Galectin-1 Promotes Lung Cancer Progression and Chemoresistance by Upregulating P38 MAPK, ERK, and Cyclooxygenase-2

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. 2012 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, 2012 American Association for Cancer Research. galectin-1 is secreted in large amounts from tumor cells and

www.aacrjournals.org OF1

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 from the donors and their relatives. Our research shown to correlate positively with the expression level of was reviewed for exempt status by the Institutional Review galectin-1 (19). However, the molecular mechanisms of Board. The detailed clinicopathologic characteristics of 82 galectin-1 in progression of lung cancer remain unclear. In patients with lung cancer are listed in Supplementary Table this study, we investigated the roles of galectin-1 in lung S1. The tissue sections were deparafinized, and then the cancer and further identified which molecular pathway slides were heated in 10 mmol/L citrate buffer (pH6.0) at participated in lung tumor progression. We found that 120 C for 20 minutes for antigen retrieval. Nonspecific knockdown of galectin-1 in lung adenocarcinoma reduced binding was blocked with 3% H2O2 for 5 minutes. Then, tumor growth in vivo and inhibited cancer migration, they were reacted with galectin-1 or COX-2 primary anti- invasion, and colony formation in vitro. Furthermore, we bodies at 4 C overnight. The subsequent steps were carried surveyed tumorigenic-associated gene profiles regulated by out using LSABþ System-HRP kit (DAKO). In addition, the

OF2 Clin Cancer Res; 2012 Clinical Cancer Research

Galectin-1 Activates COX-2 in Lung Cancer

sections were counterstained with hematoxylin. A digital Invasion and migration assay pathology system for stained cells scoring was conducted by The invasive and migratory abilities of tumor cells were Aperio ImageScope (Aperio Technologies, Inc.). The score evaluated by Transwell assay (Costar, 8-mm pore; Corning) of tumor cells staining was determined by the sum of as previously described (21). In invasion assay, Transwell percentage and intensity of stained cells to evaluate the inserts were additionally coated with Matrigel (BD protein expression level (20). Biosciences).

Galectin-1 knockdown with short hairpin RNA Anchorage-independent growth assay 5 HEK293T cells (2.4 10 /mL) were used to produce LLC-1 (1,000 cells per 6-well) or A549 (2,000 cells per 6- lentiviral-expressing short hairpin RNA (shRNA) viruses well) cells suspended in 0.33% Bacto agar (Sigma-Aldrich) against galectin-1 (shGa-1) or luciferase as a negative con- were layered over 0.5% Bacto agar. After incubation for 20 trol (shLuc) by cotransfection of pLKO.1-shGal-1, pCMV- days, these cells were fixed and stained with Giemsa stain for DR8.91, and pMD.G vectors (National RNAi core Facility, calculating the number of colonies. Academia Sinica, Taipei, Taiwan). LLC-1 and A549 cells 5 (10 cells/mL) were infected with shGal-1 or shLuc in the Reporter assay m presence of 8 g/mL protamine sulfate according to the A549 cells were cotransfected with pGL2-COX-2-luc or procedures provided from National RNAi core Facility, pGL3-NFkB-luc and pRL-SV40. After 24-hour transfec- Academia Sinica. The specific target sequences by shGal-1 tion, the medium was replaced with complete medium are shown in Supplementary Table S2. containing 5 ng/mL of TGF-b1. Forty-eight hours later, the luciferase activity was measured using the Dual-Lucif- Reverse transcription PCR and quantitative reverse erase Reporter Assay System Kit according to the manu- transcription PCR facturer’s instructions (Promega). Total cellular RNA was extracted using TRIzol reagent (Invitrogen), and 5 mg of extracted RNA samples was reverse Statistics transcribed into cDNA according to the manufacturer’s Data were expressed as mean SD, and statistical sig- protocol for reverse transcription PCR (RT-PCR; Promega). nificance was assessed by the ANOVA test. For human tissue RT-quantitative PCR (RT-qPCR) was carried out by the SYBR microarray studies, a nonparametric Mann–Whitney U test Green Mix containing Thermo-Start DNA polymerase (Bio- was used to test gene expression relevance between different Rad), according to the manufacturer’s instructions with stages. For dual staining of human tissue microarrays, the regard to an ABI7700 System (Applied Biosystems). The Spearman correlation method was used to evaluate the specific primers of RT-qPCR are shown in Supplementary association of scores. A significant difference was declared Tables S3 and S4. if the P value was <0.05. Immunoprecipitation and Western blotting The supernatant or whole-cell lysates of A549 cells Results were harvested and immunoprecipitated by indicated Galectin-1 was overexpressed in NSCLC cell lines primary antibodies (1 mg) or nonspecific IgG as a neg- Initially, to examine the expression of different types of ative control which was conjugated to protein G mag- galectin in lung cancer cells, mRNA levels of galectins in netic beads (20 mL; Millipore) at 4C overnight. The LLC-1 cells were detected and normalized against the complexes were resolved by 13% SDS-PAGE and then expression of those in normal lung isolated from mice. transferred to nitrocellulose. The membrane was blotted Elevated mRNA levels of most of galectin members were with indicated primary antibodies and then with horse- found in LLC-1 cells and the highest expression of galectin-1 radish peroxidase (HRP)-conjugated secondary antibo- among them was noted (Fig. 1A). Furthermore, we con- dies. The antibodies used in this study are shown in firmed expression of galectin-1 in 9 human lung cancer cell Supplementary Table S5. lines, including adenocarcinoma, large cell carcinoma, Cytoplasm and nucleus of cells were separated by using squamous cell carcinoma, and bronchioloalveolar cell car- the CNMCS Compartmental Protein Extraction Kit (Bio- cinoma. Most of the human lung cancer cell lines highly Chain). Whole-cell lysates (60 mg) or nuclear or cytosolic expressed galectin-1 (Fig. 1B). To investigate whether galec- fractionation of cell lysates (20 mg) were separated by 13% tin-1 expression was correlated with the progression of lung SDS-PAGE and then blotted with indicated antibodies. adenocarcinoma, immunohistochemical staining was conducted on a tissue microarray containing samples from 47 Cell proliferation in vitro patients with different stages of lung adenocarcinomas (Fig. shLuc- or shGal-1–infected LLC-1 or A549 cells (2.5 1C; Supplementary Fig. S1A). Higher levels of galectin-1 in 104 cells/mL) were seeded onto 96-well plates and cultured stage III lung adenocarcinoma were evidenced compared in complete medium up to day 5. The cell proliferation was with stage I or II. In contrast, there was no significant measured at indicated time by CellTiter 96 aqueous non- correlation between galectin-1 expressions in different radioactive cell proliferation assay (MTS assay) according to stages of squamous cell carcinomas (Supplementary Fig. the manufacturer’s instructions (Promega). S1B).

