A Krasg12d-Driven Genetic Mouse Model of Pancreatic Cancer Requires Glypican-1 for Efficient Proliferation and Angiogenesis
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Oncogene (2012) 31, 2535–2544 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE A KrasG12D-driven genetic mouse model of pancreatic cancer requires glypican-1 for efficient proliferation and angiogenesis CA Whipple1,2, AL Young1,2 and M Korc1,2 1Departments of Medicine and Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH, USA and 2The Norris Cotton Cancer Center at Dartmouth-Hitchcock Medical Center, Lebanon, NH, USA Pancreatic ductal adenocarcinomas (PDACs) exhibit Introduction multiple molecular alterations and overexpress heparin- binding growth factors (HBGFs) and glypican-1 (GPC1), Pancreatic ductal adenocarcinoma (PDAC) is a highly a heparan sulfate proteoglycan that promotes efficient metastatic malignancy that is the fourth leading cause signaling by HBGFs. It is not known, however, whether of cancer death in the United States, with an overall GPC1 has a role in genetic mouse models of PDAC. 5-year survival rate of o6% (Siegel et al., 2011). PDAC Therefore, we generated a GPC1 null mouse that exhibits a wide range of genetic and epigenetic altera- combines pancreas-specific Cre-mediated activation of tions including a high frequency (90–95%) of activating oncogenic Kras (KrasG12D) with deletion of a conditional Kras mutations, homozygous deletion (85%) and INK4A/Arf allele (Pdx1-Cre;LSL-KrasG12D;INK4A/ epigenetic silencing (15%) of the tumor suppressor Arflox/lox;GPC1À/À mice). By comparison with Pdx1- genes p16INK4A/p14ARF (INK4A), as well as an over- Cre;LSL-KrasG12D;INK4A/Arflox/lox mice that were wild abundance of heparin-binding growth factors (HBGFs) type for GPC1, the Pdx1-Cre;LSL-KrasG12D;INK4A/ (Korc, 2003; Schneider and Schmid, 2003). In spite of an Arflox/lox;GPC1À/À mice exhibited attenuated pancreatic improved understanding of these molecular alterations, tumor growth and invasiveness, decreased cancer cell there is a high incidence of treatment failure in PDAC, proliferation and mitogen-activated protein kinase activa- due, in part, to the resistance of pancreatic cancer cells tion. These mice also exhibited suppressed angiogenesis to chemotherapy and radiotherapy (Schneider and in conjunction with decreased expression of messenger Schmid, 2003; Kleeff et al., 2007; Korc, 2007). RNAs encoding several pro-angiogenic factors and mole- Many HBGFs, including fibroblast growth factor-2 cules, including vascular endothelial growth factor-A (FGF-2), vascular endothelial growth factor (VEGF), (VEGF-A), SRY-box containing gene (SOX17), chemo- platelet-derived growth factor and hepatocyte growth kine C-X3-C motif ligand 1 (CX3CL1) and integrin b3 factor, require the presence of the co-receptor glypican-1 (ITGB3). Moreover, pancreatic cancer cells isolated (GPC1) for efficient signaling (Berman et al., 1999; from the tumors of GPC1À/À mice were not as invasive Gengrinovitch et al., 1999; Chen and Lander, 2001). in response to fibroblast growth factor-2 (FGF-2) as GPC1 is a heparin sulfate proteoglycan (HSPG) that is cancer cells isolated from wild-type mice, and formed attached to the plasma membrane of epithelial and smaller tumors that exhibited an attenuated metastatic stromal cells via a glycosylphophatidylinositol anchor. potential. Similarly, VEGF-A and FGF-2 did not enhance GPC1, in conjunction with HBGFs, has a critical role in the migration of hepatic endothelial cells and immorta- cell growth, differentiation, adhesion and, possibly, lized murine embryonic fibroblasts isolated from GPC1 malignant transformation (Kleeff et al., 1998; Blackhall null mice. These data demonstrate in an oncogenic Kras- et al., 2001; Chen and Lander, 2001; Aikawa et al., driven genetic mouse model of PDAC that tumor growth, 2008). GPC1 is overexpressed in PDAC and is required angiogenesis and invasion are enhanced by GPC1, and for efficient HBGF signaling (Kleeff et al., 1998, 1999; suggest that suppression of GPC1 may be an important Aikawa et al., 2008). component of therapeutic strategies in PDAC. Using athymic mice in a GPC1 null background Oncogene (2012) 31, 2535–2544; doi:10.1038/onc.2011.430; and human pancreatic cancer cells in an orthotopic published online 26 September 2011 pancreatic cancer model, we have demonstrated that both host-derived and cancer cell-derived GPC1 contri- Keywords: pancreatic cancer; glypican-1; proliferation; bute to enhanced tumor growth and metastasis in angiogenesis; tumor microenvironment pancreatic cancer (Kleeff et al., 1998; Aikawa et al., 2008). It is not known, however, if GPC1 is important for both tumor initiation and progression in PDAC. Therefore, in the present study, we sought to investigate Correspondence: Dr M Korc, Department of Medicine, One Medical the impact of GPC1 on pancreatic tumor formation Center Drive, Dartmouth-Hitchcock Medical Center, Lebanon, NH and growth in a transgenic mouse model that combines 03756, USA. G12D E-mail: [email protected] the latent LSL-Kras knock-in allele, the