Basal‐like breast carcinoma is characterized by the expression of basal/myoepithelial markers, undifferentiated phenotype, highly aggressive behaviour and frequent triple negative status (ESR−, PR−, Her2neu−). We have previously shown that epithelial–mesenchymal transition (EMT) occurs in basal‐like breast tumours and identified Lysyl‐oxidase‐like 2 (LOXL2) as an EMT player and poor prognosis marker in squamous cell carcinomas. We now show that LOXL2 mRNA is overexpressed in basal‐like human breast carcinomas. Breast carcinoma cell lines with basal‐like phenotype show a specific cytoplasmic/perinuclear LOXL2 expression, and this subcellular distribution is significantly associated with distant metastatic incidence in basal‐like breast carcinomas. LOXL2 silencing in basal‐like carcinoma cells induces a mesenchymal‐epithelial transition (MET) associated with a decrease of tumourigenicity and suppression of metastatic potential. Mechanistic studies indicate that LOXL2 maintains the mesenchymal phenotype of basal‐like carcinoma cells by a novel mechanism involving transcriptional downregulation of Lgl2 and claudin1 and disorganization of cell polarity and tight junction complexes. Therefore, intracellular LOXL2 is a new candidate marker of basal‐like carcinomas and a target to block metastatic dissemination of this aggressive breast tumour subtype.

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 signalling pathway of the ER-stress response. LOXL2-dependent IRE1-XBP1 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.

Cell plasticity is emerging as a key regulator of tumor progression and metastasis. During carcinoma dissemination epithelial cells undergo epithelial to mesenchymal transition (EMT) processes characterized by the acquisition of migratory/invasive properties, while the reverse, mesenchymal to epithelial transition (MET) process, is also essential for metastasis outgrowth. Different transcription factors, called EMT‐TFs, including Snail, bHLH and Zeb families are drivers of the EMT branch of epithelial plasticity, and can be post‐transcriptionally downregulated by several miRNAs, as the miR‐200 family. The specific or redundant role of different EMT‐TFs and their functional interrelations are not fully understood. To study the interplay between different EMT‐TFs, comprehensive gain and loss‐of‐function studies of Snail1, Snail2 and/or Zeb1 factors were performed in the prototypical MDCK cell model system. We here describe that Snail1 and Zeb1 are mutually required for EMT induction while continuous Snail1 and Snail2 expression, but not Zeb1, is needed for maintenance of the mesenchymal phenotype in MDCK cells. In this model system, EMT is coordinated by Snail1 and Zeb1 through transcriptional and epigenetic downregulation of the miR‐200 family. Interestingly, Snail1 is involved in epigenetic CpG DNA methylation of the miR‐200 loci, essential to maintain the mesenchymal phenotype. The present results thus define a novel functional interplay between Snail and Zeb EMT‐TFs in miR‐200 family regulation providing a molecular link to their previous involvement in the generation of EMT process in vivo.

Summary: Lysyl-oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family that catalyzes the cross-linking of collagens or elastins in the extracellular matrix, thus regulating the tensile strength of tissues. However, many reports have suggested different intracellular roles for LOXL2, including the ability to regulate gene transcription and tumor progression. We previously reported that LOXL2 mediates epithelial-to-mesenchymal transition (EMT) by Snail1-dependent and independent mechanisms, related to E-cadherin silencing and downregulation of epidermal differentiation and cell polarity components, respectively. Whether or not the catalytic activity of LOXL2 is required to induce/sustain EMT is actually unknown. Here we show that LOXL2 catalytic inactive mutants collaborate with Snail1 in E-cadherin gene repression to trigger EMT and, in addition, promote FAK/Src pathway activation to support EMT. These findings reveal a non-conventional role of LOXL2 on regulating epithelial cell plasticity.