www.aacrjournals.org Clin Cancer Res; 2012 OF3

Chung et al.

Figure 1. Galectin-1 was overexpressed in NSCLC cell lines. A, the mRNA levels of galectins (Gal) in mouse LLC-1 normalized against mRNA levels of lung tissue of C57BL/6 mice were detected by RT-qPCR. B, total RNA of human NSCLC cells was extracted, and galectin-1 mRNA levels were detected using RT- PCR. Whole-cell lysates of human NSCLC cells were prepared for Western blotting using anti-Gal-1 antibody. AC, adenocarcinoma; BAC, bronchioloalveolar cell carcinoma; LC, large cell carcinoma; SC, squamous cell carcinoma. C, immunohistochemical (IHC) staining was conducted using an antibody to galectin- 1 on a tissue microarray containing samples from 47 patients with stage I (n ¼ 15), stage II (n ¼ 19), and stage III (n ¼ 13) lung adenocarcinomas. Galectni-1 expression levels were veriﬁed according to the score of tumor staining in different stages of lung adenocarcinomas. D, whole-cell lysates of A549 cells were fractionated. Nuclear (N) and cytosolic (C) galectin-1 expression was detected by Western blotting. USF-2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were the nuclear and cytosolic internal control, respectively. Right, A549 cells (8 104 cells/mL) were cultured for 2 days, and then the cultured medium was replaced with serum-free medium. After 24 hours, supernatants (Sup) of cells (5 106) were subjected to immunoprecipitate (IP) and Western blotting (WB) using anti-Gal-1 antibody. WCL, whole-cell lysate control. Results are representative of 3 independent experiments. (, P < 0.01).

Next, to identify the sublocalization of galectin-1 protein shRNA (shLuc), cells infected with shGal-1 virus expressed in A549 lung cancer, cells were fractionated into cytoplasm low levels of galectin-1 (Fig. 2A; Supplementary Fig. S1C). and nucleus (Fig. 1D, top). In addition, it is well known Galectin-1 knockdown significantly inhibited both migra- that galectin-1 protein can be secreted through nonclassi- tory and invasive abilities of infected cells (Fig. 2B). Fur- cal secretory pathway (22). The supernatant of cell culture thermore, galectin-1 silencing significantly restrained the was immunoprecipitated and Western blotted using anti- anchorage-independent growth of lung cancer cells (Fig. galectin-1 antibody to detect levels of secreted galectin-1 2C) whereas no effect on the monolayer growth in vitro was (Fig. 1D, bottom). Galectin-1 protein was expressed in the observed (Supplementary Fig. S1D). To further evaluate nucleus and cytoplasm of cells. Nevertheless, it was whether galectin-1 could promote tumor growth in vivo, revealed that quite low level of galectin-1 protein was shGal-1- or shLuc-infected LLC-1 cells were injected sub- secreted from A549 cells. The secretion level of galectin- cutaneously into mice. As shown in Fig. 2D, tumor growth 1 from A549 cells was only 1.2 pg per 106 cells (data not was reduced in mice injected with shGal-1–infected cells. shown). Therefore, galectin-1 might promote migration, invasion, and anchorage-independent growth of lung cancer cells and Suppression of galectin-1 in lung adenocarcinoma cells tumor growth in mice. resulted in reduction of the cell migration, invasion, and anchorage-independent growth in vitro and tumor Intracellular galectin-1 promoted expression of COX-2 growth in vivo and PGE2 To examine the role of galectin-1 in lung cancer progres- We further explored which mechanisms contributing to sion, lentivirus-mediated delivery of galectin-1 shRNA tumor progression were regulated by galectin-1. We ana- (shGal-1) was used to reduce galectin-1 expression in lyzed tumorigenic-associated gene profiles by RT-PCR and mouse LLC-1 and human A549 cell lines. Compared with RT-qPCR in parental, shLuc-, and shGal-1–infected cells. cells infected with control virus–expressing luciferase The mRNA levels of COX-2 were significantly

