No significant effects on mammary gland developmental stages in the absence of gal-7.

<p>Histology assessment of whole mammary glands (H&E stained) of wild-type and galectin-7-deficient mice. Three mice were used for each group of mammary gland at three distinct stages: puberty (6 weeks of age), pregnancy (5, 10 and 15 days), lactation (0, 5 and 10 days) and involution (1 and 5 days). Scale bars, 1 mm and 100 μm (insets).</p

Galectin-7 Expression Potentiates HER-2-Positive Phenotype in Breast Cancer

<div><p>HER-2 positive tumors are among the most aggressive subtypes of breast cancer and are frequently associated with metastasis and poor outcome. As with other aggressive subtypes of breast cancer, these tumors are associated with abnormally high expression of galectin-7 (gal-7), which confers metastatic breast tumor cells with increased invasive behavior. Although previous studies in the rat model of breast tumorigenesis have shown that gal-7 is also increased in primary breast tumor, its contribution to the development of the primary breast tumors remains unclear. In the present work, we have used genetically-engineered gal-7-deficient mice to examine the role of gal-7 in the development of the mammary gland and of breast cancer. Using histological and immunohistological analysis of whole mammary glands at different stages of development, we detected no significant changes between normal and gal-7-deficient mice. To test the involvement of gal-7 in breast cancer, we next examined the effects of loss of gal-7 on mammary tumor development by crossing gal-7-deficient mice with the mammary tumor transgenic mouse strain FVB-Tg(MMTV-Erbb2)NK1Mul/J. Finally, assessment of mice survival and tumor volume showed a delay of mammary tumor growth in the absence of systemic gal-7. These data suggest that gal-7 could potentiate the phenotype of HER-2 positive primary breast cancer.</p></div

Comparison between ErbB2 tumors induced in the presence or absence of gal-7 in mice.

<p>Comparison between ErbB2 tumors induced in the presence or absence of gal-7 in mice.</p

The CCAAT/Enhancer-Binding Protein Beta-2 Isoform (CEBPβ-2) Upregulates Galectin-7 Expression in Human Breast Cancer Cells

<div><p><i>Galectin-7</i> 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 <i>galectin-7</i> 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. <i>In silico</i> analysis further revealed the presence of an established CEBP element in the <i>galectin-7</i> promoter. Mutation of this binding site abolished the transcriptional activity of the <i>galectin-7</i> promoter. Chromatin immunoprecipitation analysis confirmed that C/EBPβ-2 binds to the endogenous <i>galectin-7</i> 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 <i>galectin-7</i> gene activation in breast cancer cells and highlight the different transcriptional mechanisms controlling galectin-7 in cancer cells.</p></div

C/EBPβ consensus binding site in <i>galectin-7</i> promoter.

<p>(<b>A</b>) Schematic representation of C/EBP binding sites within the 5′ flanking region of the human <i>galectin-7</i> gene. A <b>s</b>eries of 5′ deletion constructs of the 1500 bp <i>galectin-7</i> promoter region was generated and cloned into the pGL3 Basic luciferase reporter vector. The resulting plasmids were transfected in MCF-7 and MDA-MB-468 cells. Locations of the putative C/EBP binding sites in the promoter, as determined using the TFsearch computational tool, are shown as empty boxes. (<b>B</b>) Sequence analysis of the C/EBP binding sites located at positions -105-98 and the -145-140. (<b>C</b>) Mutated constructs of the C/EBPβ binding sites on the 200 bp <i>galectin-7</i> promoter region were generated and cloned into the pGL3 Basic luciferase reporter vector. The resulting plasmids were co-transfected in MCF-7 cells and were compared to the wild-type <i>p200-galectin-7</i> promoter. Transfection efficiency was normalized by co-transfection with a β-galactosidase reporter vector.</p

Immunohistological analysis of mammary tumour markers in presence or absence of gal-7.

<p>Immunohistochemical staining showing membrane-bound HER-2-positive cells and nuclear Ki67 expression in mammary tumors that were negative (<i>left panels</i>) or positive (<i>right panels</i>). Scale bars, 50 μm and 25 μm (insets).</p

Protein expression of galectin-7 in breast cancer cell lines.

<p>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).</p

High C/EBPβ expression predicts poor outcome in human breast cancer.

<p>Data obtained from the Oncomine cancer microarray database (<a href="http://www.oncomine.org" target="_blank">www.oncomine.org</a>) showing higher C/EBPβ expression in (<b>A</b>) estrogen receptor (ER)-negative and (<b>B</b>) triple negative (TN) human breast carcinomas.</p

C/EBPβ-2 induces <i>galectin-7</i> mRNA levels in breast cancer cell lines.

<p>(<b>A</b>) RT-PCR analysis showing increased expression of <i>galectin-7</i> in human breast cancer cells after transfection with an expression vector encoding C/EBPβ-2. The two lanes represent two different samples. No such increase was observed in cells transfected with an expression vector encoding C/EBPβ-3. Similar results were obtained with HaCaT cells, a keratinocyte cell line which constitutively expresses <i>galectin-7</i>. An empty pCMV5 vector was used as transfection control (CTRL) and GAPDH was used as loading control. (<b>B</b>) RT-PCR analyses showing expression of <i>galectin-7</i> mRNA levels in MCF-7 cells after transfection with increasing doses of an expression vector encoding C/EBPβ-2. GAPDH was used as loading control. (<b>C</b>) RT-PCR analysis of MCF-7 cells co-transfected with vectors encoding C/EBPβ-2 and C/EBPβ-3. Below, control Western blot analysis showing expression of C/EBPβ-2 and C/EBPβ-3 after transfection. β-actin was used as loading control.</p

Effect of quercetin on B16F1 cells overexpressing galectin-7 on apoptosis and EGR-1 expression.

<p>B16F1 cells overexpressing galectin-7 (+) or controls (–) were treated with various doses of quercetin. A) Apoptotic sensitivity was analyzed by western blotting for cleaved PARP-1 detection. B) RT-PCR analysis of galectin-7 and EGR-1 mRNA expression. C) Western blot analysis for EGR-1 detection, 293 cells transfected with EGF were used as a positive control. Actin was used as a loading and specificity control. D) Dual luciferase assay of B16F1 transfectant cells overexpressing galectin-7 (□) or controls (▪) co-transfected with luciferase reporter plasmids with an EGR-1 promoter and pRLSV40-Renilla vector as a transfection control and treated with various doses of quercetin for 24 h. A mixture of three clones of B16F1-G7 was used (G7 #5, 10 and 14) for all of these experiments.</p