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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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Claudin1 and Lgl2 are transcriptionally repressed by LOXL2A. RT-PCR of claudin1 and Lgl2 transcripts in the indicated cell lines. GAPDH is used as a loading control.B. Activity of claudin1 and Lgl2 promoters in parental MDA-MB-231 cells (WT) and in control shEGFP and shLOXL2 (clone C1) cells. The activity was determined as relative luciferase units (RLU) and normalized to the activity detected in WT cells for each promoter.C. Activity of the two Lgl2 promoters in HEK293T cells in the presence of the indicated amounts (ng) of LOXL2. The activity, as RLU, was normalized to that obtained in the presence of control pcDNA3 vector (100 ng).D.–F. Activity of claudin1 (D) and the two Lgl2 promoters (E, F) in the presence of the indicated expression vectors (50 ng). The activity, as RLU, was normalized to that obtained in the presence of control pcDNA3 vector (100 ng). Results in (B) to (F) represent the mean +/− s.d of three (B–D) and two (E, F) independent experiments performed in quadruple samples. (*p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.001)
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fig05: Claudin1 and Lgl2 are transcriptionally repressed by LOXL2A. RT-PCR of claudin1 and Lgl2 transcripts in the indicated cell lines. GAPDH is used as a loading control.B. Activity of claudin1 and Lgl2 promoters in parental MDA-MB-231 cells (WT) and in control shEGFP and shLOXL2 (clone C1) cells. The activity was determined as relative luciferase units (RLU) and normalized to the activity detected in WT cells for each promoter.C. Activity of the two Lgl2 promoters in HEK293T cells in the presence of the indicated amounts (ng) of LOXL2. The activity, as RLU, was normalized to that obtained in the presence of control pcDNA3 vector (100 ng).D.–F. Activity of claudin1 (D) and the two Lgl2 promoters (E, F) in the presence of the indicated expression vectors (50 ng). The activity, as RLU, was normalized to that obtained in the presence of control pcDNA3 vector (100 ng). Results in (B) to (F) represent the mean +/− s.d of three (B–D) and two (E, F) independent experiments performed in quadruple samples. (*p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.001)

Mentions: The above results led us to further investigate the mechanisms underlying LOXL2 mediated downregulation of tight junctions and cell polarity complexes. To this end, we chose claudin1 and Lgl2 as representative components of tight junctions and cell polarity complexes, respectively, whose expression has been previously shown to be downregulated by EMT factors, like Snail1/Snail2 and/or ZEB1 (Aigner et al, 2007; Martinez-Estrada et al, 2006; Ohkubo and Ozawa, 2004; Spaderna et al, 2008). RT-PCR analyses of claudin1 and Lgl2 transcripts showed an inverse association with LOXL2 expression in MDA-MB-231 cells (Fig 5A). We then speculated that LOXL2 could downregulate claudin1 and Lgl2 gene transcription. Promoter assays indicated that the activity of claudin1 and two independent Lgl2 promoters were upregulated in MDA-MBA-231-shLOXL2 cells compared to parental and control shEGFP cells (Fig 5B), supporting a negative influence of LOXL2. Indeed, transient promoter assays indicated that LOXL2 represses the activity of both Lgl2 promoter constructs in a dose-dependent fashion up to 60% (Fig 5C). The activity of the claudin1 promoter was also repressed by LOXL2 to about 40% (Fig 5D), a lower potency than that previously shown by Snail1 (up to 60%) (Fig 5D; Martinez-Estrada et al, 2006). Interestingly, the repressive action of LOXL2 on the claudin1 and Lgl2 promoters was independent of Snail1 and, surprisingly, also of the LOXL2 catalytic activity since a LOXL2 deletion mutant lacking the catalytic domain (ΔLOXL2; aa 548–688 deleted) showed the same repressor potency as the wild type LOXL2 on either promoter (Fig 5D–F). Taken together, these data indicate that LOXL2 downregulates tight junction and cell polarity complexes in basal-like carcinoma cells at the transcriptional level by a mechanism independent of Snail1 and LOXL2 enzymatic activity.


