Hypoxia inducible factor-1 mediates expression of galectin-1: the potential role in migration/invasion of colorectal cancer cells

Carcinogenesis vol.31 no.8 pp.1367–1375, 2010 doi:10.1093/carcin/bgq116 Advance Access publication June 4, 2010 Hypoxia inducible factor-1 mediates expression of galectin-1: the potential role in migration/invasion of colorectal cancer cells Xu-Yun Zhao1,y, Ting-Ting Chen1,y, Li Xia1, Meng Guo1, Ying Xu2, Fei Yue3, Yi Jiang1, Guo-Qiang Chen1,2 and Ke-Wen Zhao1,
1 Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Ministry of Education of China and Chemical Biology Division of Shanghai Universities E-Institutes, Ruijin Hospital, Shanghai Jiao-Tong University School of Medicine, Shanghai 200025, China, 2Institute of Health Science, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences/Shanghai Jiao-Tong University School of Medicine, Shanghai 200025, China and 3Department of General Surgery, Ruijin Hospital, Shanghai Jiao-Tong University School of Medicine, Shanghai 200025, China The expression of galectin-1, one of the most important lectins participating in the malignant tumor development, has been shown to be regulated by hypoxia, but its exact mechanism remains elusive. Here, we find that ectopically expressed hypoxiainducible factor (HIF) 1a protein, an oxygen-sensitive subunit of HIF-1 that is a master factor for cellular response to hypoxia, significantly increases galectin-1 expression in both messenger RNA and protein levels in all four colorectal cancer (CRC) cell lines tested. However, hypoxia-induced galectin-1 expression cannot be seen in sentrin/SUMO-specific protease 1 homozygous-null mouse embryonic fibroblasts that fail to accumulate HIF-1a protein. Furthermore, silence of HIF-1a or HIF-1b expression by specific short hairpin RNAs (shRNAs) antagonizes hypoxiainduced galectin-1 expression. All these results propose that galectin-1 is a direct target of transcriptional factor HIF-1. Applying luciferase reporter assay and chromatin immunoprecipitation, we identify that two hypoxia-responsive elements located at 2441 to 2423 bp upstream to transcriptional start site of galectin-1 gene are essential for HIF-1-mediated galectin-1 expression. Finally, the knockdown of galectin-1 by its specific shRNA can significantly reduce hypoxia-induced invasion and migration of CRC cell line, and the ectopic expression of galectin-1 can remarkably restore invasion and migration abilities of HIF-1a-knocked SW620 cells, proposing that galectin-1 mediates the HIF-1induced migration and invasion of CRC cells during hypoxia. Taken together, our results shed new light for understanding mechanism for hypoxia/HIF-1-mediated migration/invasion of CRC cells. Introduction Tumor hypoxia, mostly resulting from poor perfusion and anemia, is one of the key factors to induce the development of malignant cell clones with an aggressive and also treatment-resistant phenotype that leads to rapid progression and poor prognosis (1,2). It has been well Abbreviations: CRC, colorectal cancer; FBS, fetal bovine serum; Glut-1, glucose transporter 1; HIF, hypoxia-inducible factor; HRE, hypoxia-responsive element; mRNA, messenger RNA; MEF, mouse embryonic fibroblast; NC, negative control; SENP1, SUMO-specific protease 1; shRNA, short hairpin RNA; VEGF, vascular endothelial growth factor. y These authors contributed equally to this work. Materials and methods Tissue samples and immunohistochemistry Paraffin-embedded tumor tissues and normal adjacent tissues from 40 cases of CRC were collected from Ruijin hospital of Shanghai Jiao Tong University School of Medicine (SJTU-SM). The cancer stages were performed according to tumor, lymph node, metastasis classification. The immunohistochemical analysis was performed on the 4 lm thick fraction mounted on charged slides and sectioned from each clinical sample. Then, each slide was deparaffinized in 60°C, followed by treatment with xylene and graded alcohol. After the antigen retrieval and being blocked with 5% bovine serum albumin, tissue slides were immunohistochemically stained by antibodies against galectin-1 (Santa Cruz Biotechnology, Santa Cruz, CA), vascular endothelial growth factor (VEGF) and glucose transporter 1 (Glut-1) (Abcam, Cambridge, UK), respectively, then visualized by standard avidin-biotinylated peroxidase complex method. Then, hematoxylin was used for counterstaining and morphologic images were observed with Olympus BX51 microscope. All sections were evaluated systematically for galectin-1, VEGF and Glut-1 proteins according to a scale of three stages as described (18): grade 0, ,10% immunoreactive cells; grade 1, 10–50% immunoreactive cells and grade 2, .50% immunoreactive cells. Samples in grade 0 were considered as negative expression and ones in grade 1 and 2 as positive expression. Cell culture and treatment CRC cell line LS174T, RKO, SW1116 and SW620 were cultured in RPMI1640 or Dulbecco’s modified Eagle’s medium (Sigma–Aldrich, St Louis, MI) supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Gaithersburg, MD). Mouse embryonic fibroblast (MEF) cells from sentrin/SUMO-specific protease 1 (SENP1) homozygous null (SENP1/) and wild-type (SENP1þ/þ) mice, which was provided by Dr J.K.Cheng in SJTU-SM, were cultured in Dulbecco’s modified Eagle’s medium with 10% FBS. All cell lines were cultured in 5% CO2 and 95% air in a humidified atmosphere at 37°C. For hypoxic Ó The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please email: 1367
To whom correspondence should be addressed. Department of Pathophysiology, Shanghai Jiao-Tong University School of Medicine, No.280, Chong-Qing South Road, Shanghai 200025, China. Tel/Fax: þ86 21 64154900; Email: Correspondence may also be addressed to Guo-Qiang Chen. Tel/Fax: þ86 21 64154900; Email: known that the adaptive response of cell to hypoxia is mainly mediated by hypoxia-inducible factor (HIF)-1, a transcriptional heterodimer consisting of an oxygen-sensitive HIF-1a and constitutively expressed HIF-1b/aryl hydrocarbon nuclear translocator (3,4). In the normal air (normoxia), HIF-1a is rapidly degraded by E3 ligase von Hippel-Lindau tumor suppressor-mediated ubiquitin–proteasome pathway. However, the reduced oxygen availability (hypoxia) or treatment of hypoxia-mimetic agents such as cobalt chloride can stabilize HIF-1a protein, followed by its translocation into nucleus where it forms heterodimer with its partner HIF-1b (4–7). The HIF-1a/HIF-1b heterodimer binds to consensus sequence 5#-RCGTG-3# named hypoxia-responsive elements (HREs) on promoters of its target genes such as vascular endothelial growth factor (VEGF) and glucose transporter 1, which participate in angiogenesis, erythropoiesis, energy metabolism, cell proliferation, survival and/or differentiation (4,8,9). HIF-1 and hypoxia was also shown to contribute to metastasis and invasion of cancers including colorectal cancer (CRC) (10–14). Some HIF-1-targeted genes promoting extracellular matrix remodeling and penetrati (...truncated)