Sonic Hedgehog Promotes Desmoplasia in Pancreatic Cancer Jennifer M

Human Cancer Biology

Sonic Hedgehog Promotes Desmoplasia in Pancreatic Cancer Jennifer M. Bailey, Benjamin J. Swanson,Tomofumi Hamada, John P. Eggers, Pankaj K. Singh, Thomas Caffery, Michel M. Ouellette, and Michael A. Hollingsworth

Abstract Purpose: We investigated the contribution of Sonic hedgehog (SHH) to pancreatic cancer progression. Experimental Design: We expressed SHH in a transformed primary ductal-derived epithelial cell line from the human pancreas, transformed hTert-HPNE (T-HPNE), and evaluated the effects on tumor growth.We also directly inhibited the activity of SHH in vivo by administering a blocking antibody to mice challenged orthotopically with the Capan-2 pancreatic cancer cell line, which is known to express SHH and form moderately differentiated tumors in nude mice. Results: Our data provide evidence that expression of SHH influences tumor growth by contri- buting to the formation of desmoplasia in pancreatic cancer. We further show that SHH affects the differentiation and motility of human pancreatic stellate cells and fibroblasts. Conclusions: These data suggest that SHH contributes to the formation of desmoplasia in pancreatic cancer, an important component of the tumor microenvironment.

Pancreatic cancer is the fourth leading cause of cancer-related in mouse pancreatic epithelium, in concert with activated Ras deaths in the United States, resulting in 30,000 deaths each signaling, enhanced the formation of pancreatic intraepithelial year, in part because it is one of the most lethal cancers with a neoplasia and accelerated lethality, further supporting a role death-to-incidence ratio of 0.99 (1). The lethality of pancreatic for hedgehog as an early contributor to this lethal disease (6). cancer is largely due to late stage of diagnosis and resistance to We investigated the contribution of SHH to pancreatic cancer current therapies. More than 80% of patients are unresectable progression by expressing SHH in a transformed primary and most present with metastatic disease due to aggressive ductal-derived epithelial cell line from the human pancreas, localized invasion and a high incidence of early metastasis (2). transformed hTert-HPNE (T-HPNE), which was described Worldwide, the incidence of death from pancreatic cancer previously (7, 8). This cell line has been documented to has increased, which, together with the lack of current thera- represent an appropriate and important model system to pies, emphasizes the need to determine the mechanisms of delineate signaling mechanisms for Ras-mediated oncogenesis pancreatic cancer progression and better targets for diagnosis and growth of pancreatic ductal epithelial cells (8), and as such and treatment. represents a reasonable system to evaluate the superimposition The current model of pancreatic cancer progression includes of SHH expression on pancreatic cells with a defined and mutations that activate K-ras, followed by inactivating muta- limited set of transforming insults. We also directly inhibited tions or loss of expression of tumor suppressor genes including the activity of SHH in vivo using the Capan-2 pancreatic cancer INK4A p53, p16 , and SMAD4 (3). Sonic hedgehog (SHH) and cell line in an orthotopic model, which is known to express other proteins downstream of the hedgehog pathway were SHH and form moderately differentiated tumors in nude mice. recently detected in precursor lesions and samples of primary Our data provide evidence that expression of SHH contributes tumors from patients with pancreatic adenocarcinoma, which to the formation of desmoplasia in pancreatic cancer. We implicates this pathway in the initiation and progression of further show that SHH affects the differentiation and motility pancreatic cancer given its absence in normal adult pancreas of human pancreatic stellate cells and fibroblasts. These data (4, 5). Transgenic mouse models in which SHH was expressed implicate SHH as a mediator of the desmoplastic response in pancreatic cancer.

