Published OnlineFirst June 13, 2012; DOI: 10.1158/1078-0432.CCR-11-3348 Clinical Cancer Human Cancer Biology Research Galectin-1 Promotes Lung Cancer Progression and Chemoresistance by Upregulating p38 MAPK, ERK, and Cyclooxygenase-2 Ling-Yen Chung1, Shye-Jye Tang6, Guang-Huan Sun3, Teh-Ying Chou4, Tien-Shun Yeh2, Sung-Liang Yu5, and Kuang-Hui Sun1 Abstract Purpose: This study is aimed at investigating the role and novel molecular mechanisms of galectin-1 in lung cancer progression. Experimental Design: The role of galectin-1 in lung cancer progression was evaluated both in vitro and in vivo by short hairpin RNA (shRNA)-mediated knockdown of galectin-1 in lung adenocarcinoma cell lines. To explore novel molecular mechanisms underlying galectin-1–mediated tumor progression, we analyzed gene expression profiles and signaling pathways using reverse transcription PCR and Western blotting. A tissue microarray containing samples from patients with lung cancer was used to examine the expression of galectin-1 in lung cancer. Results: We found overexpression of galectin-1 in non–small cell lung cancer (NSCLC) cell lines. Suppression of endogenous galectin-1 in lung adenocarcinoma resulted in reduction of the cell migration, invasion, and anchorage-independent growth in vitro and tumor growth in mice. In particular, COX-2 was downregulated in galectin-1–knockdown cells. The decreased tumor invasion and anchorage-independent growth abilities were rescued after reexpression of COX-2 in galectin-1–knockdown cells. Furthermore, we found that TGF-b1 promoted COX-2 expression through galectin-1 interaction with Ras and subsequent activation of p38 mitogen-activated protein kinase (MAPK), extracellular signal–regulated kinase (ERK), and NF-kB pathway. Galectin-1 knockdown sensitized lung cancer cells to platinum-based chemotherapy (cisplatin). In addition, galectin-1 and COX-2 expression was correlated with the progression of lung adenocarcinoma, and high clinical relevance of both proteins was evidenced (n ¼ 47). Conclusions: p38 MAPK, ERK, and COX-2 activation are novel mediators for the galectin-1–promoted tumor progression and chemoresistance in lung cancer. Galectin-1 may be an innovative target for combined modality therapy for lung cancer. Clin Cancer Res; 1–11. Ó2012 AACR. Introduction (NSCLC; ref. 1). The 5-year survival rate of most patients Lung cancer is the leading cause of cancer-associated with advanced NSCLCs is only 9% to 15% (2). Combina- death worldwide. Approximately, 85% of lung cancer is tion cytotoxic chemotherapy results in a modest increase in histologically classified as non–small cell lung cancer survival at the cost of high toxicity (3). EGF receptor- tyrosine kinase inhibitors (EGFR-TKI), such as gefitinib and erlotinib, are commonly used for patients with advanced Authors' Affiliations: 1Department of Biotechnology and Laboratory Sci- adenocarcinoma. However, clinical reports show that only ence in Medicine, National Yang-Ming University, and Department of Edu- cation and Research, Taipei City Hospital; 2Institute of Anatomy and Cell 10% of patients with NSCLCs respond to gefitinib or Biology, National Yang-Ming University; 3Division of Urology, Department of erlotinib (4, 5). Therefore, the development of novel molec- Surgery, Tri-Service General Hospital and National Defense Medical Center; ular approaches is of particular importance for combined 4Department of Pathology and Laboratory Medicine, Taipei Veterans Gen- eral Hospital; 5Department of Clinical Laboratory Sciences and Medical modality treatments of lung cancer. Biotechnology, National Taiwan University, Taipei; and 6Institute of Marine Galectins have high affinity to b-galactoside and share a Biotechnology, National Taiwan Ocean University, Keelung, Taiwan conserved carbohydrate recognition domain (6, 7). Fifteen Note: Supplementary data for this article are available at Clinical Cancer mammalian galectins have been identified and divided into Research Online (http://clincancerres.aacrjournals.org/). 