Comprehensive Analysis of Cellular Galectin-3 Reveals No Consistent Oncogenic Function in Pancreatic Cancer Cells Alexander Hann*., Anja Gruner., Ying Chen, Thomas M. Gress", Malte Buchholz" Department of Gastroenterology, Endocrinology and Metabolism, Philipps-University of Marburg, Marburg, Germany Abstract Galectin-3 (Gal-3), a 31 kDa member of the family of beta-galactoside-binding proteins, has been implicated in the progression of different human cancers. However, the proposed roles differ widely, ranging from tumor-promoting cellular functions and negative impact on patient prognosis to tumor-suppressive properties and positive prognostic impact. We and others have previously identified Gal-3 as overexpressed in pancreatic cancer as compared to chronic pancreatitis and normal pancreatic tissue. The purpose of this study was thus the comprehensive analysis of putative cellular functions of Gal-3 by transient as well as stable silencing or overexpression of Gal-3 in a panel of 6 well-established pancreatic cancer cell lines. Our results confirm that galectin-3 is upregulated at the mRNA level in pancreatic cancer and strongly expressed in the majority of pancreatic cancer cell lines. In individual cell lines, transient knockdown of Gal-3 expression resulted in moderate inhibitory effects on proliferation, migration or anchorage-independent growth of the cells, but these effects were not consistent across the spectrum of analyzed cell lines. Moreover, functional effects of the modulation of Gal-3 expression were not observed in stable knockdown or overexpression approaches in vitro and did not alter the growth characteristics of nude mouse xenograft tumors in vivo. Our data thus do not support a direct functional role of Gal-3 in the malignant transformation of pancreatic epithelial cells, although paracrine or systemic effects of Gal-3 expression are not excluded. Citation: Hann A, Gruner A, Chen Y, Gress TM, Buchholz M (2011) Comprehensive Analysis of Cellular Galectin-3 Reveals No Consistent Oncogenic Function in Pancreatic Cancer Cells. PLoS ONE 6(6): e20859. doi:10.1371/journal.pone.0020859 Editor: Iris Schrijver, Stanford University School of Medicine, United States of America Received April 15, 2011; Accepted May 10, 2011; Published June 16, 2011 Copyright: 2011 Hann et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded in part by the German federal ministry of education and research (BMBF) within the framework of the NGFN program of medical genome research (PaCa-Net; project ID PKB-01GS08) as well as EU FP6 grant LSHB-CT-2006-018771 (Integrated Project ‘‘MolDiag-Paca’’). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: hanna@med.uni-marburg.de . These authors contributed equally to this work. " These authors also contributed equally to this work. Introduction Pancreatic ductal adenocarcinoma, the most common form of pancreatic cancer, has an overall 5-year survival rate of ,5%. Over 80% of patients present at an advanced disease stage without the option for potential curative tumor resection [1,2]. Although chemotherapy combined with a small-molecule inhibitor targeting EGF receptor signaling has recently been shown to result in a modest, but significant increase in survival in locally advanced or metastatic tumors, the prognosis remains dismal, with median survival not exceeding 6.2 months [3]. Thus, new diagnostic and therapeutic approaches that improve outcome are urgently required. The family of beta-galactoside-binding proteins (Galectins) consists of 14 members in mammals. A common feature that distinguishes Galectins from other lectins is their carbohydrate- recognition domain. Galectin-3 (Gal-3), the 31 kDa member of this family, has been linked to a variety of tumors, albeit with widely differing roles [4]. Gal-3 overexpression in cancerous tissue has been associated with a poor prognosis in different cancer types including hepatocellular carcinoma [5,6], clear cell renal carcinoma [7,8] and bladder cancer [9]. Conversely, reduced Gal-3 expression in tumor tissue compared with normal tissue was reported in ovarian cancer [10], uterine adenocarcinoma [11], breast cancer [12,13] and cervical neoplasia [14]. In colorectal cancer, reports have been contradictory. Some authors describe a positive association of high levels of Gal-3 expression with advanced tumor stages and poor prognosis [15–17], whereas others correlate decreased expression of Gal-3 with poor prognosis [18–20]. In gastric cancer, Okada et al reported that reduced Gal-3 expression correlated with lymph node metastasis and advanced tumor stage [21], whereas two other groups identified increased expression of Gal-3 in gastric cancer, but did not find a correlation with histopathological differentiation or tumor progression [22,23]. Despite the conflicting results concerning a possible prognostic role of Gal-3 expression, some reports have described tumor- promoting functions of Gal-3 in tumor cell lines in vitro. Knockdown of Gal-3 resulted in reduced cell migration and cell growth in prostate cancer cells [24], enhanced apoptosis induction in gastric cancer cells [25], and inhibited in vitro colony formation as well as nude mouse xenograft induction in breast cancer cells [26]. We and others have previously shown that Gal-3 is consistently overexpressed in pancreatic cancer as compared to both chronic pancreatitis and normal pancreas [27–30]. However, investigations into a possible functional role of Gal-3 expression in PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e20859