Authors’ Affiliation: Eppley Institute, University of Nebraska Medical Center, Materials and Methods Omaha, Nebraska Received 2/4/08; revised 4/24/08; accepted 5/14/08. Cell lines and culture conditions. The cell line we used for these The costs of publication of this article were defrayed in part by the payment of page experiments was hTert-HPNE. The cells were originally isolated from charges. This article must therefore be hereby marked advertisement in accordance the ductal structure of a human pancreas and immortalized by with 18 U.S.C. Section 1734 solely to indicate this fact. expressing the catalytic subunit of telomerase (h-Tert), along with Note: Supplementary data for this article are available at Clinical Cancer Research subsequent transductions with retroviral constructs to introduce a Kras Online (http://clincancerres.aacrjournals.org/). Requests for reprints: Michael A. Hollingsworth, University of Nebraska Medical mutation, the human papillomavirus E6 and E7 peptides, and the SV40 Center, 986805 Nebraska Medical Center, Omaha, NE 68198-6805. Phone: 402- small t antigen, as described previously (7, 8). The hTert-HPNE cells 559-8343; Fax: 402-559-3339; E-mail: [email protected]. were maintained in Medium D containing 1 volume of M3 base (InCell F 2008 American Association for Cancer Research. Corp.), 3 volumes of glucose-free DMEM, 5% fetal bovine serum (FBS), doi:10.1158/1078-0432.CCR-08-0291 5.5 mmol/L glucose, 10 ng/mL epidermal growth factor, and 50 Ag/mL

