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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Cholangiocarcinoma (CCA) cells paradoxically express the death ligand, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) and, therefore, are dependent upon potent survival signals to circumvent TRAIL cytotoxicity. CCAs are also highly desmoplastic cancers with a tumor microenvironment rich in myofibroblasts (MFBs). Herein, we examine a role for MFB-derived CCA survival signals. We employed human KMCH-1, KMBC, HuCCT-1, TFK-1, and Mz-ChA-1 CCA cells, as well as human primary hepatic stellate and myofibroblastic LX-2 cells, for these studies. In vivo experiments were conducted using a syngeneic rat orthotopic CCA model. Coculturing CCA cells with myofibroblastic human primary hepatic stellate cells or LX-2 cells significantly decreased TRAIL-induced apoptosis in CCA cells, a cytoprotective effect abrogated by neutralizing platelet-derived growth factor (PDGF)-BB antiserum. Cytoprotection by PDGF-BB was dependent upon Hedgehog (Hh) signaling, because it was abolished by the smoothened (SMO; the transducer of Hh signaling) inhibitor, cyclopamine. PDGF-BB induced cyclic adenosine monophosphate–dependent protein kinase–dependent trafficking of SMO to the plasma membrane, resulting in glioma-associated oncogene (GLI)2 nuclear translocation and activation of a consensus GLI reporter gene-based luciferase assay. A genome-wide messenger RNA expression analysis identified 67 target genes to be commonly up- (50 genes) or down-regulated (17 genes) by both Sonic hedgehog and PDGF-BB in a cyclopamine-dependent manner in CCA cells. Finally, in a rodent CCA in vivo model, cyclopamine administration increased apoptosis in CCA cells, resulting in tumor suppression. Conclusions: MFB-derived PDGF-BB protects CCA cells from TRAIL cytotoxicity by a Hh-signaling–dependent process. These results have therapeutical implications for the treatment of human CCA. (HEPATOLOGY 2011;)

Cholangiocarcinoma (CCA) is a highly lethal malignancy with limited treatment options.1-3 It is the most common biliary cancer, and epidemiologic studies suggest that its incidence is increasing in several Western countries.4 Human CCA in vivo paradoxically expresses the death ligand, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), and its cognate death receptors, 5 suggesting that these cancers are reliant on potent survival signals for tumor maintenance and progression. However, the mechanisms by which CCA evades apoptosis by TRAIL and other proapoptotic stimuli is incompletely understood.

CCAs are highly desmoplastic cancers, suggesting that cancer-associated fibroblasts within the tumor microenvironment contribute to their development and progression, as has been proposed for other cancers (e.g., breast cancer, prostate cancer, etc.).6, 7 Cancer-associated fibroblasts are perpetually “activated” and express alpha-smooth muscle actin (α-SMA); cells exhibiting this activated phenotype are often referred to as myofibroblasts (MFBs).8 In the liver, MFBs are derived from periportal fibroblasts, hepatic stellate cells (HSCs), and, perhaps, an epithelial-to-mesenchymal transition of cholangiocytes, hepatocytes, and/or the tumor itself.9, 10 A role for MFBs in carcinogenesis and tumor biology has only recently received attention.8, 11-13 Cross-talk between cancer and MFBs appears to be exploited by cancers as a tumor-promoting mechanism. For example, in CCA, the number of MFBs correlates with patient survival.14 MFBs also appear capable of providing survival signals, because they reduce the apoptosis of nonmalignant cholangiocytes in coculture experiments.15 However, information regarding the nature of the cross-talk,and, in particular, the identity of the potential survival signals, remains obscure.

Platelet-derived growth factor (PDGF) paracrine signaling between MFBs and cholangiocytes occurs in rodent models of biliary tract inflammation and fibrogenesis.15, 16 Five different ligands of PDGF exist, including PDGF-AA, -BB, -AB, -C, and -D. However, PDGF-BB appears to be the predominant isoform secreted by liver MFBs.17 Of the two cognate receptors, platelet-derived growth factor receptor (PDGFR)-α and -β, PDGFR-β is the cognate receptor for PDGF-BB. PDGFR-β is a receptor tyrosine kinase that is also known to alter plasma-membrane dynamics associated with cell migration by a cyclic adenosine monophosphate (cAMP)-dependent kinase (PKA)-dependent process18; thus, PDGF-BB effects on intracellular signaling cascades are pleiotropic. Given an emerging role for PDGF-BB in MFB-to-cholangiocyte cross-talk, a role for PDGF-BB as a survival factor for CCA warrants further investigation.

The Hedgehog (Hh)-signaling pathway has been strongly implicated in gastrointestinal tumor biology, including CCA.19, 20 Hh signaling is initiated by any of the three ligands, Sonic (SHH), Indian (IHH), and Desert (DHH) hedgehog. These ligands bind to the Hh receptor, Patched1 (PTCH1), resulting in activation of smoothened (SMO) and, subsequently, the transcription factors, glioma-associated oncogenes (GLI) 1, 2, and 3.21 How PTCH1 modulates SMO was enigmatic until quite recently, because the two proteins do not physically associate. SMO trafficking from an intracellular compartment to the plasma membrane apparently results in its activation.22 Hh ligand binding to PTCH1 increases the concentration of intracellular messengers (i.e., lipid phosphates), which, in turn, promote SMO trafficking to the plasma membrane.23, 24 PKA affects SMO trafficking and activation, raising the unexplored possibility that cues from other ligand-receptor systems, such as PDGF-BB, may also augment SMO activation by facilitating its trafficking to the plasma membrane.22 Interestingly, SHH messenger RNA (mRNA) expression is increased by PDGF-BB in immature cholangiocytes, 16 providing an additional link between Hh signaling and PDGF. Hh signaling may also be a master switch mediating the resistance of CCA cells to TRAIL cytotoxicity.25, 26 Taken together, these observations suggest that MFB-derived PDGF-BB may modulate Hh survival signaling in CCA cells.