OF4 Clin Cancer Res; 2012 Clinical Cancer Research

Galectin-1 Activates COX-2 in Lung Cancer

Figure 2. Galectin-1 knockdown in lung adenocarcinoma resulted in reduction of migration, invasion, anchorage-independent growth, and in vivo tumor growth. A, galectin-1 was knocked down in LLC-1 and A549 cell lines by lentiviral shGal-1 infection. Luciferase shRNA lentivirus (shLuc) was used as a control. The expression levels of galectin-1 were detected using RT-qPCR and Western blotting. B, shLuc- or shGal-1–infected cells (2 104) were seeded onto upper side of Transwell inserts and incubated for 24 hours at 37C to determine the migratory and invasive abilities. C, shLuc- or shGal-1–infected cells were seeded in soft agar for evaluating anchorage-independent tumor growth. D, shLuc- or shGal-1–infected LLC-1 cells were inoculated subcutaneously into C57BL/6 mice (n ¼ 6). Tumor volume was measured every 3 days. Results are representative of 3 independent experiments. (, P < 0.05; , P < 0.01). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. downregulated in both galectin-1 silencing of A549 and recovered in shGal-1/Gal-1 cells. Moreover, reexpression of LLC-1 cells (Fig. 3A; Supplementary Fig. S2A). VEGF was galectin-1 led to restore the migratory (Fig. 4C, left) and repressed in LLC-1/shGal-1 but not significantly affected in invasive abilities (Fig. 4C, right) as well as anchorage- A549/shGal-1 cells (Fig. 3A; Supplementary Fig. S2A). Fur- independent growth (Fig. 4D) of A549/shGal-1 cells. We thermore, the COX-2 protein expression and PGE2 release also used H522 cells that expressed lower levels of galectin-1 were examined in A549/shGal-1 and LLC-1/shGal-1 cells (Fig. 1B) to confirm the effects of galectin-1/COX-2 axis on (Fig. 3B; Supplementary Fig. S2B). We confirmed the reduc- tumor progression. H522 cells were transfected with galec- tion of COX-2 protein and PGE2 secretion as a result of tin-1 cDNA or pcDNA3 empty vector (Supplementary Fig. galectin-1 knockdown in lung adenocarcinoma cells. In S3A). We found that mRNA and protein levels of COX-2 addition, to explore whether exogenous galectin-1 contrib- were concordantly increased after overexpression of galec- uted to induction of COX-2 expression, A549 cells were tin-1 in H522 cells (Supplementary Fig. S3A). In addition, treated with human recombinant galectin-1 (100 ng/mL) tumor migration, invasion, and anchorage-independent and RT-PCR and Western blotting were carried out (Fig. cell growth were significantly upregulated in galectin-1– 3C). There was no significant increase in COX-2 expression overexpressed H522 cells (Supplementary Fig. S3B and upon treatment with exogenous galectin-1. Therefore, we S3C). suggest that endogenous but not exogenous galectin-1 Next, to prove whether COX-2 activation contributed regulates expression of COX-2. to galectin-1-mediated cancercellmigration,invasion, and in vivo tumor growth, shGal-1–infected A549 cells COX-2 activation contributed to galectin-1–promoted transiently expressed COX-2 cDNA or empty vector cancer cell migration, invasion, and anchorage- (shGal-1/COX-2 or shGal-1/pc). After overexpression of independent growth COX-2, expression of COX-2 (Fig. 4A) and PGE2 (Fig. 4B) To confirm the effects of galectin-1 on COX-2 activation, was both recovered in shGal-1/COX-2 cells, whereas shGal-1–infected A549 cells were transiently transfected mRNA and protein levels of galectin-1 did not change, with galectin-1 cDNA or pcDNA3 empty vector (shGal-1/ suggesting that galectin-1 was upstream of COX-2. Per- Gal-1 or shGal-1/pc). As expected, COX-2 expression (Sup- centage of migratory and invasive cells was rescued in plementary Fig. S2C) and PGE2 production (Fig. 4B) were COX-2–overexpressing A549/shGal-1 cells (Fig. 4C). The

www.aacrjournals.org Clin Cancer Res; 2012 OF5

Chung et al.

Figure 3. Intracellular galectin-1 knockdown repressed COX-2 expression. A, mRNA levels of tumorigenic-associated genes in shLuc- or shGal-1–infected A549 were detected using RT-PCR (top). The expression levels of galectin-1, COX-2, and VEGF were conﬁrmed by RT-qPCR (bottom). HIF, hypoxia-inducible factor; MMP, matrix metalloproteinase. B, the protein levels of COX-2 and PGE2 in shLuc- or shGal-1–infected A549 cells were determined by Western blotting and PGE2 Enzyme Immunoassay (EIA), respectively. C, shLuc- or shGal- 1–infected A549 cells treated with or without recombinant galectin-1 (rGal-1; 100 ng/mL) for 48 hours. The COX-2 induction in cells was evaluated by RT-PCR (top) and Western blotting (bottom). Results are representative of 3 independent experiments. (, P < 0.05; , P < 0.01). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

anchorage-independent growth was also restored in TGF-b1 induced COX-2 transcription via galectin-1 and shGal-1/COX-2 cells as shown in Fig. 4D. These results MAPK/NF-kB activation suggest that COX-2 activation contributes to the galectin- The expression of COX-2 is induced by several extracel- 1–mediated tumor progression. lular stimuli, one of which is growth factors, including TNF,

Figure 4. Galectin-1 mediated cancer cell migration, invasion, and anchorage-independent growth in a COX-2–dependent manner. A, A549/shGal-1 cells were transfected with pcDNA3 empty vector (pc) or COX-2 plasmids. The RNA and protein levels of transfectants were analyzed by RT- PCR and Western blotting. GAPDH, glyceraldehyde-3- phosphate dehydrogenase. B, PGE2 release from transfectants was examined by EIA assay. C, A549/shGal-1 cells reexpressing galectin-1 or COX-2 were subjected to migration and invasion assay. D, A549/shGal-1 cells reexpressing galectin-1 or COX-2 were seeded in soft agar for evaluating the 3-dimensional cell growth. Results are representative of 3 independent experiments. (, P < 0.05; , P < 0.01).