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stained with Diff-Quick staining kit (Allegiance)and observed by light Translational Relevance microscopy. The number of invading cells was quantified by counting the membranes at Â200 magnification. The data in this article suggest that SHH contributes Subcutaneous injections in athymic nude mice. Three million cells were injected s.c. in athymic nude mice. Before injection, the cells were in a paracrine manner to the formation of desmoplasia in trypsinized, counted, washed twice in 1Â PBS, and resuspended at a pancreatic cancer. The robust desmoplastic reaction seen density of 3 Â 106/30 AL. The cells were injected between the scapulae. in many pancreatic cancers, which includes fibroblasts, Tumor growth was monitored every 2 d by measuring the diameters endothelial cells, and other stromal cell types, is believed with a caliper. The mice were euthanized when the tumor volume to contribute to tumor progression by creating protumori- reached 1,800 mm3. genic effects in the tumor microenvironment. Desmoplasia Orthotopic implantation in athymic nude mice. One million T-HPNE also creates barriers to the delivery of therapeutic com- and T-HPNE.SHH cells were each injected into the pancreas of 12 or pounds to tumor cells. Inhibition of desmoplasia would 13 mice, respectively, to analyze tumor desmoplasia in response to inhibit tumor growth and progression, and elimination of overexpression of SHH. Preparation of cells was as described above for desmoplasia would be predicted to enhance the potential the s.c. injection model. Orthotopic implantation of T-HPNE cell lines was done as described previously (9). of delivery of some therapeutics to pancreatic tumors. SHH inhibition in orthotopic models. To inhibit SHH in the Capan-2 orthotopic models, we administered the 5E1 antibody by i.p. injections at a dosage of 500 Ag/mouse once a week for 10 wk. An IgG control gentamicin. The cells were cultured at 37jC in a humidified atmosphere antibody, 4E11, was also given to a separate group of mice at a A containing 5% CO2. concentration of 500 g/mouse, once per week. For overexpression of SHH, the cDNA of SHH was cloned into the Confocal and immunohistochemical analyses. To analyze cell mor- pWZL-blast retroviral vector [courtesy of Dr. Wade Bushman (Uni- phology in the orthotopic tumors, primary tumors were resected and versity of Wisconsin Medical School, Madison, WI)and Dr. Michel placed in formalin for 24 h before being fixed for staining. Staining Ouellette (Eppley Institute, Omaha, NE)]. Clones expressing SHH were with H&E allowed for the visualization of cells within the tumor isolated by serial dilutions and selection with blastocidin. A control sections. Fibroblasts were visually differentiated from tumor cells on the vector was transduced into the control cell lines. basis of cellular morphology and by staining with the markers The Capan-2 pancreatic cancer cell line is maintained in McCoy’s 5A described below. medium and are cultured at 37jC in a humidified atmosphere To identify SHH in the primary human tumor sections, rabbit containing 5% CO2. Pancreatic stellate cells and fibroblasts were anti-SHH primary antibody (1:200; Santa Cruz)was incubated on isolated from an islet transplant program at the University of Nebraska paraffin-embedded tissue sections for 2 h at 37jC. The secondary Medical Center and are maintained in RPMI medium at 37jCina antibody (antirabbit, 1:1,000)was incubated for 50 min at 37 jC. a humidified atmosphere containing 5% CO2. Confirmation of desmoplasia included analysis of reactivity with - Western blot analysis. Whole cell lysates were prepared by adding smooth muscle actin (SMA; 1:400; Sigma), collagen I (1:50; Abcam), 1.5-mL cell lysis buffer [10 nmol/L Tris-HCl (pH 8.0), 150 mmol/L and fibronectin (1:50; Abcam). The secondary antibody (antimouse, NaCl, 1 mmol/L phenylmethylsulfonyl fluoride and 1% Triton X-100] 1:1,000)was incubated for 60 min. Staining was developed with to cells grown to 80% confluence in T175 tissue culture flasks. Sixty Vectastain avidin-biotin complex reagent and 3,3¶-diaminobenzidine micrograms of protein were loaded onto 4% to 20% Novex Tris- substrate. glycine gradient denaturing polyacrylamide gels (Invitrogen)in a 1 Â For confocal microscopy, anti–ribonuclear protein (1:20; Chem- SDS-PAGE running buffer, containing 1 g/L SDS, 3 g/L Tris-base, and icon)and anti-SMA (1:500; Sigma)were incubated on paraffin- 14.4 g/L glycine. The proteins were transferred onto polyvinylpyrolidine embedded tissue sections for 1 h at room temperature. Secondary difluoride membranes by electrophoresis. Once transferred, the reagents, antimouse IgM-Alexa 647, and antimouse IgG-Alexa 488 membranes were blocked in Blotto [5% dry milk resuspended in 1Â (1:2,000)were incubated for 1 h. TBS (0.9% NaCl, 10 mmol/L Tris pH 7.4, and 0.5% MgCl)] overnight at Primary cell culture. Primary human pancreatic stellate cells and 4jC. Anti-SHH primary antibody was incubated at 1:200 dilution at myofibroblasts were isolated as described previously (7, 10). Cells were room temperature for 2 h. The membrane was washed thrice, 10 min monitored for 3 wk and were identified by cellular morphology and by each time, before the addition of the secondary antibody (goat anti- analysis with markers described above as cells of mesenchymal lineage rabbit HRP)at 1:5,000 dilution for 50 min. Immune complexes were or pancreatic myofibroblasts. identified with enhanced chemiluminescence Western blotting detec- In vitro wound healing assays. Wound healing assays were done tion reagents. Membranes were stripped and subsequently blotted with with the pancreatic myofibroblasts. Cells (30,000)were seeded in anti–h-actin antibody as a loading control. 24-well plates and grown into a monolayer. A pipette was used to make Invasion assay. The in vitro invasive potential of cells overexpressing a scratch through the monolayer and medium was replaced. RPMI SHH was assayed using the Biocoat Matrigel Invasion Chamber (Becton containing 7% FBS was used as a control. RPMI containing 7% FBS and Dickinson Labware). The invasion assay with pancreatic myofibroblasts 0.1 Ag/mL rhSHH and RPMI containing 7% FBS, 0.1 Ag/mL rhSHH, and included an initial seeding of 50,000, 250,000, or 500,000 T-HPNE or 0.1 Ag/mL 5E1 (Iowa hybridoma bank)served as the experimental T-HPNE.SHH cells in the lower chambers. The cells were allowed to conditions. adhere for 8 h. The medium was then removed and replaced with RPMI Statistical analysis. The in vitro colony forming, wound healing medium containing 7% FBS. In the upper chambers, 250,000 pancreatic assays, and invasion assays were analyzed with one-way ANOVA test as myofibroblasts were seeded and then allowed to incubate for 22 h at part of the Prism statistical analysis package. Significance was

37jC and 5% CO2. For invasion with the pancreatic fibroblasts with determined at the 95% confidence interval. rhSHH, 250,000 cells were seeded in the upper chamber in RPMI medium containing 7% FBS. The lower chambers included control RPMI with 7% FBS, 0.1 Ag/mL recombinant SHH (R&D Systems), and Results 1.0 Ag/mL recombinant SHH. These chambers were also allowed to sit Inhibition of SHH signaling decreases desmoplasia. We for 22 h at 37jC and 5% CO2. For analyses of all invasion assays, cells on the upper portion of the matrix membrane were wiped off with a cotton- evaluated the contribution of SHH to the growth properties of tipped swab and cells on the bottom of this membrane were fixed and pancreatic tumor cells by administering monoclonal antibody