The aim of this study was to examine the role for MFB-to-CCA cell paracrine signaling in mediating CCA resistance to TRAIL cytotoxicity. The results suggest that PDGF-BB secreted by MFBs protects CCA cells from TRAIL-induced apoptosis. PDGF-BB appears to exert its cytoprotective effects by an Hh-signaling–dependent manner. These observations have implications for the treatment of human CCA.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Cell Lines/Culture and Human Samples.

The human CCA cell lines, KMCH-1, KMBC, HuCCT-1, TFK-1, and Mz-ChA-1, as well as the erythroblastic leukemia viral oncogene homolog (ErbB-2)/neu-transformed malignant rat cholangiocyte cell line, BDEneu (in vivo experiment), and the LX-2 cells, an immortalized myofibroblast cell line derived from human HSCs, were cultured as previously described.5, 27-30 Human primary myofibroblastic HSCs were kindly provided by V.H. Shah (Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN) and cultured in Dulbecco's modified Eagle Medium, supplemented with 10% fetal bovine serum, penicillin G (100 U/mL), and streptomycin (100 μg/mL) under standard conditions. Human samples were collected in accord with the Declaration of Helsinki.

Generation of Enhanced Green Fluorescent Protein–Tagged SMO (GFP-SMO).

A pRK7 plasmid, containing the human SMO sequence (GenBank accession no.: NM_005631), was a generous gift from M. Fernandez-Zapico (Division of Oncology Research, Mayo Clinic). The pRK7-SMO plasmid was modified to accept the green fluorescent protein (GFP) tag first by inserting recognition sites for EcoRI and NotI at the C-terminus of SMO, replacing the stop codon. For this, a polymerase chain reaction (PCR)-generated EcoRI/NotI-modified SMO C-terminal coding sequence was inserted into pRK7-SMO. Next, GFP from the pEGFP-N1 protein fusion vector (catalog no.: 6085-1; GenBank accession no.: U55762; Clontech Laboratories, Inc., Mountain View, CA) was digested and inserted into the modified pRK7-SMO plasmid to generate an SMO construct fused to GFP at the C-terminal cytoplasmic domain. The plasmid was sequenced to confirm that the construct was in frame, and no PCR artifacts were introduced.

SMO Trafficking.

HuCCT-1 cells were cultured on coverslips, treated as indicated, and fixed with phosphate-buffered saline (PBS), containing 4% paraformaldehyde, for 20 minutes at 37°C. After being washed with PBS, cells were incubated with 0.5% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with PBS, containing 5% bovine serum albumin, for 60 minutes at 37°C. Cells were subsequently incubated with anti-SMO antiserum (H-300, 1:250; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. After being washed, coverslips were incubated with Texas Red-X goat antirabbit immunoglobulin G (T6391, 1:1,000; Invitrogen, Carlsbad, CA) for 1 hour in the dark. Cells were then washed three times in PBS, one time in water, and mounted using Prolong Antifade (Invitrogen). The slides were analyzed by fluorescent confocal microscopy (LSM 510; Carl Zeiss, Jena, Germany). In additional experiments, SMO trafficking was examined by total internal reflection fluorescence (TIRF) microscopy.31 KMCH-1 cells cultured on coverslips were transfected with GFP-SMO plasmid 48 hours before study. Cells were treated as indicated and fixed with ddH2O, containing 2.5% formaldehyde, 0.1 M of piperazine-N,N′-bis(2-ethanesulfonic acid), 1.0 mM of ethylene glycol tetraacetic acid, and 3.0 mM of MgSO4 for 20 minutes at 37°C. Cells were then washed three times in PBS, one time in water, and mounted using Prolong Antifade (Invitrogen). Slides were analyzed with a TIRF microscope (AxioObserver.Z1; Carl Zeiss). GFP-SMO localized to the plasma membrane was quantified using image analysis software (AxioVision 4.8.2.0; Carl Zeiss). Data are expressed as the average fluorescence intensity in the cell multiplied by the number of pixels above the background.

GLI Reporter Construct and Promoter-Reporter Assay.

To determine GLI activity, a reporter containing eight directly repeated copies of a consensus GLI-binding site (8×-GLI) downstream of the luciferase gene was employed (pδ51LucII plasmid; δ-crystalline promoter).32 The 8×-GLI reporter was kindly provided by M. Fernandez-Zapico (Division of Oncology Research, Mayo Clinic, Rochester, MN). The plasmid was transfected into normal, stable scrambled, or short-hairpin RNA targeting SMO (shSMO) KMCH-1 cells (0.5 μg/well), using FuGene HD (Roche Diagnosis, Basel, Switzerland). Cells were cotransfected with 50 ng of a plasmid expressing Renilla luciferase under the control of cytomegalovirus (pRL-CMV; Promega, Madison, WI). Twenty-four hours after transfection, cells were treated as indicated, cell lysates were prepared, and both firefly and Renilla luciferase activities were quantified using the Dual-Luciferase Reporter Assay System (Promega), according to the manufacturer's instructions. Firefly luciferase activity was normalized to Renilla luciferase activity to control for transfection efficiency and cell numbers. Data (firefly/Renilla luciferase activity) are expressed as fold increase over vehicle-treated cells transfected with the 8×-GLI/pRL-CMV reporter constructs.