OF6 Clin Cancer Res; 2012 Clinical Cancer Research

Galectin-1 Activates COX-2 in Lung Cancer

Figure 5. TGF-b1 drove galectin-1–regulated COX-2 transcription though p38 MAPK and ERK pathway in lung adenocarcinoma. A, shLuc- or shGal-1–infected cells were treated with or without TGF-b1. One hour later, Western blotting was carried out. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B, shLacZ- or shGal-1–infected A549 cells were cotransfected with pcDNA3 (pc) or galectin-1 (Gal-1), pNF-kB-luc, and pRL-SV40. Twenty-four hours later, cells were treated with TGF-b1 for 48 hours. Luciferase reporter assay was conducted to investigate NF-kB promoter activity regulated by galectin-1. C, shLacZ- or shGal-1–infected A549 cells were cotransfected with pcDNA3 (pc) or NF-kB-p65 (p65), pXC918/COX-2-luc, and pRL-SV40. Twenty-four hours later, cells were treated with or without TGF-b1 for 48 hours. Luciferase reporter assay was conducted to examine COX-2 promoter activity regulated by NF-kB. RLU, relative luciferase units. D, shLuc- or shGal-1–infected A549 cells were subjected to immunoprecipitation (IP) using anti-Ras antibody (right) or anti-Gal-1 antibody (left) and then immunoblotted using anti-Gal-1 antibody or anti-Ras antibody. IgG, negative control. WCL, whole-cell lysate control. Results are representative of 3 independent experiments. (, P < 0.05; , P < 0.01). WB, Western blotting.

TGF, and EGF (23). Recent reports indicate that TGF-b1 shGal-1–knockdown A549 cells followed by treatment regulates COX-2 promoter activity through several signaling with or without recombinant TGF-b1proteinfor60 routes including activation of p38 MAPK, ERK1/2, or Akt minutes. The downstream mediators involved in TGF- pathways (24, 25). NF-kB is involved in p38 MAPK- and b1 signaling, including p38 MAPK, NF-kB, ERK1/2, c-jun- ERK1/2-induced COX-2 transcription by directly binding to NH2-kinase (JNK), and Akt, were analyzed by Western COX-2 promoter. Furthermore, it has been indicated that blotting. As shown in Fig. 5A, p38 MAPK and ERK1/2 TGF-b1 may trigger a Smad-dependent pathway to control phosphorylation was induced by TGF-b1, but the galectin-1 expression in metastatic mammary adenocarci- increased levels induced by TGF-b1 were much lower in noma (26). Therefore, we investigated whether galectin-1 shGal-1–infected cells than those in shLuc-infected cells. knockdown affected the COX-2 expression induced by TGF- The common downstream gene of p38 MAPK and ERK b1 in lung cancer cells. COX-2 promoter activity was ana- signaling pathways, NF-kB/p65, was also reduced upon lyzed using luciferase reporter assay upon treatment of TGF- suppression of galectin-1 expression. Nevertheless, Akt b1 in shLacZ- or shGal-1–infected A549 cells. TGF-b1 and JNK phosphorylation was not affected by TGF-b1 increased the COX-2 promoter activity (Supplementary Fig. (Fig. 5A). S2D) and protein levels of galectin-1 and COX-2 (Fig. 5A). To confirm the role of NF-kB in galectin-1–mediated However, suppression of galectin-1 in A549 cells signifi- expression of COX-2, shLacZ- or shGal-1–infected cells were cantly blocked TGF-b1–induced COX-2 promoter activity transfected with pGL3/NF-kB-luc reporter plasmid and (Supplementary Fig. S2D) and protein levels of galectin-1 treated with TGF-b1. The luciferase activity of NF-kB report- and COX-2 (Fig. 5A). er was markedly reduced by suppression of galectin-1 while We next defined the TGF-b1 signaling pathways in rescued by reexpression of galectin-1 (Fig. 5B). Moreover, galectin-1–induced COX-2 expression. We prepared the promoter activity of COX-2 suppressed by galectin-1

www.aacrjournals.org Clin Cancer Res; 2012 OF7

Chung et al.

knockdown was signiﬁcantly increased when NF-kB/p65 galectin-1 knockdown blocked the cisplatin-induced PGE2 was overexpressed in shGal-1–infected A549 cells (Fig. 5C). production (Fig. 6A, bottom). Furthermore, shGal-1– These results suggest that TGF-b1 induces COX-2 expression infected A549 cells were more sensitive to cisplatin treat- through galectin-1 and MAPK/NF-kB activation. ment than shLuc-infected cells (Fig. 6B). After cisplatin treatment for 72 hours, the percentage inhibition of cell Galectin-1 interacted with H-Ras in lung cancer cells growth was 92% in shGal-1–infected cells whereas only It has been reported that intracellular galectin-1 binds 55% inhibition in shLuc-infected cells (Fig. 6B). These oncogenic H-Ras to mediate Ras membrane anchorage and suggest that COX-2 inhibition via galectin-1 knockdown cell transformation (13). TGF-b1 promotes H-Ras–activat- sensitizes lung cancer cells to cisplatin treatment. ed tumor migration and invasion in breast cancer cells (27). Therefore, we investigated whether galectin-1 interacted Galectin-1 promoted lung cancer progression and with Ras in lung cancer cells. The association of galectin- chemoresistance by upregulating p38, ERK, and COX-2 1 and Ras was conducted by co-immunoprecipitation fol- in EGFR-mutated adenocarcinoma cells lowed by Western blotting. As shown in Fig. 5D, galectin-1 Because A549 cells harbor KRAS mutation, to explore directly interacted with Ras and galectin-1 knockdown whether galectin-1–regulated tumor progression is KRAS- reduced the association of galectin-1 and Ras in lung cancer speciﬁc or universal, the PC-9 cell line, which is known to cells. carry an EGFR exon 19 deletion and KRAS wild-type, was used. Upon knockdown of galectin-1, mRNA and protein Galectin-1 knockdown sensitized lung cancer cells to levels of both galectin-1 and COX-2 were reduced in PC-9 cisplatin treatment cells (Supplementary Fig. S4A). Galectin-1 knockdown in COX-2 induction may reduce the response of malignant PC-9 cells resulted in suppression of tumor migration, cells to cytotoxic therapy (28). To investigate the effects of invasion, and anchorage-independent cell growth (Supple- galectin-1 knockdown on chemosensitivity of lung cancer mentary Fig. S4B). The expression of p-p38 MAPK, p-ERK, cells, we treated A549/shLuc and A549/shGal-1 cells with and NF-kB/p65 was downregulated in shGal-1–infected ¼ m cisplatin (IC50 20 mol/L) for up to 48 hours (Fig. 6A and PC-9 cells (Supplementary Fig. S4C). Furthermore, shGal- B). A549/shLuc cells increased expression of COX-2 protein 1–infected lung cancer cells were more sensitive to cisplatin time dependently after cisplatin treatment. Galectin-1 treatment than shLuc-infected cells (Supplementary Fig. knockdown in A549 (A549/shGal-1) attenuated cisplatin- S4D). We suggest that either in KRAS-mutated A549 cells induced COX-2 expression (Fig. 6A, top). In addition, or in EGFR-mutated PC-9 cells, galectin-1 appears to