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5E1 (11), a blocking antibody to SHH, to mice challenged desmoplastic reaction in tumors derived from the Capan-2 cell orthotopically with Capan-2, which expresses moderately high line with and without inhibition of SHH by treatment with levels of SHH and forms moderately differentiated pancreatic antibody 5E1. Tumors in animals treated with the control tumors with desmoplasia (12). One million Capan-2 cells were antibody, 4E11, showed dense desmoplastic reactions. Tumors implanted orthotopically into the pancreas of athymic nude derived from mice treated with the 5E1 showed minimal mice and 500 Ag of 5E1 were administered once per week. evidence of desmoplasia. We confirmed that areas of desmoplasia An isotype control IgG antibody, 4E11, was given to a control contained myofibroblasts by staining the tumor sections with group of mice. Treatment with 5E1 resulted in a decreased SMA (Fig. 1B). Morphometric analysis revealed a significant tumor volume from an average of f0.45 cm3 (Capan-2 decrease in the number of SMA+ cells between control tumors controls)to f0.1 cm3 (5E1-treated mice). and tumors from mice treated with 5E1 (Fig. 1C). Histologic and immunohistologic analyses of tumor sections SHH expression promotes a desmoplastic reaction when over- revealed that inhibiting SHH with 5E1 significantly decreased the expressed in transformed HPNE cells. To further investigate the degree of desmoplasia. Figure 1 shows staining of tumor sections role of SHH in the production of pancreatic cancer desmo- with H&E and with the mesenchymal marker for differentiated plasia, we overexpressed SHH in a transformed primary fibroblasts, a-SMA. The images in Fig. 1A are representative of the pancreatic epithelial cell line, T-HPNE. SHH overexpression

Fig. 1. Inhibition of SHH signaling decreases desmoplasia in Capan-2 orthotopic tumors. A, histologic analysis of tumors derived following orthotopic challenge with Capan-2 and treatment with a blocking antibody for SHH (5E1), an isotype control (4E11), or no treatment (Capan-2). H&E staining, at Â100 magnification, shows reactive stroma and desmoplasia in the Capan-2 and control 4EII-treated Capan-2 tumors (see black arrows).The 5E1-treated Capan-2 tumors showed little or no evidence of desmoplasia by histologic examination. B, immunohistochemistry with the mesenchymal marker SMA. Paraffin-embedded sections of tumors that arose from orthopic implantation of Capan-2 cells and treatment with a blocking antibody for SHH (5E1), an isotype control (4E11), or no treatment (Capan-2). Brown coloration, positive staining for SMA. Counterstaining was done with hematoxylin (blue). C, morphometric quantification of the relative amount of SMA+ desmoplasia in the Capan-2 control, 4E11-treated, and 5E1-treated tumors. The 5E1tumors showed a significant decrease in the amount of SMA+ desmoplasia. Relative SMA+ areas on the tumor sections were quantified by calculating the total cellular content of the section and expressing the results as a percentage: (SMA+ desmoplasia / all cell types in total area) Â 100. Two representative results of 5E1-treated Capan-2 primary tumors showed some staining with SMA, but the areas of staining were individualized fibroblasts representing <20% of the section. Inhibition of SHH significantly decreased the amount of SMA+ desmoplasia in Capan-2 tumors (n = 5 per group).

www.aacrjournals.org 5997 Clin Cancer Res 2008;14(19) October 1,2008 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. Human Cancer Biology in this cell line was confirmed by Western blot (Supplemen- and the reactivity was restricted to the area around pancreatic tary Fig. S1). T-HPNE and T-HPNE cells expressing SHH (T- ducts, which have previously been shown to stain positive. HPNE.SHH)were implanted orthotopically into the pancreas Figure 2C shows morphometric analysis of tumors derived of athymic nude mice. Both T-HPNE cells and T-HPNE.SHH from the T-HPNE and T-HPNE.SHH cells and indicates a signi- formed poorly differentiated tumors at the orthotopic site. ficant increase in desmoplasia in the tumors derived from the Expression of SHH in the T-HPNE cells resulted in an increase T-HPNE.SHH cells. in mean tumor weight from f1 g (T-HPNE)to f3 g (T- We stained adjacent sections with collagen I and fibronectin HPNE.SHH). There was dense desmoplasia in tumors produced to determine if the myofibroblasts synthesized these two well- by the T-HPNE.SHH cell line, whereas desmoplasia was characterized components of desmoplasia. Figure 3A shows minimal in tumors derived from T-HPNE cells not expressing immunohistochemical staining of tumors derived from the SHH, as evidenced by morphologic evaluation and immuno- T-HPNE.SHH cell line and confirms that the area containing histochemical analysis of the orthotopic tumors derived from myofibroblasts and desmoplasia also stained positive for the T-HPNE.SHH cell line with SMA to confirm that cells in the fibronectin and collagen I. Moreover, there were few myofi- desmoplastic reaction were myofibroblasts. Our analysis broblasts in the tumors derived from the T-HPNE cell line showed a significant increase in cells that stained positive for (Fig. 3A), and immunohistochemical analysis of these sections this mesenchymal marker within the tumor and at the showed an absence of collagen I and fibronectin. periphery (Fig. 2A and B). Tumors derived from the T-HPNE In Fig. 3B, we confirmed the presence of collagen I and cell line showed minimal reactivity with a-SMA (Fig. 2A), fibronectin by immunohistochemistry to stain for these