3 subtypes: proto-type, chimera, and tandem-repeat type Corresponding Author: Kuang-Hui Sun, Department of Biotechnology (6). Galectins are involved in a wide range of physiologic and Laboratory Science in Medicine, National Yang-Ming University; Department of Education and Research, Taipei City Hospital, No. 155, process such as cell adhesion, cell-cycle progression, and Sec. 2, Lie-Nong St., Taipei 112, Taiwan. Phone: 886-2-2826-7228; Fax: apoptosis. Galectin-1, classified as a proto-type galectin, is 886-2-2826-4092; E-mail: [email protected] well known to be involved in the initiation, amplification, doi: 10.1158/1078-0432.CCR-11-3348 and resolution of inflammatory responses (8). In addition, Ó2012 American Association for Cancer Research. galectin-1 is secreted in large amounts from tumor cells and www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst June 13, 2012; DOI: 10.1158/1078-0432.CCR-11-3348 Chung et al. galectin-1 in lung cancer. Galectin-1 could enhance expres- Translational Relevance sion of COX-2, and its metabolite, prostaglandin E2 Targeted therapeutic approaches have been indicated (PGE2), to promote tumor progression in lung cancer. to improve standard chemotherapy. EGF receptor We suggest that TGF-b1 may promote COX-2 expression (EGFR) inhibitors have been commonly used in lung via Ras/galectin-1 to activate mitogen-activated protein cancer treatment. Unfortunately, clinical data show that kinase (MAPK)/NF-kB pathway. Our results indicate that only 10% of patients with non–small cell lung cancer MAPK/COX-2 activation is a novel mechanism that con- (NSCLC) respond to EGFR inhibitors. Our studies pro- tributes to galectin-1–promoted cancer progression and vide insight into the influence of galectin-1, which is chemoresistance in lung adenocarcinoma and that galec- increasingly recognized as an important factor of regu- tin-1 may be a potential target for drug design in lung latory proteins, in lung cancer development. Suppres- cancer therapy. sion of galectin-1 elicits antitumor activity both in vivo in vitro and and inhibits cancer-relevant signaling path- Materials and Methods ways in NSCLCs. Furthermore, galectin-1 is highly expressed in lung cancer tissues from patients with stage Cell lines III NSCLCs, indicating that galectin-1 may be a predictor Mouse Lewis lung carcinoma-1 [LLC-1; H-2b; American for outcome of patients with NSCLCs. In addition, Type Cell Collection (ATCC no. CRL-1642)] and human galectin-1 knockdown sensitized lung cancer cells to lung adenocarcinoma A549 (ATCC no. CCL-185) were platinum-based chemotherapy (cisplatin). These results obtained from BioResources Collection and Research Cen- suggest that galectin-1 may be a biomarker for prognosis ter. Human NSCLC cell lines including adenocarcinoma and an innovative target for combined modality therapy (EKVX, HOP62, NCI-H23, and NCI-H522), large cell car- for lung cancer. cinoma (HOP92 and NCI-H460), squamous cell carcinoma (NCI-H226), and bronchioloalveolar cell carcinoma (NCI- H322M) cell lines were purchased from the National Can- cer Institute (Bethesda, MD). PC-9 lung adenocarcinoma cell line was a kind gift from Dr. A. Maan-Yuh Lin (National promotes immunosuppression by inducing apoptosis of Yang-Ming University, Taipei, Taiwan). HEK293T cell line activated T cells (9). It also contributes to various steps in was a kind gift from Dr. Jason C. Huang (National Yang- tumor progression such as transformation, angiogenesis, Ming University). and metastasis (10). It has been reported that galectin-1 exhibits opposing biologic effects, depending on the phys- icochemical properties of the protein and the target cell type Mouse model b and status (11). Extracellular galectin-1–induced antiproli- Male C57BL/6 (H-2 ; 6–8 weeks of age) mice were ferative effects result from the inhibition of the Ras/mito- purchased from National Laboratory Animal Center (Tai- gen-activated protein (MAP)-ERK (MEK)/extracellular sig- pei, Taiwan). These animals were raised under specific nal–regulated kinase (ERK) pathway and transcriptional pathogen-free conditions in the Animal Center of National induction of p27 (12), whereas intracellular galectin-1 Yang-Ming University in accordance with the regulations of exhibits specificity toward H-Ras and triggers the ERK path- the Animal Care Committee of National Yang-Ming Uni- in vivo way for tumor transformation (13, 14). Moreover, galectin- versity. To determine the tumor growth , C57BL/6  1 accumulation in the peritumoral stroma of ovary and mice were subcutaneously inoculated with tumor cells (2 5 m breast carcinomas regulates both cancer cell proliferation 10 /100 L cells). Tumor volume was measured with a   3 and invasiveness (15–17). Recent studies report that caliper and calculated as length width height (in cm )at galectin-1 activates carcinoma-associated fibroblasts and intervals of 3 days. increases MCP-1 secretion of oral squamous cell carcinoma, which promotes tumor progression and metastasis (15). Tissue microarrays and immunohistochemistry The contradictory effects of galectin-1 on regulating cancer Tissue array slides were purchased from Biomax (US progression still remain to be elucidated. Biomax, Inc.). The company provided certified documents Galectin-1–expressing lung tumors have been shown to that all human tissue samples were collected with informed have poor prognosis (18). Invasiveness of tumor cells has consent