Lysyl oxidase-like 2 (LOXL2), a new regulator of cell polarity required for metastatic dissemination of basal-like breast carcinomas

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.


Supplemental Experimental Procedures
RNA extraction, probe synthesis and hybridization on oligonucleotide arrays of tumour samples .
Total RNA from breast tumour samples was isolated using TRIZOL reagent (Life Technologies) and RNaesy Extraction Kit (QIAGen) as indicated by the manufacturer.
Purity of isolated RNA was evaluated spectrophotometrically by the A260/A280 absorbance ratio. Microarray experiments were performed using Human Whole Genome V2 4*44K array G4845A (Agilent technologies). RNA was labelled and array hybridized 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 analyzed using Feature Extraction Software 10.0 (Agilent technologies). RNA samples from independent tumours were labelled with Cy5-dUTP and as control a pooled RNA obtained from equal concentration of each RNA tumor sample labelled with Cy3-dUTP was used. A hierarchical clustering method was applied to group the genes and samples on the basis of the similarities in expression and the unsupervised analyses were visualized using the SOTA

Immunofluorescence and confocal analyses
Inmunofluorescence analysis was performed on cell grown on coverslips and fixed under conditions for antibodies and fixation procedures described in Table S4.
Secondary antibodies were anti-mouse or anti-rabbit Alexa 488/546, depending on the primary antibodies. Phalloidin-647 (Amersham) was used to stain F-actin. Confocal microscopy analyses were performed using a Leica Spectral TCS SP2, x63 objective.
Maximal projections were obtained for x-y planes; for z-x planes a z level color coded projection was used to show the position of membrane structures in the different sections.

Generation of ΔLOXL2 (mutant catalytic domain) vector
The LOXL2

Promoter assays
Cotransfections were carried out in the presence of the 50 ng of Snail1, LOXL2 or ΔLOXL2 cDNAs (or the indicated amounts of LOXL2 cDNA) and 200 ng of the indicated promoters and 10 ng of β-gal as control of transfection efficiency. The amount of total DNA was normalized with empty pcDNA3 vector (up to 100 ng). Luciferase and β-galactosidase activities were measured using the luciferare and β-Glo assay substrates (Promega) and normalized to the wild-type promoter activity detected in cells transfected with pcDNA3 empty vector or in wild-type cells.

Barrier assays
Barrier assay was performed as described (Van Horssen et al, 2006). Basically, cells were grown on coverslips to confluence and then the coverslips were transferred to a new culture plate with fresh medium and photographed at different time points.

Invasion assays
Invasion of the indicated cell lines on modified Boyden chambers (8 m pore filters) coated with 100 g/ml collagen type IV gel were performed as previously described

Proliferation assays
To study the proliferation index, 2.5x10 4 cells were grown into 96-wells plate according to Cell Proliferation ELISA, BrdU (colorimetric) kit, (Roche Diagnostic SL, Basel, Switzerland) using the manufacturer's recommended conditions.  Table and figure legends   Supplemental Table S1. Genes with differential expression between basal (B) and

non-basal (NB) Grade 3 breast tumors with significant association (FDR<0.2).
Gene  Table S3. Relationship between LOXLl2 expression and clinicopathological e immunohistochemical features in IDC grade 3.

Unsupervised analysis
Unsupervised analysis using Basal Breast Signature (BBS) in public databases n=311