LOXL2 drives epithelial-mesenchymal transition via activation of IRE1-XBP1 branch of unfolded protein response.. Canis lupus familiaris

Epithelial-to-Mesenchymal Transition (EMT) is a key process contributing to the aggressiveness of cancer cells. EMT is triggered by activation of different transcription factors collectively known as EMT-TFs. Different cellular cues and cell signalling networks activate EMT at transcriptional and posttranscriptional level in different biological and pathological situations. Among them, overexpression of LOXL2 (lysyl oxidase-like 2) induces EMT independent of its catalytic activity. Remarkably, perinuclear/cytoplasmic accumulation of LOXL2 is a poor prognosis marker of squamous cell carcinomas and is associated to basal breast cancer metastasis by mechanisms no yet fully understood. Here, we report that overexpression of LOXL2 promotes its accumulation in the Endoplasmic Reticulum where it interacts with HSPA5 leading to activation of the IRE1-XBP1-branch of the Unfolded Protein Response (UPR). LOXL2-dependent UPR activation induces the expression of several EMT-TFs: SNAI1, SNAI2, ZEB2 and TCF3 that are direct transcriptional targets of XBP1. Remarkably, inhibition of IRE1 blocks LOXL2-dependent upregulation of EMT-TFs thus hindering EMT induction. LOXL2 relationship to Endoplasmic Reticulum Stress Overall design: Microarray experiments were performed using Human Whole Genome V2 4*44K array G4845A (Agilent technologies). Three independent passages from MDCK-II-LOXL2 and MDCK-II-DLOXL2 cells were used, and MDCK-II transfected with empty pcDNA3 vector were used as control. Total RNA was extracted and purified using RNAextraction kit (Qiagen). Microarray labelling and hybridization was performed using the Low RNA Linear Amplification Kit and the In Situ Hybridization Kit Plus (Agilent technologies), respectively, following manufacturer’s protocol. After hybridization and washing, the slides were scanned in an Axon GenePix Scanner (Axon Instruments) and analysed using Feature Extraction Software 10.0 (Agilent technologies). RNA samples from independent MDCK-II-LOXL2 and -DLOXL2-stably transfected cells were labelled with Cy5-dUTP and equal concentrations of RNA from control cells were labelled with Cy3-dUTP. Differentially expressed genes were selected using the False Discovery Rate (FDR) method with an adjusted p-value <0.2.