Figure 6. Effects of galectin-1 knockdown on drug sensitivity and clinical relevance of galectin-1 and COX-2 expression in lung adenocarcinoma. A, COX-2 expression and PGE2 production were examined by Western blotting and EIA assay upon treatment of 20 mmol/L (IC50) of cisplatin for up to 48 hours. CDDP, cisplatin; GAPDH, glyceraldehyde-3- phosphate dehydrogenase. B, shLuc- or shGal-1–infected A549 cells were cultured in 96-well plates and treated with 20 mmol/L of cisplatin for up to 72 hours. Cell growth rate was examined by MTS assay. C, statistic analysis of galectin-1 expression was compared with COX-2 in lung adenocarcinoma tissues (n ¼ 47). D, COX-2 expression levels were veriﬁed in different stages of lung adenocarcinomas (n ¼ 47), (, P < 0.01).

OF8 Clin Cancer Res; 2012 Clinical Cancer Research

Galectin-1 Activates COX-2 in Lung Cancer

regulate p38 MAPK, ERK, and COX-2 expression to promote tin-1. Intracellular galectin-1 might play much more impor- tumor progression. tant roles to regulate lung tumor progression. In this article, we showed that COX-2 activation contrib- Clinical relevance of galectin-1 and COX-2 expression uted to galectin-1–promoted lung cancer progression. in tissues of lung cancer COX-2 augments tumor angiogenesis, invasion, and resis- COX-2 is highly expressed in 70% of lung adenocarci- tance to apoptosis (39). The expression of COX-2 is induced nomas and can be detected throughout the progression of by several extracellular signals including proinflammatory a premalignant lesion to the metastatic phenotype (29). and growth-promoting stimuli. All signals converge to the To examine the clinical relevance of galectin-1 and COX-2 activation of MAPK and PI3K pathways that regulate COX-2 expression, immunohistochemical staining was con- mRNA expression (23, 24). After treatment of TGF-b1, p38 ducted on tissue microarrays containing samples from MAPK and ERK1/2 was induced but abrogated by suppres- 82 patients with lung cancer, including small cell lung sion of galectin-1, indicating that galectin-1 may be an cancer and NSCLC. A positive correlation between galec- upstream mediator of p38 MAPK and ERK signaling. In tin-1 and COX-2 expression in lung cancer was shown addition, the transcriptional activity of COX-2 was induced (Supplementary Fig. S5A). In addition, galectin-1 expres- under TGF-b1 treatment and inhibited by galectin-1 knock- sion was correlated with COX-2 expression (Fig. 6C) in 47 down (Fig. 5A). Therefore, TGF-b1–induced COX-2 mRNA patients with lung adenocarcinomas. Higher levels of expression was galectin-1–dependent. COX-2 in stage III lung adenocarcinoma were addressed Galectin-1 is a critical scaffolding protein and a major than in stage I (Fig. 6D; Supplementary Fig. S5B). How- regulator of H-Ras nanoclusters that contributes to Ras ever, in squamous cell carcinomas (n ¼ 17), although membrane anchorage and tumor transformation (13). It clinical relevance between galectin-1 and COX-2 expres- accords with our finding that galectin-1 and H-Ras are sion was revealed (Supplementary Fig. S5C), neither directly associated in A549 cells (Fig. 5D). Furthermore, galectin-1 nor COX-2 expression was correlated with the TGF-b1 activates H-Ras to promote malignant progression progression of squamous cell carcinoma (Supplementary of cancer (27). Therefore, we suggest that galectin-1–acti- Figs. S1A and S5D). Taken together, expression levels of vated p38 MAPK and ERK pathways, which were induced by bothgalectin-1andCOX-2werecorrelatedwiththe TGF-b1, may result from interaction with H-Ras and sub- progression of lung adenocarcinoma, and furthermore, sequently upregulated COX-2 expression (Supplementary a significant positive correlation between galectin-1 and Fig. S5E). It has been investigated that COX-2 promoter COX-2 expression in lung cancer was revealed. activity is positively regulated by NF-kB translocation. COX- 2 expression is induced by TNF-a depending heavily on Discussion activation of NF-kB (23, 40). We also found that under the In this study, we showed that endogenous galectin-1 may treatment of TGF-b1, galectin-1 would promote NF-kB promote lung cancer progression and chemoresistance by expression; NF-kB was bound to the response element of increasing p38 MAPK, ERK, and COX-2 expression. Several COX-2 promoter region to activate COX-2 mRNA transcrip- reports indicate that galectin-1 exhibits opposing effects in tion (Fig. 5A–C). Besides from promoter deletion analysis, that endogenous galectin-1 has growth-promoting whereas we noted that galectin-1 may induce promoter activity exogenous galectin-1 has growth-inhibiting effects (30, 31). within 330 to 1 proximal region of COX-2 (data not Galectin-1 has been found to induce activated T-cell apo- shown). Of note, this promoter region of COX-2 contains ptosis (9, 32–34). However, it remains highly controversial NF-kB–binding element. According to these results, we as these cells treated with high concentration of recombi- suggest that COX-2 transcription was regulated by TGF- nant galectin-1 (35, 36) or concentrated supernatant of b1/Ras/galectin-1/NF-kB axis (Supplementary Fig. S5E). tumor cell culture (37) in previous studies. The conditioned Ineffective response and high resistance of lung cancer supernatant obtained from tumor cell culture contains cells to chemotherapy result from induction of COX-2 and detectable but quite low level of galectin-1 that is not PGE2 (28, 41). We found that galectin-1 knockdown may sufficient for induction of apoptosis. More importantly, block the cisplatin-induced COX-2 and PGE2 expression induction of T-cell apoptosis requires cell–cell physical and increase the response of cancer cells to cisplatin treat- contact between T cells and tumor cells (9). In this study, ment (Fig. 6A and B). These results suggest that suppression we found that lung cancer cells secreted quite low amount of of galectin-1 may reduce the effects of COX-2–induced drug galectin-1 protein and intracellular galectin-1 promoted resistance. Moreover, galectin-1 knockdown in lung ade- COX-2 expression. Intriguingly, A549 cells could express nocarcinoma cells reduced p38 MAPK and ERK1/2 phos- a-2,3-sialyltransferase 1 (data not shown) that blocks O- phorylation induced by TGF-b1 (Fig. 5A). It has been linked glycan elongation to render resistance of exogenous reported that p38 MAPK could induce tumor dormancy galectin-1 (38). Treatment of recombinant galectin-1 did that maintains cells in a quiescent state related to drug not induce cell death of A549 cells. We hypothesize that resistance (42, 43). p38 MAPK activation induces phos- tumor cells may disrupt galectin-1–induced itself cell death phorylation of Hsp27 leading to resistance to chemother- and promote tumor progression resulting from loss of apeutic agents in myeloma (44). STAT1, a downstream susceptibility to exogenous galectin-1 via altering cell sur- mediator of p38 MAPK, has been indicated in the regulation face glycosylation and overexpression of endogenous galec- of platinum resistance in ovarian cancer (45). In addition,