Animal Experiments.

All animal studies were performed in accord with and approved by the institutional animal care and use committee. In vivo intrahepatic cell implantation (a syngeneic rat orthotopic CCA model) was carried out in male adult Fischer 344 rats (Harlan, Indianapolis, IN) with initial body weights between 190 and 220 g, as previously described.28-30 Cyclopamine (2.5 mg/kg body weight; 0.5 mL), complexed with 2-hydroxypropyl-β-cyclodextrin (Tocris, Ellisville, MO), as previously described, 33, 34 or vehicle was given intraperitoneally every day for 1 week (first injection: postoperative day 7; seventh injection: postoperative day 13). Twenty-four hours after receiving the last injection, rats were euthanized and livers were removed for further analysis, including histopathology and mRNA extraction. To assess the numbers of metastases-free and metastases-bearing rats, abdominal cavities, retroperitoneal spaces, and thoracic cavities were thoroughly examined as previously described.29

Supplemental Methods.

Materials, generation of shSMO KMCH-1 cells, quantitation of PDGF-BB and cAMP, coculture experiments, quantitation of apoptosis, immunoblotting analysis, immunohistochemistry for α-SMA, PDGFR-β, PDGF-BB, and cytokeratin 7 (CK7), as well as reverse-transcriptase polymerase chain reaction (RT-PCR), the genome-wide mRNA expression assay, and statistical analysis are described in the Supporting Information.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Expression of PDGF-BB by MFBs and PDGFR-β by CCA Cells.

Initially, we assessed basal PDGF-BB secretion by two human CCA cell lines, KMCH-1 and KMBC, primary HSC cells, and the human MFB cell line, LX-2, by enzyme-linked immunosorbent assay (ELISA) (monoculture conditions; Fig. 1A). The MFB cells secreted significantly higher levels of PDGF-BB than the CCA cell lines. Because many cancer cells do not express PDGF receptors, 35 we next examined KMCH-1 cells for the presence of PDGFR-β and its activating phosphorylation by PDGF-BB (Fig. 1B). Immunoblotting analysis confirmed the protein expression of PDGFR-β in KMCH-1 cells (Fig. 1B, lower), whereas PDGFR-α was not detectable (data not shown). PDGFR-β also displayed receptor phosphorylation (Tyr857) upon PDGF-BB treatment (Fig. 1B, upper). In addition, we confirmed the mRNA expression of PDGFR-β in KMCH-1 cells and four other human CCA cell lines (KMBC, HuCCT-1, TFK-1, and MzChA-1), as well as in the ErbB-2/neu-transformed malignant rat cholangiocyte cell line, BDEneu (employed in the in vivo CCA model; Supporting Fig. 1). To characterize the expression of α-SMA, PDGFR-β, and PDGF-BB in vivo, we performed immunohistochemistry for these proteins in human CCA specimens (Fig. 1C). Numerous α-SMA-positive MFBs were present in the stromal tumor microenvironment in all human CCA samples examined (Fig. 1C, left). Moreover, PDGFR-β immunoreactivity was confirmed in CCA cell glands in approximately half of the samples (Fig. 1C, middle), whereas PDGF-BB was expressed in MFBs in two-thirds of the samples (Fig. 1C, right). Thus, PDGF-BB was shown to be secreted by MFBs and its receptor was expressed by CCA cells.

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Figure 1. Cellular expression of PDGF-BB, PDGFR-β, and α-SMA in CCA. (A, B) PDGF-BB is secreted by MFBs and phosphorylates PDGFR-β in CCA cells in vitro. (A) Basal PDGF-BB secretion (24 hours; corrected for total protein expression) by two human CCA cell lines, KMCH-1 and KMBC, a human MFB cell line, LX-2, and myofibroblastic human primary HSC cells (Hu.Pr.) was assessed in serum-free supernatant by ELISA (monoculture conditions). Mean ± standard error of the mean (SEM) (n = 3). (B) KMCH-1 cells (serum-starved for 2 days) were treated with vehicle or PDGF-BB at the concentrations and time intervals indicated. Cell treatment was followed by immunoblotting analysis for PDGFR-β and phospho-PDGFR-β (Tyr857). Upper bands show the N-linked glycosylated, and lower bands show the unglycosylated forms of PDGFR-β. (C) α-SMA (MFB marker; left), PDGFR-β (middle), and PDGF-BB (right) protein expression (brown) was examined by immunohistochemistry (counterstaining with Mayers' hematoxylin) in human CCA specimens.

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MFB-Derived PDGF-BB Promotes Resistance to TRAIL Cytotoxicity.

