Induction of galectin-7 in MDA-MB-231 cells is CRD-dependent.

<p>Expression of galectin-7 was measured by Western blot at different times following stimulation with rhGal-7.</p

Western blot analysis showing endocytosis of FITC-labeled galectin-7.

<p>(<b>A</b>) Western blot analysis of cell lysates collected from MDA-MB-231 cells treated with rh-Gal-7 or FITC-rhGal-7 (15 min. post-treatment) (<b>B</b>) Expression of cytosolic or mitochondrial galectin-7 following treatment with rhGal-7 or FITC-rhGal-7 (15 min. post-treatment).</p

Real-time analysis showing internalization of FITC-rhGal-7.

<p><b>Time-lapse imaging of FITC-rhGal-7 endocytosis in</b> MDA-MB-231 cells. T0: 0 sec, T1 to T5: 210 at 270 sec (at 15 seconds intervals).</p

Endocytosis FITC-labeled galectin-7 viewed by confocal microscopy.

<p>Confocal microscopy of MDA-MB-231 cells treated with FITC-rhGal-7 during 25 minutes. Staining with DAPI (a) and WGA (c), which target the nucleus and plasma membrane respectively, are shown. In (b), staining of FITC-gal-7. (e) and (f) show cross sections of the cell.</p

Increase mRNA levels of human gal-7 in cancer cells following stimulation with human recombinant galectin-7 (rhGal-7).

<p>(<b>A</b>) Levels of transcripts were measured by RT-PCR 16 h after addition of rhGal-7. (<b>B</b>) Kinetic analysis of <i>lgals7</i> mRNA expression induced by rhGal-7 (5μM) in OVCAR and A2780 cells. (<b>C</b>) mRNA level of <i>lgals7</i> in MDA-MB-231 cells after treatment with rhGal-7 (5μM). (<b>D</b>) Quantitative analyses of <i>lgals7</i> mRNA levels in MDA-MB-231 as measured by imaging densitometry. (* <i>p</i> ≤ 0.05). (<b>E</b>) Luciferase activity measured in protein extracts collected from MDA-MB-231 cells transfected with a luciferase reporter vector containing <i>p200-gal7</i> promoter following treatment with rhGal-7 (** <i>p</i> ≤ 0.005). (<b>F</b>) <i>lgals7</i> expression induced by rhGal-7 at different concentrations. Data are representative of at least three independent experiments.</p

Confocal microscopy of 3D sections of MDA-MB-231 cells treated with FITC-rhGal-7.

<p>(<b>A</b>) 3D reconstruction of endocytosed FITC-rhGal-7 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187194#pone.0187194.g005" target="_blank">Fig 5</a>. In (<b>B</b>), multiple cross sections in the thickness of the cell.</p

Mitochondrial localization of galectin-7.

<p>Western blot analysis in MDA-MB-231 cells showing expression of cytosolic and mitochondrial galectin-7 15 min after stimulation with rhGal-7 at 0°C or 37°C. Tubulin and CoxIV were used as controls for cytosolic and mitochondrial extracts respectively.</p

Expression of Galectin-7 Is Induced in Breast Cancer Cells by Mutant p53

<div><p>Galectin-7 was initially described as a marker of epithelial differentiation expressed in the stratified epithelium of various tissues. Like other members of the galectin family, its expression level is often significantly altered in cancer cells. In breast cancer, its expression is significantly augmented in aggressive molecular subtypes, most notably in estrogen receptor-negative tumors and in cell lines with a basal-like phenotype. Studies using experimental mouse models have further shown high expression of galectin-7 was sufficient to increase the metastatic behavior of poorly metastatic breast cancer cells, rendering them more resistant to apoptosis. This expression pattern in breast cancer cells is unexpected because galectin-7 was originally identified as a <i>p53</i>-induced gene. To address this paradox, we have examined the molecular mechanisms regulating galectin-7 in breast cancer cells. Our results showed that transfection of breast cancer cells with expression vectors encoding mutant p53 was sufficient to induce galectin-7 at both mRNA and protein levels. Doxorubicin treatment of breast cancer cells harboring a mutant p53 also induced galectin-7. This induction was specific since knockdown of endogenous mutant p53 inhibited doxorubicin-induced galectin-7 expression. The p53-induced galectin-7 expression in breast cancer cells correlated with increased NF-κB activity and was inhibited by NF-κB inhibitors, indicating that the ability of mutant p53 to induce galectin-7 was dependent on NF-κB activity. The implication of NF-κB was further supported by data showing that NF-κB bound to the endogenous galectin-7 promoter and that TNFα-induced <i>galectin-7</i> expression was abolished by NF-κB inhibitors. Taken together, our data provide an explanation to the observed high galectin-7 expression levels in cancer cells and suggest that galectin-7 could be part of a common pathway used by mutant p53 to promote cancer progression.</p> </div

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