www.aacrjournals.org Clin Cancer Res; 2012 OF9

Chung et al.

ERK signaling also governs drug resistance in human cancer Analysis and interpretation of data (e.g., statistical analysis, biosta- tistics, computational analysis): L.-Y. Chung, S.-J. Tang, T.-Y. Chou, T.-S. (46). Therefore, inhibition of the common upstream reg- Yeh, K.-H. Sun ulator of p38 MAPK, ERK, and COX-2 signaling, galectin-1, Writing, review, and/or revision of the manuscript: L.-Y. Chung, G.-H. will be more effective for cancer therapy. In conclusion, Sun, K.-H. Sun Administrative, technical, or material support (i.e., reporting or orga- galectin-1 not only is a critical scaffolding protein and a nizing data, constructing databases): L.-Y. Chung, S.-J. Tang, T.-S. Yeh, major regulator of H-Ras nanoclusters for tumor transfor- S.-L. Yu, K.-H. Sun mation (13) but also may contribute to tumor progression Study supervision: S.-J. Tang, G.-H. Sun, K.-H. Sun and drug resistance through p38, ERK, and COX-2 path- Acknowledgments ways. We suggest that a nontoxic anti-galectin-1 drug (galec- The authors thank Drs. Jason C. Huang for kindly providing H293T cell tin-1 knockdown) in combination with a toxic chemother- line, A. Maan-Yuh Li for kindly providing PC-9 cell line, Wolfgang Dubiel for the pcDNA3-COX-2-Flag plasmid, L. P. Ting for the pFLAG-p65 plasmid, and apeutic agent (cisplatin) may serve as a novel therapeutic Ju-Ming Wang for the pGL2-COX-2-luc reporter. modality for lung cancer. Grant Support This work was supported by National Science Council (NSC98-2320-B- Disclosure of Potential Conﬂicts of Interest 010-001-MY3, NSC100-2321-B-010-017), Taipei City Hospital, and the No potential conﬂicts of interest were declared. Ministry of Education (Aim for the Top University Plan), Taiwan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Authors' Contributions advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Conception and design: L.-Y. Chung, S.-J. Tang, G.-H. Sun, T.-S. Yeh, K.-H. this fact. Sun Acquisition of data (provided animals, acquired and managed patients, Received January 4, 2012; revised May 25, 2012; accepted May 31, 2012; provided facilities, etc.): T.-Y. Chou, S.-L. Yu, K.-H. Sun published OnlineFirst June 13, 2012.