Next, we examined the effect of coculturing KMCH-1 cells with PDGF-BB-secreting myofibroblastic human primary HSCs (Fig. 2A,C) or LX-2 cells (Fig. 2B,D) on TRAIL-induced CCA cell apoptosis. As assessed by either nuclear morphology (Fig. 2A,B) or the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay (Fig. 2C,D), KMCH-1 cells were more resistant to TRAIL-induced apoptosis when cocultured with human primary HSCs or LX-2 cells, as compared to monoculture conditions. Interestingly, KMCH-1 cells were resensitized to TRAIL (10 ng/mL) when cocultured in the presence of neutralizing antihuman PDGF-BB antiserum (Fig. 2A-D). Similar results were obtained from coculturing KMBC cells with LX-2 cells (Supporting Fig. 1A). These findings were somewhat selective for TRAIL-induced apoptosis, because less cytotoxic potentiation by the neutralizing antihuman PDGF-BB antiserum was observed in etoposide-treated cells (Supporting Fig. 1B). Thus, PDGF-BB secreted by cocultured MFB cells reduces the susceptibility of CCA cells to TRAIL-induced apoptosis.

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Figure 2. PDGF-BB promotes resistance to TRAIL cytotoxicity. KMCH-1 cells were plated alone (monoculture) or together with PDGF-BB-secreting human primary HSCs (Hu.Pr.; A and C) or LX-2 (B and D) cells in a Transwell insert coculture system (KMCH-1 cells in the bottom and human primary HSCs or LX-2 cells in the top wells; 1:1 ratio) for 6 days. Cells were treated as indicated with vehicle (V), recombinant human (rh)TRAIL (T; 10 ng/mL for 6 hours on day 6) or rhTRAIL plus antihuman PDGF-BB antiserum (T+AB-P; rhTRAIL: 10ng/mL for 6 hours on day 6; antihuman PDGF-BB antiserum: 10 μg/mL for 24 hours on day 5). After rhTRAIL treatment for 6 hours, KMCH-1 cells were analyzed for apoptotic nuclear morphology by DAPI staining (A, B) and for DNA fragmentation by TUNEL staining (C, D), with quantitation of apoptotic nuclei by fluorescence microscopy. Mean ± SEM (n = 5). (C, D, left) Representative fluorescent photomicrographs of TUNEL-positive KMCH-1 cells are depicted.

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PDGF-BB Cytoprotection Is Dependent on Hh Signaling.

Given that PDGF-BB modulates antiapoptotic Hh signaling in immature cholangiocytes16 and Hh signaling appears to be a potent survival signal for CCA cells, 25, 26 we explored the effect of Hh-signaling inhibition on PDGF-BB-mediated cytoprotection against TRAIL cytotoxicity. Apoptosis was assessed morphologically after 4′-6-diamidino-2-phenylindole (DAPI) staining (Fig. 3A, upper, and B) and biochemically by a caspase-3/-7 activity assay (Fig. 3A, lower). Exogenous PDGF-BB protected KMCH-1 cells from TRAIL-induced apoptosis (Fig. 3A). In contrast, cyclopamine, an inhibitor of SMO (the transducer of Hh signaling)36 sensitized KMCH-1 cells to TRAIL-induced apoptosis (Fig. 3A). Moreover, cyclopamine completely abrogated PDGF-BB inhibition of TRAIL-induced apoptosis (Fig. 3A). Likewise, shSMO KMCH-1 cells also underwent TRAIL-mediated apoptosis, despite exogenous PDGF-BB treatment (Fig. 3B, lower; compare with stable scrambled KMCH-1 cells; Fig. 3B, upper). Taken together, these observations suggest that PDGF-BB-mediated protection from TRAIL-induced apoptosis is dependent upon an intact Hh-signaling pathway.

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Figure 3. Cytoprotection by PDGF-BB is dependent on Hh signaling. (A) First, KMCH-1 cells (serum-starved for 2 days) were treated with vehicle or PDGF-BB (200 ng/mL) for 8 hours. PDGF-BB was retained in culture media, and cells were then treated with rhTRAIL (5 ng/mL), cyclopamine (an Hh [SMO] inhibitor; 5 μM) or TRAIL plus cyclopamine (5 ng/mL; 5 μM) for an additional 6 hours. (B) Similar experiments (same PDGF-BB and TRAIL treatment) were performed with shSMO KMCH-1, instead of cyclopamine-treated KMCH-1 cells (lower), and stable scrambled KMCH-1, instead of normal KMCH-1 cells (controls, upper). (C) Stable knockdown of SMO only in shSMO KMCH-1 cells was confirmed by immunoblotting analysis. Apoptosis was measured by DAPI staining, with quantitation of apoptotic nuclei by fluorescence microscopy (A upper and B; mean ± SEM; n = 3) or fluorescent analysis of caspase-3/-7 activity (A lower; mean ± SEM; n = 6; RFU, relative fluorescence unit).

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PDGF-BB Induces Translocation of SMO to the Plasma Membrane.