Fig. 2. SHH promotes desmoplasia in an orthotopic model of pancreatic cancer. A and B, histologic analysis of orthotopic pancreatic tumors derived from theT-HPNE.SHH cell line. Tumor sections were stained for the marker SMA to determine if there was a difference in the amount of SMA+ desmoplasia in the SHH-expressing tumor cells. Brown, positive staining for SMA. A, peripheral sections of T-HPNE and T-HPNE.SHH pancreatic tumors, at Â40 magnification, representing the large desmoplastic reaction in the periphery of theT-HPNE.SHH orthotopic tumors (note black arrows). Infiltrating SMA+ cells are also indicated with a black arrow. B, immunohistochemical analysis with SMA to stain for myofibroblasts in theT-HPNE and T-HPNE.SHH tumor sections; Â200 magnification. Brown, positive staining; black arrows, SMA+ cells. C, morphometric analysis of desmoplasia inT-HPNE versusT-HPNE.SHH orthotopic tumors. Analysis was done by quantifying SMA+ areas on the tumor sections relative to the total cellular content and expressing the results as a percentage: (SMA+ desmoplasia / total area) Â 100. Tumors derived from theT-HPNE.SHH cell line showed significantly more desmoplasia than the tumors derived from theT-HPNE cells (n = 13 per group).

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Fig. 3. Expression of collagen I and fibronectin in orthotopic tumor sections. A, representative immunohistochemical analysis ofT-HPNE andT-HPNE.SHH tumor sections to determine if fibronectin and collagen I are expressed in either of the tumor sections. Areas of desmoplasia were identified by staining for SMA (described in Figs.1and 2). Evidence that the SMA+ areas were expressing fibronectin and collagen I is indicated by brown staining in the areas of the tumor sections designated desmoplasia in the T-HPNE.SHH tumor sections.We did not observe positive staining in tumors derived from theT-HPNE cell line. H&E staining of the tumor sections is also shown. B, representative immunohistochemical staining for collagen I and fibronectin in the Capan-2 orthotopic tumors with no treatment, treated with 4E11, or treated with 5E1. There was a decrease in the intensity and amount of staining for both collagen I and fibronectin in the tumor sections treated with 5E1when compared with the controls.

components in tumors derived from untreated mice challenged Myofibroblasts express Gli-1. We sought to determine if with the Capan-2 cell line. There is a decrease in both the SHH was stimulating myofibroblasts by a paracrine mechanism. intensity and quantity of both collagen I and fibronectin staining Figure 4A shows immunohistochemical analysis of human in the Capan-2 tumors derived from the mice treated with 5E1. pancreatic cancer tissue obtained from autopsy, showing that