References 1. Herbst RS, Heymach JV, Lippman SM. Lung cancer. N Engl J Med 15. van den Bru^le F, Califice S, Garnier F, Fernandez PL, Berchuck A, 2008;359:1367–80. Castronovo V. Galectin-1 accumulation in the ovary carcinoma peri- 2. Collins LG, Haines C, Perkel R, Enck RE. Lung cancer: diagnosis and tumoral stroma is induced by ovary carcinoma cells and affects both management. Am Fam Physician 2007;75:56–63. cancer cell proliferation and adhesion to laminin-1 and fibronectin. Lab 3. Breathnach OS, Freidlin B, Conley B, Green MR, Johnson DH, Gon- Invest 2003;83:377–86. dara DR, et al. Twenty-two years of phase III trials for patients with 16. Jung EJ, Moon HG, Cho BI, Jeong CY, Joo YT, Lee YJ, et al. Galectin-1 advanced non-mall cell lung cancer: sobering results. J Clin Oncol expression in cancer-associated stromal cells correlates tumor inva- 2001;19:1734–42. siveness and tumor progression in breast cancer. Int J Cancer 4. Janne PA, Engelman JA, Johnson BE. Epidermal growth factor recep- 2007;120:2331–8. tor mutations in non-small-cell lung cancer: implications for treatment 17. Wu MH, Hong HC, Hong TM, Chiang WF, Jin YT, Chen YL. Targeting and tumor biology. J Clin Oncol 2005;23:3227–34. galectin-1 in carcinoma-associated fibroblasts inhibits oral squamous 5. Kosaka T, Yatabe Y, Endoh H, Kuwano H, Takahashi T, Mitsudomi T. cell carcinoma metastasis by downregulating MCP-1/CCL2 expres- Mutations of the epidermal growth factor receptor gene in lung sion. Clin Cancer Res 2011;17:1306–16. cancer: biological and clinical implications. Cancer Res 2004;64: 18. Szoke T, Kayser K, Baumhakel JD, Trojan I, Furak J, Tiszlavicz L, et al. 8919–23. Prognostic significance of endogenous adhesion/growth-regulatory 6. Liu FT, Rabinovich GA. Galectins as modulators of tumour progres- lectins in lung cancer. Oncology 2005;69:167–74. sion. Nat Rev Cancer 2005;5:29–41. 19. Wu MH, Hong TM, Cheng HW, Pan SH, Liang YR, Hong HC, et al. 7. Thijssen VL, Poirier F, Baum LG, Griffioen AW. Galectins in the tumor Galectin-1-mediated tumor invasion and metastasis, up-regulated endothelium: opportunities for combined cancer therapy. Blood matrix metalloproteinase expression, and reorganized actin cyto- 2007;110:2819–27. skeletons. Mol Cancer Res 2009;7:311–8. 8. Almkvist J, Karlsson A. Galectins as inflammatory mediators. Glyco- 20. Zhu ZH, Sun BY, Ma Y, Shao JY, Long H, Zhang X, et al. Three conj J 2004;19:575–81. immunomarker support vector machines-based prognostic classi- 9. Kovacs-Solyom F, Blasko A, Fajka-Boja R, Katona RL, Vegh L, Novak fiers for stage IB non-small-cell lung cancer. J Clin Oncol 2009;27: J, et al. Mechanism of tumor cell-induced T-cell apoptosis mediated by 1091–9. galectin-1. Immunol Lett 2010;127:108–18. 21. Ho MY, Leu SJ, Sun GH, Tao MH, Tang SJ, Sun KH. IL-27 directly 10. Camby I, Le Mercier M, Lefranc F, Kiss R. Galectin-1: a small protein restrains lung tumorigenicity by suppressing cyclooxygenase-2-medi- with major functions. Glycobiology 2006;16:137R–57R. ated activities. J Immunol 2009;183:6217–26. 11. Rabinovich GA, Ilarregui JM. Conveying glycan information into T-cell 22. Nickel W. Unconventional secretory routes: direct protein export homeostatic programs a challenging role for galectin-1 in inflammatory across the plasma membrane of mammalian cells. Traffic 2005;6: and tumor microenvironments. Immunol Rev 2009;230:144–59. 607–14. 12. Fischer C, Sanchez-Ruderisch H, Welzel M, Wiedenmann B, Sakai T, 23. Tsatsanis C, Androulidaki A, Venihaki M, Margioris AN. Signalling Andre S, et al. Galectin-1 interacts with the a5b1 fibronectin receptor to networks regulating cyclooxygenase-2. Int J Biochem Cell Biol restrict carcinoma cell growth via induction of p21 and p27. J Biol 2006;38:1654–61. Chem 2005;280:37266–77. 24. Rodriguez-Barbero A, Dorado F, Velasco S, Pandiella A, Banas B, 13. Paz A, Haklai R, Elad-Sfadia G, Ballan E, Kloog Y. Galectin-1 binds Lopez-Novoa JM. TGF-beta1 induces COX-2 expression and PGE2 oncogenic H-Ras to mediate Ras membrane anchorage and cell synthesis through MAPK and PI3K pathways in human mesangial transformation. Oncogene 2001;20:7486–93. cells. Kidney Int 2006;70:901–9. 14. Belanis L, Plowman SJ, Rotblat B, Hancock JF, Kloog Y. Galectin-1 is a 25. Chun KS, Surh YJ. Signal transduction pathways regulating cycloox- novel structural component and a major regulator of H-ras nanoclus- ygenase-2 expression: potential molecular targets for chemopreven- ters. Mol Biol Cell 2008;19:1404–14. tion. Biochem Pharmacol 2004;68:1089–100.