We next sought to explore how PDGF-BB stimulates Hh signaling to promote CCA cell survival. Initially, we analyzed the direct effect of PDGF-BB on mRNA expression of the Hh-signaling ligands, SHH, IHH, and DHH, as well as PTCH1, SMO, and GLI1-3, by quantitative RT-PCR (Supporting Fig. 3A). PDGF-BB did not significantly alter mRNA expression of the three Hh ligands nor that of PTCH1, SMO, or GLI1-3 in KMCH-1 and HuCCT-1 cells. To investigate whether PDGF-BB promotes Hh signaling by down-regulation of known negative regulators of this pathway, we also measured mRNA levels of hedgehog-interacting protein (HIP) and suppressor of fused (SUFU). PDGF-BB had no effect on these negative regulators in KMCH-1 cells (Supporting Fig. 3B). Because translocation of SMO from intracellular vesicles to the plasma membrane results in its activation during Hh signaling, 22 we next examined the cellular localization of SMO upon PDGF-BB treatment by immunocytochemistry (Fig. 4A). PDGF-BB significantly induced the translocation of SMO from intracellular compartments to the plasma membrane (arrows; Fig 4A, middle). This process appears to be PKA dependent, because it was effectively attenuated by the PKA inhibitor, H-89. Similar results were obtained when we employed KMCH-1 cells transiently transfected with a plasmid expressing GFP-tagged human SMO and analyzed GFP-SMO localized at the plasma membrane by TIRF microscopy26 (Fig. 4B). Moreover, PDGF-BB derived from cocultured LX-2 cells also induced SMO trafficking, as assessed by TIRF microscopy (Supporting Fig. 4A). As a further indicator for Hh-signaling activation, we examined the effect of PDGF-BB on GLI2 nuclear translocation in KMCH-1 cells by immunoblotting analysis (Fig 4C). PDGF-BB treatment increased GLI2 abundance in nuclear protein extracts, an effect that, again, was attenuated by the PKA inhibitor, H-89. Consistent with these results, KMCH-1 cells transiently transfected with a GLI reporter construct displayed GLI activation upon PDGF-BB treatment. The SMO inhibitor, cyclopamine, effectively blocked PDGF-BB-mediated GLI activation (Fig. 4D, upper). Likewise, stable scrambled KMCH cells also displayed PDGF-BB-induced GLI activation, whereas no PDGF-BB effect was observed in shSMO KMCH-1 cells (Fig. 4D, lower).

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Figure 4. PDGF-BB induces SMO trafficking to the plasma membrane, resulting in GLI2 nuclear translocation and transcriptional activation of a GLI reporter gene. (A) HuCCT-1 cells were treated with vehicle, PDGF-BB (200 ng/mL), or PDGF-BB plus the PKA inhibitor, H-89 (5 μM), for 2 hours. SMO cellular localization was analyzed by immunocytochemistry. Note that PDGF-BB induces the translocation of SMO from intracellular compartments (left) to the plasma membrane (yellow arrows, middle), an effect inhibited by the PKA inhibitor, H-89 (right). Cells with membranes positive for SMO immunocytochemistry were counted in each of these conditions. Mean ± SEM (n = 7). (B) KMCH-1 cells were transiently transfected with a plasmid expressing GFP-tagged human SMO (GFP-SMO; 48 hours), then treated with vehicle or PDGF-BB (200 ng/mL) with and without H-89 (5 μM) for 2 hours. GFP-SMO localized at the plasma membrane was analyzed by TIRF microscopy, and fluorescent intensity was quantified using image analysis software. Mean ± SEM (n = 20). (C) GLI2 nuclear translocation in KMCH-1 cells was assessed by immunoblotting analysis after treating cells with vehicle or PDGF-BB (200 ng/mL) with and without H-89 (5 μM) for 8 hours. Lamin B was used as the loading control for nuclear protein extracts. (D) KMCH-1 cells (normal, stable scrambled [scr.], or shSMO) were transiently transfected (24 hours) with a reporter construct, containing eight consecutive consensus GLI-binding sites (8×-GLI), and cotransfected with pRL-CMV. Cells were then treated as indicated with vehicle or PDGF-BB (200 ng/mL) with and without cyclopamine (5 μM) for 24 hours. Both firefly and Renilla luciferase activities were quantified, and data (firefly/Renilla luciferase activity) are expressed as fold increase over vehicle-treated cells transfected with the 8×-GLI/pRL-CMV reporter constructs. Mean ± SEM (n = 3; ***P < 0.001).

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Because PKA function is cAMP dependent, 37 we measured the effect of PDGF-BB on intracellular cAMP levels (Supporting Fig. 4B). Indeed, PDGF-BB-treated cells rapidly displayed significant increases of cAMP levels, as compared to controls, an effect that was blocked by the PDGFR(-β) inhibitor, imatinib. Thus, PDGF-BB appears to promote Hh-signaling–dependent cytoprotection by inducing cAMP/PKA-mediated SMO trafficking to the plasma membrane. To further confirm the PDGF-BB-stimulated, SMO-dependent gene regulation, we identified 67 target genes to be commonly up-regulated (50 genes) or down-regulated (17 genes) by both SHH and PDGF-BB in a cyclopamine-dependent manner in KMCH-1 cells via an Affymetrix U133 Plus 2.0 GeneChip analysis (Table 1).

Table 1. Gene Targets Regulated by Both SHH and PDGF-BB in a Cyclopamine-Dependent Manner in KMCH-1 Cells*
Gene SymbolGene NameGenBank Accession No.
  • *

    Alphabetical order.

  • Affymetrix U133 Plus 2.0 GeneChip analysis was performed. Genes were considered to be up-regulated when they (1) displayed significant up-regulation (P < 0.05, compared with the control group) upon SHH (single treatment), as well as PDGF-BB (single treatment) stimulation, and (2) displayed significant down-regulation (P < 0.05, compared with the SHH and PDGF-BB groups, respectively) upon the addition of Hh inhibitor cyclopamine to the SHH as well as to the PDGF-BB treatment. Down-regulated genes were regulated vice versa to (1) and (2).

