Overexpressed Galectin-3 in Pancreatic Cancer Induces Cell Proliferation and Invasion by Binding Ras and Activating Ras Signaling

<div><p>Pancreatic cancer (PDAC) is a lethal disease with a five-year survival of 3–5%. Mutations in K-Ras are found in nearly all cases, but K-Ras mutations alone are not sufficient for the development of PDAC. Additional factors contribute to activation of Ras signaling and lead to tumor formation. Galectin-3 (Gal-3), a multifunctional β-galactoside-binding protein, is highly expressed in PDAC. We therefore investigated the functional role of Gal-3 in pancreatic cancer progression and its relationship to Ras signaling. Expression of Gal-3 was determined by immunohistochemistry, Q-PCR and immunoblot. Functional studies were performed using pancreatic cell lines genetically engineered to express high or low levels of Gal-3. Ras activity was examined by Raf pull-down assays. Co-immunoprecipitation and immunofluorescence were used to assess protein-protein interactions. In this study, we demonstrate that Gal-3 was highly up-regulated in human tumors and in a mutant K-Ras mouse model of PDAC. Down-regulation of Gal-3 by lentivirus shRNA decreased PDAC cell proliferation and invasion in vitro and reduced tumor volume and size in an orthotopic mouse model. Gal-3 bound Ras and maintained Ras activity; down-regulation of Gal-3 decreased Ras activity as well as Ras down-stream signaling including phosphorylation of ERK and AKT and Ral A activity. Transfection of Gal-3 cDNA into PDAC cells with low-level Gal-3 augmented Ras activity and its down-stream signaling. These results suggest that Gal-3 contributes to pancreatic cancer progression, in part, by binding Ras and activating Ras signaling. Gal-3 may therefore be a potential novel target for this deadly disease.</p> </div

Gal-3 expression in human pancreatic tumor tissues.

<p>A, Tissue microarray slides consisting of 125 pancreatic ductal adenocarcinomas and paired non-neoplastic pancreatic tissue samples were immunohistochemically stained using a monoclonal Gal-3 antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042699#s2" target="_blank">Materials and Methods</a>. Gal-3 expression was increased along the disease sequence-normal (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042699#pone-0042699-g001" target="_blank">Figure 1A, a,b</a>), pancreatitis (c,d) and pancreatic ductal adenocarcinoma (e,f). B. Summary of Gal-3 IHC of the PDAC tissue microarrays. The tumors were categorized into Gal3-low (combined scores ≤3) and Gal3-high (combined score ≥4) based on the percentage of Gal-3 staining in tumor cells (0, no staining; 1, ≤10%; 2, 10–50% and 3, >50%) and the staining intensity (0-negative, 1-weak, 2-moderate and 3- strong).</p

Gal-3 expression in pancreatic tumor tissues and cells from a K-RAS mutant mouse model.

<p>A. Gal-3 expression was analyzed in cells derived from normal pancreas and pancreatic tumors from K-Ras mutant mice by western blotting as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042699#s2" target="_blank">Materials and Methods</a>. B. Real-time quantitative PCR for Gal-3 RNA expression using Gal-3-specific primers after total RNA extraction from normal pancreas, pancreatitis tissues and pancreatic tumors and from isolated cells from different tumors arising in a K-Ras mutant mouse model as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042699#s2" target="_blank">Materials and Methods</a>. Fold change from determinations performed in triplicate.</p

Gal-3 binds to Ras and mediates Ras activity in PDAC cells.

<p>A. Co-Immunoprecipitation was performed in Panc-1 and MPanc96 with knock down Gal-3 using either anti-Ras or anti-Gal-3 antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042699#s2" target="_blank">Materials and Methods</a>. B. Equal amounts of protein from Panc-1 and Mpanc96 GN10 control cells or shRNA A3 cells were incubated with agarose beads coated with Raf1-RBD, and active Ras-GTP was detected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042699#s2" target="_blank">Materials and Methods</a>. C. Active Ras GTP activity was determined in BXPC-3 L-gal3 cells and control cells (V) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042699#s2" target="_blank">Materials and Methods</a>. D. Cell plasma membrane and Cytoplasmic fractions of GN10 and A3 cells from both Panc-1 and MPan96 were subjected to SDS-PAGE and then immunoblotted with anti-Ras antibody. E. Indirect immunofluorescence was performed on BXPC-3-V and BXPC-3 L-gal3 cells using anti-Gal-3 antibody (TIB166 1∶100, red) and anti-Ras antibody (1∶100, green), followed by DAPI counterstaining (blue). The merge of Gal-3 (red) and Ras (green) with DAPI (blue) is also shown.</p

Effects of Gal-3 on PDAC cell colony formation and the growth of PDAC tumor in vivo.