OF10 Clin Cancer Res; 2012 Clinical Cancer Research

Galectin-1 Activates COX-2 in Lung Cancer

26. Daroqui CM, Ilarregui JM, Rubinstein N, Salatino M, Toscano MA, 36. Hahn HP, Pang M, He J, Hernandez JD, Yang RY, Li LY, et al. Galectin- Vazquez P, et al. Regulation of galectin-1 expression by transforming 1 induces nuclear translocation of endonuclease G in caspase- and growth factor b1 in metastatic mammary adenocarcinoma cells: impli- cytochrome c-independent T cell death. Cell Death Differ 2004;11: cations for tumor-immune escape. Cancer Immunol Immunother 2006; 1277–86. 56:491–9. 37. Rubinstein N, Alvarez M, Zwirner NW, Toscano MA, Ilarregui JM, Bravo 27. Kim E, Kim M, Moon A. Transforming growth factor (TGF)-b in con- A, et al. Targeted inhibition of galectin-1 gene expression in tumor cells junction with H-ras activation promotes malignant progression of results in heightened T cell-mediated rejection A potential mechanism MCF10A breast epithelial cells. Cytokine 2005;29:84–91. of tumor-immune privilege. Cancer Cell 2004;5:241–51. 28. Altorki NK, Port JL, Zhang F, Golijanin D, Thaler HT, Duffield-Lillico AJ, 38. Valenzuela HF, Pace KE, Cabrera PV, White R, Porvari K, Kaija H, et al. et al. Chemotherapy induces the expression of cyclooxygenase-2 in O-glycosylation regulates LNCaP prostate cancer cell susceptibility to non-small cell lung cancer. Clin Cancer Res 2005;11:4191–7. apoptosis induced by galectin-1. Cancer Res 2007;67:6155–62. 29. Hida T, Yatabe Y, Achiwa H, Muramatsu H, Kozaki K, Nakamura S, 39. Castelao JE, Bart RD, DiPerna CA, Sievers EM, Bremner RM. Lung et al. Increased expression of cyclooxygenase 2 occurs frequently in cancer and cyclooxygenase-2. Ann Thorac Surg 2003;76:1327–35. human lung cancers, specifically in adenocarcinomas. Cancer Res 40. Nakao S, Ogtata Y, Shimizu E, Yamazaki M, Furuyama S, Sugiya H. 1998;58:3761–4. Tumor necrosis factor a (TNF-a)-induced prostaglandin E2 release is 30. Yamaoka K, Mishima K, Nagashima Y, Asai A, Sanai Y, Kirino T. mediated by the activation of cyclooxgenase-2 (COX-2) transcription Expression of galectin-1 mRNA correlates with the malignant potential via NFkB in human gingival fibroblasts. Mol Cell Biochem 2002;238: of human gliomas and expression of antisense galectin-1 inhibits the 11–8. growth of 9 glioma cells. J Neurosci Res 2000;59:722–30. 41. Duarte ML, de Moraes E, Pontes E, Schluckebier L, de Moraes JL, 31. Kopitz J, von Reitzenstein C, Andre S, Kaltner H, Uhl J, Ehemann V, Hainaut P, et al. Role of p53 in the induction of cyclooxygenase-2 by et al. Negative regulation of neuroblastoma cell growth by carbohy- cisplatin or paclitaxel in non-small cell lung cancer cell lines. Cancer drate-dependent surface binding of galectin-1 and functional diver- Lett 2009;279:57–64. gence from galectin-3. J Biol Chem 2001;276:35917–23. 42. Aguirre-Ghiso JA. Models, mechanisms and clinical evidence for 32. He J, Baum LG. Presentation of galectin-1 by extracellular matrix cancer dormancy. Nat Rev Cancer 2007;7:834–46. triggers T cell death. J Biol Chem 2004;279:4705–12. 43. Feng Y, Wen J, Chang CC. p38 Mitogen-activated protein kinase and 33. Rabinocich GA, Ariel A, Hershkoviz R, Hirabayashi J, Kasai K-I, Lider O. hematologic malignancies. Arch Pathol Lab Med 2009;133:1850–6. Specific inhibition of T-cell adhesion to extracellular matrix and proin- 44. Wen J, Cheng HY, Feng Y, Rice L, Liu S, Mo A, et al. P38 MAPK flammatory cytokine secretion by human recombinant galectin-1. inhibition enhancing ATO-induced cytotoxicity against multiple mye- Immunology 1999;97:100–6. loma cells. Br J Haematol 2008;140:169–80. 34. Galvan M, Tsuboi S, Fukuda M, Baum LG. Expression of a specific 45. Stronach EA, Alfraidi A, Rama N, Datler C, Studd JB, Agarwal R, et al. glycosyltransferase enzyme regulates T cell death mediated by galec- HDAC4-regulated STAT1 activation mediates platinum resistance in tin-1. J Biol Chem 2000;275:16730–7. ovarian cancer. Cancer Res 2011;71:4412–22. 35. Brandt B, Buchse T, Abou-Eladab EF, Tiedge M, Krause E, Jeschke U, 46. McCubrey JA, Steelman LS, Chappell WH, Abrams SL, Wong EW, et al. Galectin-1 induced activation of the apoptotic death-receptor Chang F, et al. Roles of the Raf/MEK/ERK pathway in cell growth, pathway in human Jurkat T lymphocytes. Histochem Cell Biol malignant transformation and drug resistance. Biochim Biophys Acta 2008;129:599–609. 2007;1773:1263–84.

www.aacrjournals.org Clin Cancer Res; 2012 OF11

Galectin-1 Promotes Lung Cancer Progression and Chemoresistance by Upregulating p38 MAPK, ERK, and Cyclooxygenase-2

Ling-Yen Chung, Shye-Jye Tang, Guang-Huan Sun, et al.

Clin Cancer Res Published OnlineFirst June 13, 2012.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-11-3348

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2012/06/13/1078-0432.CCR-11-3348.DC1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2012/06/28/1078-0432.CCR-11-3348. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.