www.aacrjournals.org 5999 Clin Cancer Res 2008;14(19) October 1,2008 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. Human Cancer Biology tumor cells stain positive for SHH, whereas the surrounding in the human tumors, raising the possibility that these were fibroblasts and areas of desmoplasia were positive for Gli-1, stellate cells that were progenitors to the myofibroblasts whose expression is known to be induced following stimulation observed in Capan-2 tumors when SHH signaling was not with SHH. These data support the hypothesis that fibroblasts inhibited (Fig. 5A). Capan-2 tumors were stained for cyclin D1, that make up the desmoplastic reaction in human pancreatic which was detected in the myofibroblasts, supporting the hypo- cancer show evidence of paracrine signaling in response to SHH thesis that these represented stellate cells within the tumor that produced by human pancreatic cancer cells. were proliferating in response to SHH stimulation (Fig. 5B). We also analyzed adjacent sections of T-HPNE.SHH tumors We isolated pancreatic stellate cells from the human pancreas with antibodies to SMA and Gli-1, which showed evidence that using previously described techniques (7, 10)and confirmed SMA+ myofibroblasts expressed Gli-1, supporting the hypothesis their lineage by PCR to identify markers of both epithelial and that these cells were responding to SHH produced by the mesenchymal lineage. Consistent with the anticipated pheno- T-HPNE-SHH cells in a paracrine manner (Fig. 4B). type for pancreatic stellate cells, the isolated cells expressed Myofibroblasts and SMA+ cells are of murine origin. We used higher levels of SMA and vimentin and displayed a spindle- a human-specific antibody to a ribonuclear protein and an like morphology (Supplementary Fig. S2). We stimulated these antibody that binds to both murine and human SMA to cells with rhSHH at 1 and 10 Ag/mL for 24 hours and observed determine if the SMA+ cells were of human or murine origin. an increase in the mesenchymal markers SMA, vimentin, and Figure 5A presents confocal analysis of expression of these desmin and a decrease in the epithelial marker cytokeratin 19 markers in orthotopic tumors derived from human T-HPNE, (Fig. 5C). These data are the first to suggest that SHH induces the T-HPNE.SHH, Capan-2, and 5E1-treated Capan-2 tumors. The differentiation of pancreatic stellate cells into myofibroblasts. majority of these cells were clearly of murine origin and showed SHH in vitro promotes the invasion of human pancreatic myofibroblastic morphology. myofibroblasts. We analyzed the ability of human myofibro- SHH promotes the differentiation and proliferation of pan- blasts from the pancreas to respond to SHH in Matrigel creatic stellate cells. Confocal analysis of the 5E1-treated invasion assays to determine if SHH contributes to increased Capan-2 tumors (Fig. 5A)revealed single SMA + cells embedded desmoplasia by enhancing the motility and invasive capacity of

Fig. 4. SHH paracrine signaling between tumor and stroma. A, representative immunohistochemical analysis of primary pancreatic ductal adenocarcinoma tissue sections from patients with pancreatic cancer. Brown color, positive staining for Gli-1or SHH.We observed positive staining for SHH in the tumor cells, whereas there was minimal staining in the tumor cells for Gli-1 (black arrows, positive staining; white arrows, negative staining).We observed minimal staining for Gli-1in the tumor cells and observed reactivity with the Gli-1antibody in areas of desmoplasia. B, immunohistochemical analysis of T-HPNE.SHH tumors, showing both SMA and Gli-1staining; Â200 magnification. The brown staining in adjacent sections indicates that the SMA+ myofibroblasts infiltrating theT-HPNE.SHH tumor section are expressing Gli-1 (n =3).