Up-regulated
C7orf40Chromosome 7 open reading frame 40AI937446
CARD10Caspase recruitment domain family, member 10AY028896
CCL2Chemokine (C-C motif) ligand 2S69738
CYR61Cysteine-rich, angiogenic inducer, 61AF003114
DKK1Dickkopf homolog 1 (Xenopus laevis)NM_012242
ENC1Ectodermal-neural cortex (with BTB-like domain)NM_003633
EPHA2EPH receptor A2NM_004431
F3Coagulation factor III (thromboplastin, tissue factor)NM_001993
GPATCH4G patch domain containing 4BE794289
HAS3Hyaluronan synthase 3AF232772
HBEGFHeparin-binding EGF-like growth factorNM_001945
HEATR1HEAT repeat containing 1NM_018072
ISG20L2Interferon-stimulated exonuclease gene 20 kDa-like 2AW294587
JAG1Jagged 1 (Alagille syndrome)U77914
LAMB3Laminin, beta 3L25541
LUC7LLUC7-like (Saccharomyces cerevisiae)BE049621
LYARLy1 antibody reactive homolog (mouse)AW958593
LYARLy1 antibody reactive homolog (mouse)AL136750
MAT2AMethionine adenosyltransferase II, alphaBC001686
METAP2Methionyl aminopeptidase 2AW003997
MKI67IPMKI67 (FHA domain) interacting nucleolar phosphoproteinAL577809
MORC4MORC family CW-type zinc finger 4NM_024657
MRPS2Mitochondrial ribosomal protein S2NM_016034
NOB1NIN1/RPN12 binding protein 1 homolog (S. cerevisiae)BC000050
NOC3LNucleolar complex associated 3 homolog (S. cerevisiae)NM_022451
NSUN2NOL1/NOP2/Sun domain family, member 2BC001041
NUP35Nucleoporin 35 kDaAL529634
PFKFB36-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3NM_004566
PLK2Polo-like kinase 2 (Drosophila)NM_006622
PNNPinin, desmosome-associated proteinBF508848
PNPT1Polyribonucleotide nucleotidyltransferase 1AI967971
POLR1EPolymerase (RNA) I polypeptide E, 53 kDaNM_022490
POP1Processing of precursor 1, ribonuclease P/MRP subunit (S. cerevisiae)D31765
PRKCHProtein kinase C, etaNM_024064
PUS3Pseudouridylate synthase 3NM_031307
PWP2PWP2 periodic tryptophan protein homolog (yeast)U56085
RBM34RNA-binding motif protein 34AA887480
RND3Rho family GTPase 3BG054844
RRADRas-related associated with diabetesNM_004165
SERPINE1Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1AL574210
SLC25A32Solute carrier family 25, member 32NM_030780
SLC41A1Solute carrier family 41, member 1AW439816
SNHG7Small nucleolar RNA host gene 7 (nonprotein coding)AL533103
SYTL2Synaptotagmin-like 2AB046817
TGFB2Transforming growth factor, beta 2M19154
UTP14AUTP14, U3 small nucleolar ribonucleoprotein, homolog A (yeast)BC001149
VEGFCVascular endothelial growth factor CU58111
WDR73WD repeat domain 73AF161382
ZNF239Zinc finger protein 239NM_005674
ZYXZyxinBC002323
Down-regulated
BNIP3LBCL2/adenovirus E1B 19 kDa interacting protein 3-likeAL132665
CCNG1Cyclin G1BC000196
CCNG2Cyclin G2AW134535
ELMOD2ELMO/CED-12 domain containing 2BG477315
IFIT2Interferon-induced protein with tetratricopeptide repeats 2AA131041
INSIG1Insulin-induced gene 1BG292233
LMO4LIM domain only 4BC003600
LQK1Hypothetical LOC642946N51468
MEGF9Multiple EGF-like domains 9W68084
NBR1Neighbor of BRCA1 gene 1NM_005899
NHSL1NHS-like 1AA503387
PCMTD1Protein-L-isoaspartate (D-aspartate) O-methyltransferase domain containing 1AA453163
SCDStearoyl-CoA desaturase (delta-9-desaturase)AB032261
SCDStearoyl-CoA desaturase (delta-9-desaturase)AF132203
TCEAL8Transcription elongation factor A (SII)-like 8AI743979
TMEM135Transmembrane protein 135AK000684
TP53INP1Tumor protein p53-inducible nuclear protein 1AW341649

Hh Signaling Inhibition Is Tumor Suppressive In Vivo.

To determine whether the proapoptotic in vitro effect of Hh-signaling inhibition by cyclopamine observed in cocultures would be translatable to an in vivo model, we employed a syngeneic rat orthotopic CCA model (BDEneu malignant cells injected into the liver of male Fischer 344 rats). Like human CCA, the BDEneu cells also express TRAIL in vivo.28-30 We confirmed that BDEneu cells express mRNA of members of the Hh-signaling pathway (i.e., SHH, IHH, DHH, PTCH1, SMO, and GLI 1-3) (Supporting Fig. 5). This rodent model of CCA also duplicates the desmoplasia characteristic of the human disease, with numerous α-SMA-positive MFBs present in the stromal tumor microenvironment (Fig. 5A). We confirmed that the tumor samples (including MFBs and CCA cells) in this in vivo model also richly expresses mRNA for PDGF-BB and its cognate receptor, PDGFR-β, as compared nontumor liver tissue (Fig. 5B). Moreover, PDGFR-β immunoreactivity was identified in CCA cells (Fig. 5C), whereas PDGF-BB expression was apparent in the MFBs and at the margin of CCA glands (Fig. 5D). Thus, this preclinical, rodent model of CCA mimics the characteristic features observed in human CCA tissue and cell lines.