<p>A. Colony formation assays were performed in MPanc96 GN10 control cells and Gal-3 shRNA knock down A3 cells (top panel) or BXPC-3 V control cells and BXPC-3 Gal3 cDNA overexpressed L-gal3 cells (lower panel) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042699#s2" target="_blank">Materials and Methods</a>. MPanc96-A3 cells formed smaller and fewer colonies compared with the vector transfected control cells (GN10). In contrast, BXPC-3 L-gal3 cells formed larger and a greater number of colonies than control V cells. B. Bar graph in the right panel demonstrates the mean colony numbers after plating either MPanc96 GN10 control cells and shRNA knock down A3 cells (top) or BXPC-3 V and BXPC-3 L-gal3 (lower); p<0.001. C. Representative bioluminescence images of athymic mice 3 weeks after orthotopic implantation of GN10 and A3 pancreatic cancer cells orthotopically into the pancreas of athymic mice. D. Measurements of photons/s/cm²/steridian depicting bioluminescence area at 10% peak margin (mean ± SE) at week 3 using Xenogen IVIS as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042699#s2" target="_blank">Materials and Methods</a> (<i>n</i> = <i>5</i>). E. Tumor weights from control mice (GN10, n = 5) and Gal3 knockdown group (A3, n = 5) were weighted after mice were sacrifice at week four. F. Mouse tumor tissues from control (GN10) and Gal-3 knockdown group (A3) were immunohistochemically stained using Gal-3, Ras and phospho-ERK antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042699#s2" target="_blank">Materials and Methods</a>.</p

Increased sensitivity of pancreatic cancer cells to treatment with gemcitabine <i>in vivo</i> resulting from TM4SF1 silencing.

<p>Quantitative RT-PCR and western blotting showed the silencing of TM4SF1 in MIA PaCa-2 stably transfected with shControl or shTM4SF1. (B) Tumors induced by MIA PaCa-2 cells stably expressing shControl or shTM4SF1 and expressing the luciferase gene developed in nude mice. At the end of the experiment, the animals were analyzed using bioluminescent imaging. Weights of tumors induced by MIA PaCa-2 cells stably expressing shRNA with or without gemcitabine (100mg/kg) treatment in nude mice.The tumor weights (g) of shCtrl, shCtrl+GEM, shTM4SF1 and shTM4SF1+GEM were 0.934±0.132, 0.792±0.101, 0.750±0.149 and 0.398±0.080. TM4SF1 silencing by gemcitabine resulted in markedly lower tumor weights than in the control mice (*P < 0.05). (C) <i>In vivo</i> proliferation was measured by PCNA assay on paraffin sections from shControl and shTM4SF1 animals treated with gemcitabine. A representative image at a magnification of ×200 indicating cell proliferation is shown.</p

Expression of TM4SF1 in pancreatic cancer cell lines.

<p>Quantitative RT-PCR analysis resulted regarding the expression of TM4SF1 mRNA in human pancreatic cancer cell lines.TM4SF1 mRNA was more highly expressed in the cancer cell lines than in HPDE cells. The mRNA expression of TM4SF1 was lower in three gemcitabine-sensitive cell lines (L3.6pl, BxPC-3, SU86.86) than that in four gemcitabine-resistant cell lines (MIA PaCa-2, PANC-1, Hs766T, AsPC-1).</p

TM4SF1 Promotes Gemcitabine Resistance of Pancreatic Cancer <i>In Vitro</i> and <i>In Vivo</i>

<div><p>Background</p><p>TM4SF1 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and affects the development of this cancer. Also, multidrug resistance (MDR) is generally associated with tumor chemoresistance in pancreatic cancer. However, the correlation between TM4SF1 and MDR remains unknown. This research aims to investigate the effect of TM4SF1 on gemcitabine resistance in PDAC and explore the possible molecular mechanism between TM4SF1 and MDR.</p><p>Methods</p><p>The expression of TM4SF1 was evaluated in pancreatic cancer cell lines and human pancreatic duct epithelial (HPDE) cell lines by quantitative RT-PCR. TM4SF1 siRNA transfection was carried out using Hiperfect transfection reagent to knock down TM4SF1. The transcripts were analyzed by quantitative RT-PCR, RT-PCR and western blotting for further study. The cell proliferation and apoptosis were obtained to investigate the sensitivity to gemcitabine of pancreatic cancer cells after silencing TM4SF1 <i>in vitro</i>. We demonstrated that cell signaling of TM4SF1 mediated chemoresistance in cancer cells by assessing the expression of multidrug resistance (MDR) genes using quantitative RT-PCR. <i>In vivo</i>, we used orthotopic pancreatic tumor models to investigate the effect of proliferation after silencing TM4SF1 by a lentivirus-mediated shRNA in MIA PaCa-2 cell lines.</p><p>Results</p><p>The mRNA expression of TM4SF1 was higher in seven pancreatic cancer cell lines than in HPDE cell lines. In three gemcitabine-sensitive cell lines (L3.6pl, BxPC-3, SU86.86), the expression of TM4SF1 was lower than that in four gemcitabine-resistant cell lines (MIA PaCa-2, PANC-1, Hs766T, AsPC-1). We evaluated that TM4SF1 was a putative target for gemcitabine resistance in pancreatic cancer cells. Using AsPC-1, MIA PaCa-2 and PANC-1, we investigated that TM4SF1 silencing affected cell proliferation and increased the percentages of cell apoptosis mediated by treatment with gemcitabine compared with cells which were treated with negative control. This resistance was associated with the expression of multidrug resistance genes including ABCB1 and ABCC1. <i>In vivo</i>, silencing of TM4SF1 in MIA PaCa-2 cell lines increased the effectiveness of gemcitabine-based treatment in orthotopic pancreatic tumor models evaluated using noninvasive bioluminescent imaging.</p><p>Conclusion</p><p>These findings suggest that TM4SF1 is a surface membrane antigen that is highly expressed in pancreatic cancer cells and increases the chemoresistance to gemcitabine. Thus, TM4SF1 may be a promising target to overcome the chemoresistance of pancreatic cancer.</p></div