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Fig. 5. SHH enhances pancreatic stellate cell proliferation and differentiation and enhances pancreatic myofibroblast invasion. A, confocal microscopy ofT-HPNE and T-HPNE.SHH tumors (as indicated) using antihuman ribonuclear protein (blue)andanti-SMA(green). Differential interference contrast microscopy (DIC)plusoverlay shows that in theT-HPNE.SHH tumors, there are cells positive for SMA but not human ribonuclear protein, which indicates that the majority of the myofibroblasts are of murine origin. Confocal analysis of anti ^ ribonuclear protein (RNP) or SMA expression on Capan-2 control tumors and tumors treated with the 5E1inhibitor (as indicated). Blue, antihuman ribonuclear protein; green, SMA. In the 5E1-teated Capan-2 tumors, isolated SMA+ cells can be identified as green in the overlay. B, serial sections showing SMA and cyclin D1staining of SMA+ myofibroblasts in Capan-2 tumors (brown stain). C, real-time PCR analysis on markers of mesenchymal and epithelial differentiation done on pancreatic stellate cells after 24-h stimulation with recombinant human SHH at either 1or 10 Ag/mL. SHH stimulation increased the expression of desmin, vimentin, and SMA and decreased the expression of cytokeratin 19. these cells. Human pancreatic myofibroblasts, which express We also conducted an in vitro wound healing assay to the markers vimentin and SMA and lack expression of determine if SHH increased the ability of the myofibroblasts in cytokeratin 19 (Supplementary Fig. S3A), were isolated and a monolayer to migrate into a wound. Figure 6C and D shows shown to express Gli-1 when stimulated with SHH (Supple- the visual and quantitative results from this experiment. After mentary Fig. S3B). Figure 6A shows the results of a Matrigel 48 hours, SHH significantly increased the ability of the invasion assay in which increasing numbers of T-HPNE and myofibroblasts to recover from the wound (90% recovery with T-HPNE.SHH cells were seeded in the lower chamber and SHH versus 45% recovery in the control cells and cells with myofibroblasts were seeded in the upper chamber. For cultures SHH and the 5E1 SHH inhibitor). These experiments support containing 250,000 and 500,000 T-HPNE cells expressing the hypothesis that SHH increases the migration and invasion SHH, there was a significant increase in the invasive capacity of human pancreatic myofibroblasts in vitro. of myofibroblasts as compared with T-HPNE controls. We repeated this experiment with recombinant SHH in the lower Discussion chamber (rhSHH, R&D Systems). Figure 6B shows that there was a concentration-dependent and significant increase in the Hedgehog signaling was first identified and characterized in ability of the myofibroblasts to invade through a Matrigel Drosophila and has been shown to play a role in organogenesis membrane in response to exposure to rhSHH. and differentiation. During vertebrate patterning, hedgehog

www.aacrjournals.org 6001 Clin Cancer Res 2008;14(19) October 1,2008 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. Human Cancer Biology signaling induces differentiation of the skeleton, central ultimately SHH transduces a signal that induces nuclear nervous system, early gut endoderm, limb axes and ventral translocation of Gli, a target transcription factor, and the somite buds (13, 14). Interestingly, SHH has not been shown transcriptional activation of hedgehog target genes, including to contribute to normal differentiation of the pancreas, and Ptch, Gli, and hedgehog interaction protein, Hhip (18, 19). ectopic expression in the epithelia of the embryonic pancreatic Several reports have implicated the misregulation of the mesoendoderm leads to malformation of the pancreatic hedgehog signaling pathway in the initiation and progression mesoderm and an increase in the number of mesenchymal of pancreatic cancer (5, 20). Mutations in this pathway are cells in the developing pancreatic compartment (4). associated with cancer initiation and progression, as mutations There are three known human hedgehog family members: in the tumor suppressor Patched are characterized in basal Sonic (SHH), Indian (Ihh), and Desert (Dhh). After translation cell carcinoma, medulloblastoma, and rhabdomyosarcoma, of the 45-kDa protein, the hedgehog proteins undergo an whereas oncogenic mutations in the downstream transcrip- autocatalytic cleavage facilitated by a cholesterol modification, tion factor Gli have been identified in glioblastoma and basal an event that yields a 19-kDa NH2-terminal protein that is cell carcinoma (21, 22). Pancreatic cancer presents a different secreted and a 25-kDa COOH-terminal protein that remains oncogenic insult within the hedgehog pathway, as SHH is up- associated with the cell (15, 16). The secreted ligand binds to a regulated in the tumor cells but absent in the normal pancreas. 12-pass transmembrane protein, Patched (Ptch), which relieves We observed that expression of SHH was correlated with the inhibitory effect of Patched on another transmembrane desmoplasia in two cell lines grown orthotopically in nude protein, Smoothened (SMO; reefs. 15–17), which in turn mice. Desmoplasia, a common and well-noted histologic enables hedgehog signaling. The complex signaling cascade feature of pancreatic cancer, has been proposed to contribute downstream of SMO activation is not well characterized, yet to progression of the disease (23); however, the molecular