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Figure 5. Hh-signaling inhibition promotes apoptosis in PDGFR-β-expressing CCA cells in vivo. A syngeneic rat orthotopic model of CCA (BDEneu cells; Fischer 344 rats) was employed for this examination. (A-D) The rodent CCA model recapitulates cellular expression patterns of α-SMA, PDGF-BB, and PDGFR-β observed in human CCA. (A) α-SMA protein expression (brown) in tumors of untreated tumor-bearing rats was examined by immunohistochemistry (counterstaining with Mayers' hematoxylin). (B) CCA and normal liver specimens of untreated tumor-bearing rats (14 days after tumor cell implantation into the left lateral liver lobe) were analyzed for the mRNA expression of PDGF-B and PDGFR-β by quantitative RT-PCR. Mean ± SEM (n = 3). (C) PDGFR-β protein expression (brown) in tumors of untreated tumor-bearing rats was examined by immunohistochemistry. PDGFR-β-positive CCA cell nests are surrounded by a paucicellular stromal matrix. (D) PDGF-BB immunoreactivity (brown) was similarly analyzed, and the most intense signal was observed in stromal cells and the tumor-stromal interface. (E) Apoptosis of CCA cells was assessed in tumor tissues by TUNEL staining (green), and the identity of cells was determined by costaining via immunohistochemistry for CK7 (a CCA marker; red). Animals were treated with vehicle (upper) or cyclopamine (lower; 2.5 mg/kg body weight intraperitoneally daily for 1 week: first injection: postoperative day 7; seventh injection: postoperative day 13). Quantitation of TUNEL- and CK7-positive cells (expressed as number per HPF) 14 days after CCA cell implantation demonstrated that in cyclopamine-treated animals, CCA cell apoptosis was increased, as compared to controls (bar graph). Mean ± SEM (n = 10). HPF, high-power field.

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Next, we examined the potential therapeutic effects of the Hh-signaling inhibitor, cyclopamine, in this in vivo model of CCA. In cyclopamine-treated animals, CCA cell apoptosis was increased, as compared to controls. Apoptosis of CCA cells was confirmed by demonstrating the colocalization of TUNEL-positive cells with cells displaying CK7 (a biliary epithelial cell marker expressed by CCA cells; Fig. 5E). Consistent with the proapoptotic effects of cyclopamine in this model, cyclopamine also had an effect on tumor size. Indeed, tumor weight and tumor/liver as well as tumor/body-weight ratios were significantly decreased in cyclopamine-treated rats (Fig. 6A,B). In addition, animals treated with cyclopamine displayed no extrahepatic metastases, whereas 43% of vehicle-treated animals had extrahepatic metastases, predominantly occurring in the greater omentum and peritoneum (Fig. 6C; inset Fig. 6A, left upper). In aggregate, these data suggest that cyclopamine promotes CCA cell apoptosis and decreases tumor growth as well as metastasis in an in vivo rodent model of CCA.

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Figure 6. Hh-signaling inhibition reduces tumor growth and metastasis in vivo. A syngeneic rat orthotopic model of CCA (BDEneu cells; Fischer 344 rats) was employed for this examination. In cyclopamine-treated (2.5 mg/kg body weight intraperitoneally daily for 1 week; first injection: postoperative day 7; seventh injection: postoperative day 13) or vehicle-treated tumor-bearing rats (n = 7 rats/group), tumor/liver/body weight and extrahepatic metastasis were assessed 14 days after tumor cell implantation into the left lateral liver lobe. (A) Depicted are representative abdominal cavities (left), explanted livers (middle), and hematoxylin/eosin sections (right) of vehicle-treated (upper) and cyclopamine-treated (lower) rats. Arrows indicate the liver tumors, whereas the arrowhead (insert in the upper left photomicrograph) displays a representative example of extrahepatic metastasis (peritoneum). N.l., healthy liver; CCA, cholangiocarcinoma. (B) Changes in tumor weight as well as tumor/liver and tumor/body-weight ratios are depicted as box-and-whisker plots showing lowest value, 25th percentile, median, 75th percentile, highest value, and, in some cases, outliers. (C) The stacked column plot indicates the numbers of animals with and without metastases for vehicle- and cyclopamine-treated groups (P = 0.068, by chi-square test).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The results of this study provide new mechanistic insights regarding cytoprotective MFB-to-tumor cell paracrine signaling in CCA. These data indicate that MFB-derived PDGF-BB does the following: (1) protects CCA cells from TRAIL-induced cell death in vitro; (2) exerts this cytoprotection in an Hh-signaling–dependent manner by inducing cAMP/PKA-mediated SMO trafficking to the plasma membrane, resulting in GLI2 nuclear translocation and GLI transcriptional activity; and (3) and appears to act similarly in a rodent in vivo model of CCA, where Hh-signaling inhibition by cyclopamine promotes CCA cell apoptosis and is tumor suppressive. These findings are illustrated in Fig. 7 and discussed in greater detail below.

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Figure 7. Schematic diagram illustrating the role of PDGF-BB in promoting apoptosis resistance in CCA cells. PDGF-BB, mainly secreted by MBFs, promotes Hh signaling by inducing the trafficking of Hh-signaling mediator SMO to the plasma membrane and subsequent GLI activation, resulting in apoptosis resistance. Hh-signaling inhibitors, such as cyclopamine, block Hh survival signaling and, thus, promote apoptosis in CCA cells.

