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The CCAAT/enhancer-binding protein beta-2 isoform (CEBPβ-2) upregulates galectin-7 expression in human breast cancer cells.

Campion CG, Labrie M, Grosset AA, St-Pierre Y - PLoS ONE (2014)

Bottom Line: In silico analysis further revealed the presence of an established CEBP element in the galectin-7 promoter.Mutation of this binding site abolished the transcriptional activity of the galectin-7 promoter.Chromatin immunoprecipitation analysis confirmed that C/EBPβ-2 binds to the endogenous galectin-7 promoter.

View Article: PubMed Central - PubMed

Affiliation: INRS-Institut Armand-Frappier, Laval, Québec, Canada.

ABSTRACT
Galectin-7 is considered a gene under the control of p53. However, elevated expression of galectin-7 has been reported in several forms of cancer harboring an inactive p53 pathway. This is especially true for breast cancer where galectin-7 expression is readily expressed in a high proportion in basal-like breast cancer tissues, conferring cancer cells with increased resistance to cell death and metastatic properties. These observations suggest that other transcription factors are capable of inducing galectin-7 expression. In the present work, we have examined the role of CCAAT/enhancer-binding protein beta (C/EBPβ) in inducing expression of galectin-7. C/EBP proteins have been shown to contribute to breast cancer by upregulating pro-metastatic genes. We paid particular attention to C/EBPβ-2 (also known as LAP2), the most transcriptionally active of the C/EBPβ isoforms. Our results showed that ectopic expression of C/EBPβ-2 in human breast cancer cells was sufficient to induce expression of galectin-7 at both the mRNA and protein levels. In silico analysis further revealed the presence of an established CEBP element in the galectin-7 promoter. Mutation of this binding site abolished the transcriptional activity of the galectin-7 promoter. Chromatin immunoprecipitation analysis confirmed that C/EBPβ-2 binds to the endogenous galectin-7 promoter. Analysis of galectin-7 protein expression in normal epithelia and in breast carcinoma by immunohistochemistry further showed the expression pattern of C/EBPβ closely micmicked that of galectin-7, most notably in mammary myoepithelial cells and basal-like breast cancer where galectin-7 is preferentially expressed. Taken together, our findings suggest that C/EBPβ is an important mediator of galectin-7 gene activation in breast cancer cells and highlight the different transcriptional mechanisms controlling galectin-7 in cancer cells.

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Protein expression of galectin-7 in breast cancer cell lines.MCF-7 or MDA-MB-231 cells were transfected with an expression vector encoding C/EBPβ-2 before cell fixation and permeabilization. A goat anti-human galectin-7 polyclonal antibody was used in combination with an Alexa Fluor 488-conjugated donkey anti-goat IgG to detect endogenous galectin-7 (green). Nuclei were stained with DAPI (blue).
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pone-0095087-g002: Protein expression of galectin-7 in breast cancer cell lines.MCF-7 or MDA-MB-231 cells were transfected with an expression vector encoding C/EBPβ-2 before cell fixation and permeabilization. A goat anti-human galectin-7 polyclonal antibody was used in combination with an Alexa Fluor 488-conjugated donkey anti-goat IgG to detect endogenous galectin-7 (green). Nuclei were stained with DAPI (blue).

Mentions: We first set out to determine if the C/EBPβ-2 isoform could induce galectin-7 gene expression in human breast cancer cell lines. Using semi-quantitative RT-PCR, we found that galectin-7 mRNA increased in all cell lines tested following transfection of a vector encoding the C/EBPβ-2 isoform (Figure 1A). This expression by C/EBPβ-2 was independent of the p53 status of the cells. It was observed in cells expressing a wild-type form of p53, such as MCF-7, or in cells harboring an inactive p53 pathway, such as the p53 MDA-MB-453 or MDA-MB-231, which express the p53R280K mutant protein. It was also found in HaCaT cells, which express the transcriptionally inactive TP53H179Y/R282W alleles. In all cases, de novo expression of galectin-7 was not induced following transfection of a vector encoding C/EBPβ-3 (also known as LIP). This increased expression of galectin-7 by C/EBPβ-2 was also dose-dependent (Figure 1B). C/EBPβ-3, which lacks the N-terminal transactivation domains but represses the transcription activity of other C/EBPs by competing for C/EBP consensus binding sites or by forming inactive heterodimers with other C/EBPs [12], [19], did not repress the constitutive expression of galectin-7 in MDA-MB-468 or HaCaT cells, nor did it repress C/EBPβ-2-induced galectin-7 (Figure 1C). The ability of C/EBPβ-2 to induce galectin-7 was confirmed at the protein level by confocal microscopy in both MDA-MB-231 and MCF-7 cells (Figure 2). Taken together, these results indicate that increased expression of C/EBPβ-2 is sufficient to induce galectin-7 in breast cancer cell lines and possibly other types of cells.