conditional lox/lox Received 16 June 2011; revised 30 July 2011; accepted 18 August 2011; INK4A knockout allele, and either a GPC1 wild- published online 26 September 2011 type (Pdx1-Cre;LSL-KrasG12D;INK4A/Arflox/lox;GPC1 þ / þ ) Role of glypican-1 in pancreatic cancer progression CA Whipple et al 2536 or knockout (Pdx1-Cre;LSL-KrasG12D;INK4A/Arflox/lox; GPC1 þ / þ mice was significantly greater than in the GPC1À/À) allele. GPC1À/À mice (Figure 1b). At 65 days, 14 of 14 GPC1 þ / þ We now report that the absence of GPC1 results in a transgenic mice harbored large and invasive pancreatic significant decrease in tumor growth, invasiveness, tumors that adhered to and invaded surrounding organs, angiogenesis, pancreatic cancer cell proliferation and whereas 4 of 20 GPC1À/À mice did not have any tumors, mitogen-activated protein kinase (MAPK) activation and the remaining 16 mice exhibited significantly smaller in vivo. In vitro, pancreatic cancer cells isolated from tumors that were not grossly invasive or adherent tumors arising in GPC1À/À transgenic mice exhibited (Figures 1a and b). None of the mice exhibited grossly attenuated invasion in response to FGF-2 by compar- evident distant metastases. ison with pancreatic cancer cells established from Next, we carried out histological and immunohisto- transgenic GPC1 þ / þ mice. Additionally, primary hepa- chemical analysis of pancreata collected from GPC1 þ / þ tic endothelial cells and murine embryonic fibroblasts and GPC1À/À mice, using 10 pancreata per group from isolated from GPC1À/À mice were completely resistant to the 30-day-old mice, and 14 pancreata per group from FGF-2-mediated migration. These findings indicate that the 65-day-old mice. Half of the compound transgenic endogenous GPC1 contributes to pancreatic cancer mice developed pancreatic intraepithelial neoplasia progression, even when driven by oncogenic Kras in (PanIN) lesions by 30 days of age and most of these conjunction with loss of INK4A, two highly prevalent were PanIN-1, -2 lesions (Supplementary Table 1). By alterations in PDAC. contrast, 3 of the 10 GPC1 þ / þ mice exhibited PanIN-3 lesions (Supplementary Figure 1), and 1 mouse had 2 separate invasive PDACs (Supplementary Table 1), whereas only 1 of the 10 GPC1À/À mice displayed a Results PanIN-3 lesion, and none had a cancer. By day 65, 14 of 14 GPC1 þ / þ and 13 of 14 GPC1À/À mice harbored Creation of a novel transgenic mouse model invasive carcinomas. Although one GPC1À/À mouse did We generated a compound mouse model in which not have any PanIN or carcinoma, all of the transgenic pancreas-specific activation of endogenous KrasG12D and mice displayed focal areas of acinar to ductal metaplasia the loss of pancreatic INK4A expression were combined (ADM lesions). with the complete absence of GPC1. To maintain a PanIN lesions in both GPC1 þ / þ and GPC1À/À mice similar genetic background for both the GPC1 wild-type were histologically similar across all PanIN stages. (GPC1 þ / þ ) and knockout (GPC1À/À) transgenic mouse Moreover, PanIN-1A lesions in both groups of mice lines and to avoid lethality caused by homozygosity of displayed abundant mucin production as evidenced either the mutant Kras or the Pdx1-Cre alleles (Johnson by the presence of strong MUC1 immunoreactivity et al., 1997), generation of the mouse model was carried and Alcian blue staining (Figure 1c). In addition, out in a step-wise manner (Supplementary Figure 1). trichrome staining revealed that the stroma was First, to create the GPC1À/À transgenic mouse, the similarly abundant in the tumors of both groups of mouse line carrying either the conditionally expressed G12D lox/lox mice (Figure 1c). By contrast, Ki67 and CD34, which LSL-Kras mutation or the INK4A knockout are markers for proliferation and angiogenesis, respec- allele was bred separately with GPC1À/À mice. In tively (Vilar et al., 2007; Le Mercier et al., 2009), were parallel, the mice harboring the INK4Alox/lox knockout markedly decreased in tumors from GPC1À/À by com- allele and the Pdx1-Cre allele were bred to the GPC1À/À parison with GPC þ / þ mice (Figure 2). Immunofluo- mouse line. Two rounds of breeding for each line rescent staining for Ki67 indicated that proliferation was produced mice that were either Pdx1-Cre;INK4ALox/Lox; significantly reduced in both the PanIN (CK19-positive) GPC1À/À or LSL-KrasG12D;INK4ALox/Lox;GPC1À/À. The and stromal cells of tumors from the 65-day-old GPC1À/À offspring from these separate breeding strategies were transgenic mice (Figure 3). Immunofluorescent staining crossed to produce Pdx1-Cre;LSL-KrasG12D; for pMAPK was also decreased in the cancer cells within INK4ALox/Lox;GPC1À/À mice (Supplementary Figure 1). the tumors in GPC1À/À mice (Figure 4), whereas caspase-3 A similar breeding strategy was used to create the staining was similar in both groups (data not shown). GPC1 þ / þ transgenic mouse. Loss of INK4A and recom- Quantitative real-time RT–PCR (Q–PCR) of tumor bination of the mutant Kras allele were confirmed by RNA isolated from 65-day-old GPC þ / þ and GPC1À/À genotyping all the mice.