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)

Claudin1 and Lgl2 are transcriptionally repressed by LOXL2A. RT-PCR of claudin1 and Lgl2 transcripts in the indicated cell lines. GAPDH is used as a loading control.B. Activity of claudin1 and Lgl2 promoters in parental MDA-MB-231 cells (WT) and in control shEGFP and shLOXL2 (clone C1) cells. The activity was determined as relative luciferase units (RLU) and normalized to the activity detected in WT cells for each promoter.C. Activity of the two Lgl2 promoters in HEK293T cells in the presence of the indicated amounts (ng) of LOXL2. The activity, as RLU, was normalized to that obtained in the presence of control pcDNA3 vector (100 ng).D.–F. Activity of claudin1 (D) and the two Lgl2 promoters (E, F) in the presence of the indicated expression vectors (50 ng). The activity, as RLU, was normalized to that obtained in the presence of control pcDNA3 vector (100 ng). Results in (B) to (F) represent the mean +/− s.d of three (B–D) and two (E, F) independent experiments performed in quadruple samples. (*p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.001)
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fig05: Claudin1 and Lgl2 are transcriptionally repressed by LOXL2A. RT-PCR of claudin1 and Lgl2 transcripts in the indicated cell lines. GAPDH is used as a loading control.B. Activity of claudin1 and Lgl2 promoters in parental MDA-MB-231 cells (WT) and in control shEGFP and shLOXL2 (clone C1) cells. The activity was determined as relative luciferase units (RLU) and normalized to the activity detected in WT cells for each promoter.C. Activity of the two Lgl2 promoters in HEK293T cells in the presence of the indicated amounts (ng) of LOXL2. The activity, as RLU, was normalized to that obtained in the presence of control pcDNA3 vector (100 ng).D.–F. Activity of claudin1 (D) and the two Lgl2 promoters (E, F) in the presence of the indicated expression vectors (50 ng). The activity, as RLU, was normalized to that obtained in the presence of control pcDNA3 vector (100 ng). Results in (B) to (F) represent the mean +/− s.d of three (B–D) and two (E, F) independent experiments performed in quadruple samples. (*p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.001)
Mentions: The above results led us to further investigate the mechanisms underlying LOXL2 mediated downregulation of tight junctions and cell polarity complexes. To this end, we chose claudin1 and Lgl2 as representative components of tight junctions and cell polarity complexes, respectively, whose expression has been previously shown to be downregulated by EMT factors, like Snail1/Snail2 and/or ZEB1 (Aigner et al, 2007; Martinez-Estrada et al, 2006; Ohkubo and Ozawa, 2004; Spaderna et al, 2008). RT-PCR analyses of claudin1 and Lgl2 transcripts showed an inverse association with LOXL2 expression in MDA-MB-231 cells (Fig 5A). We then speculated that LOXL2 could downregulate claudin1 and Lgl2 gene transcription. Promoter assays indicated that the activity of claudin1 and two independent Lgl2 promoters were upregulated in MDA-MBA-231-shLOXL2 cells compared to parental and control shEGFP cells (Fig 5B), supporting a negative influence of LOXL2. Indeed, transient promoter assays indicated that LOXL2 represses the activity of both Lgl2 promoter constructs in a dose-dependent fashion up to 60% (Fig 5C). The activity of the claudin1 promoter was also repressed by LOXL2 to about 40% (Fig 5D), a lower potency than that previously shown by Snail1 (up to 60%) (Fig 5D; Martinez-Estrada et al, 2006). Interestingly, the repressive action of LOXL2 on the claudin1 and Lgl2 promoters was independent of Snail1 and, surprisingly, also of the LOXL2 catalytic activity since a LOXL2 deletion mutant lacking the catalytic domain (ΔLOXL2; aa 548–688 deleted) showed the same repressor potency as the wild type LOXL2 on either promoter (Fig 5D–F). Taken together, these data indicate that LOXL2 downregulates tight junction and cell polarity complexes in basal-like carcinoma cells at the transcriptional level by a mechanism independent of Snail1 and LOXL2 enzymatic activity.

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