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

Moreno-Bueno G, Salvador F, Martín A, Floristán A, Cuevas EP, Santos V, Montes A, Morales S, Castilla MA, Rojo-Sebastián A, Martínez A, Hardisson D, Csiszar K, Portillo F, Peinado H, Palacios J, Cano A - EMBO Mol Med (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, UAM, Instituto de Investigaciones Biomédicas "Alberto Sols", CSIC-UAM, IdiPAZ, Instituto de Investigación Sanitaria La Paz, Madrid, Spain. gmoreno@iib.uam.es

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LOXL2 silencing in basal carcinoma cells induces expression and reorganization of tight junctions and cell polarity complexesA, B. Confocal immunofluorescence analyses in MDA-MB-231-shEGFP and -shLOXL2 (Clone 1) (A) and MCF7 and MCF-LOXL2 (B) cells for E-cadherin (a, b); claudin1 (c, d), ZO1, (e, f), Par3 (g, h) and Lgl2 (i, j). x–y maximal projections are shown for E-cadherin (A, B) and claudin1 (B) stain; z–x planes are shown in the rest of panels. Insets in panels d, f, h, j, indicate enlarged areas (right) showing positive immunostaining along the lateral membrane. Colour scale indicates the intensity of staining from basal (bottom, purple) to apical (top, red) localization. Bars, 50 µm.
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fig04: LOXL2 silencing in basal carcinoma cells induces expression and reorganization of tight junctions and cell polarity complexesA, B. Confocal immunofluorescence analyses in MDA-MB-231-shEGFP and -shLOXL2 (Clone 1) (A) and MCF7 and MCF-LOXL2 (B) cells for E-cadherin (a, b); claudin1 (c, d), ZO1, (e, f), Par3 (g, h) and Lgl2 (i, j). x–y maximal projections are shown for E-cadherin (A, B) and claudin1 (B) stain; z–x planes are shown in the rest of panels. Insets in panels d, f, h, j, indicate enlarged areas (right) showing positive immunostaining along the lateral membrane. Colour scale indicates the intensity of staining from basal (bottom, purple) to apical (top, red) localization. Bars, 50 µm.

Mentions: The striking induction of MET by LOXL2 silencing in basal-like carcinoma cells led us to analyse in further detail the expression/organization of cell junctions. As demonstrated above, the cell phenotype induced by LOXL2 silencing in basal-like carcinoma cells was apparently independent of E-cadherin (Fig 2E, Supporting Information Fig S3B). Indeed, confocal analyses indicated that the low E-cadherin protein levels detected in MDA-MBA-231-shLOXL2 cells were not properly organized at cell–cell contacts in most adherent cells (Fig 4A, a,b). In contrast, MCF7-LOXL2 cells, although showing reduced E-cadherin levels (Supporting Information Fig S6C), organized E-cadherin at cell–cell contacts in most adherent cells in a pattern similar to that present in parental MCF7 cells (Fig 4B, a,b). The expression and organization of other adhesion complexes, in particular tight junctions, was then investigated in both cell systems. Western blot analyses indicated increased expression of claudin1 in MDA-MBA-231-shLOXL2 cells while the soluble ZO1 protein levels decreased slightly compared to parental cells (Supporting Information Fig S6A). On the other hand, strongly decreased expression of ZO1 was detected in MCF7-LOXL2 cells compared to parental MCF7 cells, while no expression of claudin1 was detected in parental or MCF7-LOXL2 cells (Supporting Information Fig S6A). Importantly, confocal immunofluorescence analyses showed that ZO1 and claudin1 were properly organized at cell–cell contacts at the upper lateral membrane in MDA-MBA-231-shLOXL2 cells (Fig 4A, d,f and insets) in a pattern strikingly similar to that present for ZO1 in MCF7 cells (Fig 4B, f and inset). In agreement with these observations, ZO1 and claudin1 were fully disorganized or not detected in MDA-MB-231-shEGFP and MCF7-LOXL2 cells (Fig 4A/B, c,e). Together, these data indicate a negative role for LOXL2 in the expression and organization of tight junction complexes of basal-like carcinoma cells.