The CCAAT/enhancer-binding protein beta-2 isoform (CEBPβ-2) upregulates galectin-7 expression in human breast cancer cells.

Campion CG, Labrie M, Grosset AA, St-Pierre Y - PLoS ONE (2014)

Protein expression of galectin-7 in breast cancer cell lines.MCF-7 or MDA-MB-231 cells were transfected with an expression vector encoding C/EBPβ-2 before cell fixation and permeabilization. A goat anti-human galectin-7 polyclonal antibody was used in combination with an Alexa Fluor 488-conjugated donkey anti-goat IgG to detect endogenous galectin-7 (green). Nuclei were stained with DAPI (blue).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4008383&req=5

pone-0095087-g002: Protein expression of galectin-7 in breast cancer cell lines.MCF-7 or MDA-MB-231 cells were transfected with an expression vector encoding C/EBPβ-2 before cell fixation and permeabilization. A goat anti-human galectin-7 polyclonal antibody was used in combination with an Alexa Fluor 488-conjugated donkey anti-goat IgG to detect endogenous galectin-7 (green). Nuclei were stained with DAPI (blue).
Mentions: We first set out to determine if the C/EBPβ-2 isoform could induce galectin-7 gene expression in human breast cancer cell lines. Using semi-quantitative RT-PCR, we found that galectin-7 mRNA increased in all cell lines tested following transfection of a vector encoding the C/EBPβ-2 isoform (Figure 1A). This expression by C/EBPβ-2 was independent of the p53 status of the cells. It was observed in cells expressing a wild-type form of p53, such as MCF-7, or in cells harboring an inactive p53 pathway, such as the p53 MDA-MB-453 or MDA-MB-231, which express the p53R280K mutant protein. It was also found in HaCaT cells, which express the transcriptionally inactive TP53H179Y/R282W alleles. In all cases, de novo expression of galectin-7 was not induced following transfection of a vector encoding C/EBPβ-3 (also known as LIP). This increased expression of galectin-7 by C/EBPβ-2 was also dose-dependent (Figure 1B). C/EBPβ-3, which lacks the N-terminal transactivation domains but represses the transcription activity of other C/EBPs by competing for C/EBP consensus binding sites or by forming inactive heterodimers with other C/EBPs [12], [19], did not repress the constitutive expression of galectin-7 in MDA-MB-468 or HaCaT cells, nor did it repress C/EBPβ-2-induced galectin-7 (Figure 1C). The ability of C/EBPβ-2 to induce galectin-7 was confirmed at the protein level by confocal microscopy in both MDA-MB-231 and MCF-7 cells (Figure 2). Taken together, these results indicate that increased expression of C/EBPβ-2 is sufficient to induce galectin-7 in breast cancer cell lines and possibly other types of cells.

Bottom Line: In silico analysis further revealed the presence of an established CEBP element in the galectin-7 promoter.Mutation of this binding site abolished the transcriptional activity of the galectin-7 promoter.Chromatin immunoprecipitation analysis confirmed that C/EBPβ-2 binds to the endogenous galectin-7 promoter.

View Article: PubMed Central - PubMed

Affiliation: INRS-Institut Armand-Frappier, Laval, Québec, Canada.

ABSTRACT
Galectin-7 is considered a gene under the control of p53. However, elevated expression of galectin-7 has been reported in several forms of cancer harboring an inactive p53 pathway. This is especially true for breast cancer where galectin-7 expression is readily expressed in a high proportion in basal-like breast cancer tissues, conferring cancer cells with increased resistance to cell death and metastatic properties. These observations suggest that other transcription factors are capable of inducing galectin-7 expression. In the present work, we have examined the role of CCAAT/enhancer-binding protein beta (C/EBPβ) in inducing expression of galectin-7. C/EBP proteins have been shown to contribute to breast cancer by upregulating pro-metastatic genes. We paid particular attention to C/EBPβ-2 (also known as LAP2), the most transcriptionally active of the C/EBPβ isoforms. Our results showed that ectopic expression of C/EBPβ-2 in human breast cancer cells was sufficient to induce expression of galectin-7 at both the mRNA and protein levels. In silico analysis further revealed the presence of an established CEBP element in the galectin-7 promoter. Mutation of this binding site abolished the transcriptional activity of the galectin-7 promoter. Chromatin immunoprecipitation analysis confirmed that C/EBPβ-2 binds to the endogenous galectin-7 promoter. Analysis of galectin-7 protein expression in normal epithelia and in breast carcinoma by immunohistochemistry further showed the expression pattern of C/EBPβ closely micmicked that of galectin-7, most notably in mammary myoepithelial cells and basal-like breast cancer where galectin-7 is preferentially expressed. Taken together, our findings suggest that C/EBPβ is an important mediator of galectin-7 gene activation in breast cancer cells and highlight the different transcriptional mechanisms controlling galectin-7 in cancer cells.

Show MeSH
Related in: MedlinePlus