Expression and functions of galectin-7 in human and murine melanomas.

International audienceThe identification of galectin-7 as a p53-induced gene and its ability to induce apoptosis in many cell types support the hypothesis that galectin-7 has strong antitumor activity. This has been well documented in colon cancer. However, in some cases, such as breast cancer and lymphoma, its high expression level correlates with aggressive subtypes of cancer, suggesting that galectin-7 may have a dual role in cancer progression. In fact, in breast cancer, overexpression of galectin-7 alone is sufficient to promote metastasis to the bone and lung. In the present work, we investigated the expression and function of galectin-7 in melanoma. An analysis of datasets obtained from whole-genome profiling of human melanoma tissues revealed that galectin-7 mRNA was detected in more than 90% of biopsies of patients with nevi while its expression was more rarely found in biopsies collected from patients with malignant melanoma. This frequency, however, was likely due to the presence of normal epidermis tissues in biopsies, as shown our studies at the protein level by immunohistochemical analysis. Using the experimental melanoma B16F1 cell line, we found that melanoma cells can express galectin-7 at the primary tumor site and in lung metastasis. Moreover, we found that overexpression of galectin-7 increased the resistance of melanoma cells to apoptosis while inducing de novo egr-1 expression. Overexpression of galectin-7, however, was insufficient to modulate the growth of tumors induced by the subcutaneous injection of B16F1 cells. It also failed to modulate the dissemination of B16F1 cells to the lung

Validation of B16F1 transfectants overexpressing luciferase and/or galectin-7.

<p>A) RT-PCR analysis for galectin-7 mRNA expression in transfectant cells overexpressing galectin-7 with or without luciferase in comparison with a control B16F1 cell line (F1). The aggressive murine lymphoma cell line S19 (+) was used as a positive control. Actin was used as a loading and specificity control. B) Luciferase assay of three B16F1 transfectant cell lines overexpressing luciferase and cotransfected with pRc-CMV2-galectin-7 in comparison with the control B16F1 cell line. C) Confocal microscopy for galectin-7 in control B16F1 cells (iii) and galectin-7 transfectant cells (G7#5) (iv); these cells were also visualized (i, ii).</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

Galectin-7 expression in human melanoma tissues.

<p>A) Percentage of positive biopsies of normal skin (n = 7), nevus (n = 18) and malignant melanoma (n = 45), as determined by the <i>in silico</i> analysis of galectin-7 expression from a microarray of human biopsies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063307#pone.0063307-Robbins1" target="_blank">[26]</a>. B) Representative graph of galectin-7 mRNA expression in each biopsy described in (A). C) Detection of galectin-7 by immunohistochemistry in representative biopsies of nevi and malignant melanoma. Overall, 13 malignant melanomas and 47 nevi were tested.</p

Effect of galectin-7 in B16F1 cells on survival and metastasis in lungs.

<p>A) Survival curve of C57BL/6 mice injected i.v. with B16F1 transfectant cells overexpressing galectin-7 (□) or controls (♦) (2×10⁵ cells) (n = 8−10). B) Luciferase assay of lungs of C57BL/6 mice sacrificed at 9, 12, 15 or 18 days after the i.v. injection of a mixture of B16F1 luciferase transfectant cells overexpressing galectin-7 (+) or control (–) (n = 3−8). Normal lungs were used as a negative control (T: 0; n = 2). C) <i>Ex vivo</i> imaging of a lung metastasis, as seen in (B), of B16F1 cells overexpressing galectin-7 (iii and v) or control cells (ii and iv) at 9 and 18 days in comparison with control lungs (i).</p

Effect of galectin-7 on B16F1 cell migration.

<p>A) Images of the motility assay after the wounding of confluent B16F1 cells overexpressing galectin-7 (B16-G7) or controls (B16-Srα) at T = 0 and T = 16 hr. A mixture of three clones of B16F1-G7 was used (G7 #5, 10 and 14). B) Representative graph of the results obtained in (A).</p

Increased galectin-7 expression in B16F1 primary tumors and lung metastases.

<p>Primary tumors were collected at necropsy 18 days after the subcutaneous injection of B16F1 cells (5×10⁴ cells) in C57BL/6 (WT) and galectin-7-deficient mice (KOG7). (A) RT-PCR analysis of galectin-7 mRNA expression in two B16F1 primary tumors from WT and KOG7 mice in comparison with the B16F1 cell line. Actin was used as a loading and specificity control. B) Immunohistochemistry for galectin-7 in normal skin (i, ii) and B16F1 primary tumors (iii, iv) in WT and KOG7 mice. These galectin-7-positive structures located in the suprabasal epidermis have been reported before and likely represent suprabasal keratinocytes, which are known to express galectin-7 constitutively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063307#pone.0063307-Magnaldo1" target="_blank">[13]</a>. Control stained without HRP and in absence of Abs did not show any detectable staining in both wt and KOG7 mice. C) Immunohistochemistry for galectin-7 in a lung collected 20 days post-injection from one KOG7 mouse injected intravenously via the tail vein with B16F1 cells (2×10⁵ cells) in comparison with a normal lung. Scale bars in all immunohistochemistry images represent 600 µM.</p

Overexpression of Galectin-7, A Myoepithelial Cell Marker, Enhances Spontaneous Metastasis of Breast Cancer Cells

Galectins are members of a family of β-galactosides-binding proteins that have recently emerged as novel modulators in different aspects of cancer. The expression of galectins in tumors and/or the tissue surrounding them has been well documented. Since galectin-7 expression has been associated with epithelial tissues and varies significantly in various types of cancer, we have investigated for the first time its role in breast cancer. Using two preclinical mouse models, high levels of galectin-7 expression in breast cancer cells drastically increased their ability to metastasize to lungs and bones. Significant increases in the number of pulmonary metastases and osteolytic lesions were induced by overexpression of galectin-7 compared with control cells. In human tissues, galectin-7 was specifically found in myoepithelial cells of normal human breast tissue, but not in luminal cells. Its expression was severely altered in breast carcinoma, many samples showing greater than 70% of galectin-7 positive cells. High expression levels of galectin-7 were restricted to high-grade breast carcinomas, including HER2 overexpressing and basal-like groups. In HER2 overexpressing cases, galectin-7 expression was associated with lymph node axillary metastasis. Taken together, our results indicate that galectin-7 may represent a potential target for both specific detection and therapeutic inhibition of metastatic breast cancer