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

Moreno-Bueno G, Salvador F, Martín A, Floristán A, Cuevas EP, Santos V, Montes A, Morales S, Castilla MA, Rojo-Sebastián A, Martínez A, Hardisson D, Csiszar K, Portillo F, Peinado H, Palacios J, Cano A - EMBO Mol Med (2011)

LOXL2 silencing in basal carcinoma cells induces expression and reorganization of tight junctions and cell polarity complexesA, B. Confocal immunofluorescence analyses in MDA-MB-231-shEGFP and -shLOXL2 (Clone 1) (A) and MCF7 and MCF-LOXL2 (B) cells for E-cadherin (a, b); claudin1 (c, d), ZO1, (e, f), Par3 (g, h) and Lgl2 (i, j). x–y maximal projections are shown for E-cadherin (A, B) and claudin1 (B) stain; z–x planes are shown in the rest of panels. Insets in panels d, f, h, j, indicate enlarged areas (right) showing positive immunostaining along the lateral membrane. Colour scale indicates the intensity of staining from basal (bottom, purple) to apical (top, red) localization. Bars, 50 µm.
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fig04: LOXL2 silencing in basal carcinoma cells induces expression and reorganization of tight junctions and cell polarity complexesA, B. Confocal immunofluorescence analyses in MDA-MB-231-shEGFP and -shLOXL2 (Clone 1) (A) and MCF7 and MCF-LOXL2 (B) cells for E-cadherin (a, b); claudin1 (c, d), ZO1, (e, f), Par3 (g, h) and Lgl2 (i, j). x–y maximal projections are shown for E-cadherin (A, B) and claudin1 (B) stain; z–x planes are shown in the rest of panels. Insets in panels d, f, h, j, indicate enlarged areas (right) showing positive immunostaining along the lateral membrane. Colour scale indicates the intensity of staining from basal (bottom, purple) to apical (top, red) localization. Bars, 50 µm.
Mentions: The striking induction of MET by LOXL2 silencing in basal-like carcinoma cells led us to analyse in further detail the expression/organization of cell junctions. As demonstrated above, the cell phenotype induced by LOXL2 silencing in basal-like carcinoma cells was apparently independent of E-cadherin (Fig 2E, Supporting Information Fig S3B). Indeed, confocal analyses indicated that the low E-cadherin protein levels detected in MDA-MBA-231-shLOXL2 cells were not properly organized at cell–cell contacts in most adherent cells (Fig 4A, a,b). In contrast, MCF7-LOXL2 cells, although showing reduced E-cadherin levels (Supporting Information Fig S6C), organized E-cadherin at cell–cell contacts in most adherent cells in a pattern similar to that present in parental MCF7 cells (Fig 4B, a,b). The expression and organization of other adhesion complexes, in particular tight junctions, was then investigated in both cell systems. Western blot analyses indicated increased expression of claudin1 in MDA-MBA-231-shLOXL2 cells while the soluble ZO1 protein levels decreased slightly compared to parental cells (Supporting Information Fig S6A). On the other hand, strongly decreased expression of ZO1 was detected in MCF7-LOXL2 cells compared to parental MCF7 cells, while no expression of claudin1 was detected in parental or MCF7-LOXL2 cells (Supporting Information Fig S6A). Importantly, confocal immunofluorescence analyses showed that ZO1 and claudin1 were properly organized at cell–cell contacts at the upper lateral membrane in MDA-MBA-231-shLOXL2 cells (Fig 4A, d,f and insets) in a pattern strikingly similar to that present for ZO1 in MCF7 cells (Fig 4B, f and inset). In agreement with these observations, ZO1 and claudin1 were fully disorganized or not detected in MDA-MB-231-shEGFP and MCF7-LOXL2 cells (Fig 4A/B, c,e). Together, these data indicate a negative role for LOXL2 in the expression and organization of tight junction complexes of basal-like carcinoma cells.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, UAM, Instituto de Investigaciones Biomédicas "Alberto Sols", CSIC-UAM, IdiPAZ, Instituto de Investigación Sanitaria La Paz, Madrid, Spain. gmoreno@iib.uam.es

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Related in: MedlinePlus