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In this study, we explored a role for PDGF-BB as an MFB-derived survival factor for CCA cells. Indeed, in coculture experiments, MFB cytoprotection against TRAIL-induced apoptosis was abrogated by neutralizing antisera to PDGF-BB, suggesting MFB-derived PDGF-BB is a potent anti-TRAIL survival factor for CCA cells. Although many cancer cells may not express PDGF receptors, 35 our data indicate CCA cells express PDGFR-β and respond to PDGF-BB by activating (via phosphorylation) this receptor. These observations suggest the existence of a distinctive paracrine survival-signaling pathway between MFB and CCA cells.

Coactivation networks are being increasingly recognized in cancer biology.38 We had previously implicated a major role for Hh-signaling–directed survival signals against TRAIL cytotoxicity of CCA cells in vitro.25, 26 Also, others have suggested PDGF-BB increases Hh ligand generation in immature bile ductular cells.16 Given this information, we posited that a PDGF-BB- and Hh-signaling coactivation network could contribute to survival signaling in CCA cells. Somewhat surprisingly, we found that PDGF-BB does not induce Hh ligand expression.15, 16 Instead, PDGF-BB appears to increase Hh signaling by promoting SMO trafficking to the plasma membrane (an event known to increase SMO activation22). Moreover, these processes were blocked by H89 (an inhibitor of the cAMP-regulated kinase PKA), suggesting that PDGF-BB-induced SMO trafficking is PKA mediated. We note that the role of PKA in the Hh pathway is complex and likely depends upon cell type and cellular context. For example, although PKA has been reported to promote Hh signaling at the level of SMO, it may act as a negative regulator by promoting the cleavage of GLI proteins into their repressor forms.22 However, in CCA cells treated with PDGF-BB, PKA does not repress PDGF-BB-mediated GLI transcriptional activity, because we observed the activation of a GLI reporter gene assay, as well as common gene expression between SHH and PDGF-BB stimulation in a cyclopamine-inhibitable manner.

Consistent with a requirement for PKA during PDGF-BB stimulation of SMO trafficking, we also were able to demonstrate an increase of intracellular cAMP by PDGF-BB. Because receptor tyrosine kinases—as opposed to G-protein-coupled receptors—do not directly stimulate adenylate cyclase (the enzyme generating cAMP), the mechanism by which PDGFR-β signaling enhances PKA activity in CCA cells will require further elucidation. A plausible mechanism would be the PDGF-BB/mitogen-activated protein kinase (MAPK)/prostaglandin E2/cAMP axis described in arterial smooth muscle cells.39

The SMO inhibitor, cyclopamine, significantly increased apoptosis in CCA cells and achieved suppression of CCA tumor growth and metastasis in a preclinical rodent model of CCA. The orthotopic rodent model of CCA employed in these studies reflects a similar molecular signature and TRAIL expression as human CCA, 29, 30 exhibits a tumor microenvironment rich in activated α-SMA-secreting MFBs, and also recapitulates the cellular expression patterns of PDGF-BB and PDGFR-β found in many human CCA samples. Berman et al. also reported that cyclopamine suppresses digestive tract tumors, including CCA in vivo (in a xenograft tumor model).19 Herein, we expand this observation and provide evidence of functional interactions between tumor microenvironment and CCA cells. Moreover, we demonstrate that Hh-signaling inhibition increases the apoptosis of CCA cells in vivo. The mechanism by which cyclopamine induces apoptosis in vivo likely involves TRAIL expression in tumor tissue, because cyclopamine does not increase the apoptosis of monocultured CCA cells in the absence of TRAIL. Hh signaling has also been implicated in altering tumor microenvironment.40 For example, Hh inhibitors increase the efficiency of cytotoxic chemotherapy in rodent models of pancreatic cancer by modulating the microenvironment of the cancer.13 In contrast to those studies, we did not observe a decrease in α-SMA-positive MFBs in cyclopamine-treated tumors (data not shown). Moreover, the importance of Hh signaling in cancer cells, as opposed to stromal cells, has recently been emphasized.41 Our observations are most consistent with a direct effect of cyclopamine on tumor cells in vivo, although we cannot exclude a noncytotoxic effect of cyclopamine on MFB function.

In conclusion, MFB-derived PDGF-BB protects CCA cells from TRAIL-induced apoptosis. This cytoprotection is exerted through a coactivation network involving Hh signaling. These observations support the examination of selective Hh inhibitors (currently in clinical development42, 43) in human CCA.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The Affymetrix U133 Plus 2.0 GeneChip analysis was performed in collaboration with the Genomics Technology Center Core and Dr. Y. Li from the Division of Biomedical Statistics and Informatics (both Mayo Clinic, Rochester, MN). The assistance of Dr. U. Yaqoob with the immunoblotting for (phospho-)PDGFR-β is also gratefully acknowledged, as well as the superb secretarial service of C. Riddle.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_24588_sm_suppfig1.tif1351KSupporting Figure 1.
HEP_24588_sm_suppfig2.tif2469KSupporting Figure 2.
HEP_24588_sm_suppfig3.tif3966KSupporting Figure 3.
HEP_24588_sm_suppfig4.tif4400KSupporting Figure 4.
HEP_24588_sm_suppfig5.tif501KSupporting Figure 5.
HEP_24588_sm_supptable1.doc44KSupporting Table 1.

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