Fig. 6. SHH increases the in vitro invasive capacity of primary cells from the human pancreas. A, invasion assay in Matrigel invasion chambers. T-HPNE cells orT-HPNE.SHH cells were seeded in the lower chambers at densities of 50,000, 250,000, and 500,000 per well.With 250,000 and 500,000 cells, SHH secreted from theT-HPNE.SHH cell line significantly increased the invasive potential of an immortalized human pancreatic myofibroblast cell line (*, P < 0.05). Columns, mean number of invasive cells for each cell line (n = 3); bars, SE. B, invasion assay in Matrigel invasion chambers. Recombinant SHHin the lower chamber of the invasion assay significantly increased the invasive potential of an immortalized human pancreatic myofibroblast cell line (**, P < 0.01). Columns, mean number of invasive cells for each cell line (n = 3); bars, SE. C and D, in vitro wound healing assay. C, pictures of initial time point controls and 48-h time points showing the effects of SHH stimulation on pancreatic myofibroblasts. D, quantitative analysis of the in vitro wound healing assay (percent closure). SHH-stimulated immortalized human pancreatic myofibroblasts showed a significant increase in their ability to recover from the wound (*, P = 0.05). The addition of the 5E1blocking antibody significantly inhibited the SHH-mediated wound healing ability back to control levels (*, P = 0.05).Values are shown for each cell line; bars, SD.

ClinCancerRes2008;14(19)October1,2008 6002 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. SHH, Desmoplasia, and Pancreatic Cancer processes that cause the desmoplastic reaction are not well proliferation of human pancreatic stellate cells, which can also characterized. Previous experiments in prostate cancer have be a mechanism whereby SHH induces desmoplasia. determined that SHH contributes to desmoplasia and tumor In summary, our data are significant in that they identify growth (24); thus, we propose that SHH has a similar SHH as a regulator of one critical pathologic outcome of mechanistic role in pancreatic cancer. pancreatic ductal adenocarcinoma: the production of desmo- We explored the requirement for SHH in the induction of plasia, or the infiltration of fibrous tissue into neoplasms, desmoplasia by administering a SHH-neutralizing antibody, which results from the proliferation of fibroblasts and the 5E1, to mice with Capan-2 cells injected orthotopically into increase in fibrosis within the stroma (28–30). Desmoplasia their pancreas. Capan-2 tumors express SHH and produce a is a common histologic feature of many adenocarcinomas desmoplastic reaction when grown orthotopically in mice. (31, 32). The stroma, or the complex microenvironment sur- Capan-2 tumors treated with the SHH-inhibiting antibody were rounding tumor cells, includes inflammatory cells, fibroblasts, smaller and did not elicit a desmoplastic response when extracellular matrix, and small blood and lymphatic vessels compared with the control tumors derived from the Capan-2 (33, 34). Previous experiments have shown that TGFh is a cell line that received no treatment or an isotype control mediator of fibrosis (12)in pancreatic cancer. In data not antibody. Similarly, SHH expression in T-HPNE cells (T- shown, we conducted a microarray on T-HPNE versus T- HPNE.SHH)produced larger tumors that showed significant HPNE.SHH cells and determined that the expression of SHH differences in cellular morphology. Within the tumor sections did not up-regulate the expression of TGFh. Thus, it is possible of the T-HPNE.SHH cell line, we noted the presence of that SHH directly influences the desmoplastic reaction, but it infiltrating fibroblasts as long, extended cells with elongated remains possible that SHH acts by inducing signals that interact nuclei. This fibrotic infiltration was accompanied by expression with TGFh signaling pathways (35). We have also shown that of collagen I and fibronectin, and represented further evidence SHH can act as a chemoattractant for myofibroblasts in of a desmoplastic reaction within tumors from the SHH invasion assays and in wound healing assays, where SHH overexpressing transformed cell line (25, 26). This reaction increased the ability of pancreatic myofibroblasts to recover was not observed in the tumor sections from the T-HPNE from a wound. These data suggest that SHH is a critical player cell line. in the tumor microenvironment. Further elucidation of its We furthered evaluated the effects of SHH signaling in vitro functions will lead to a better understanding of the patho- on primary pancreatic myofibroblasts and found that SHH genesis of this lethal disease and the potential for therapeutic acted as a chemoattractant. This result complements previous targeting of SHH to disrupt the microenvironment in pan- studies, which showed that cancer cells recruit fibroblasts, and creatic cancer. provides the first reported evidence that SHH, secreted from transformed pancreatic epithelia, contributes to the recruitment Disclosure of Potential Conflicts of Interest and organization of fibroblasts to enhance tumor progression (27). We also show that SHH can induce the differentiation and No potential conflicts of interest were disclosed.

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Jennifer M. Bailey, Benjamin J. Swanson, Tomofumi Hamada, et al.

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