. In parallel, the highly expressing L1-MET cells showed an increased rate of early and late apoptosis together with a strong reduction of MET gene and its protein expression. On the contrary, in MDA-MB231 cells L1-MET silencing induced only a slight MET gene and protein impairment. Overall, L1-MET knock-down caused a decrease expression of a conserved gene cluster, including a marked reduction of EGFR protein expression. Moreover, L1-MET silenced cells showed lower MET and EGFR phosphorylation, with a downstream silencing effect on pERK and pAKT. Results of cell treatment with the inhibitors of lysosome and proteasome activity bafilomycin and MG-132 ruled out the interaction of L1-MET silencing with protein degradation pathway. This is the first study investigating the function of the L1-MET transcript in cancer models. Our results show that although L1-MET is unable to encode for a protein, its silencing exerts a strong phenotypic effect on different tumor cell types, suggesting potential regulations at the transcriptional level. Note: This abstract was not presented at the meeting. Citation Format: Enrico Berrino, Umberto Miglio, Valentina Miano, Letizia Lanzetti, Silvia Benvenuti, Carla Debernardi, MIchele De Bortoli, Caterina Marchio, Tiziana Venesio, Anna Sapino. L1-MET transcription silencing modulates MET and EGFR gene and their protein expression and induces apoptosis and cell-death in different types of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 261.