Myofibroblast-derived PDGF-BB Promotes Hedgehog Survival Signaling in Cholangiocarcinoma Cells

Cell lines/culture and human samples

The human CCA cell lines KMCH-1, KMBC, HuCCT-1, TFK-1, and Mz-ChA-1 and 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} The 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 (DMEM) supplemented with 10% fetal bovine serum, penicillin G (100 U/mL), and streptomycin (100 μg/mL) under standard conditions. Human samples were collected in accordance 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, Rochester, MN). The pRK7-SMO plasmid was modified to accept the GFP tag first by inserting recognition sites for EcoRI and NotI at the C-terminus of SMO, replacing the stop codon. For this, a PCR-generated EcoRI/NotI modified SMO C-terminal coding sequence was inserted into pRK7-SMO. Next, GFP from the pEGFP-N1 protein fusion vector (Clontech Laboratories, Inc., Mountain View, CA; Catalog no.: 6085-1; GenBank accession no.: U55762) was digested and inserted into the modified pRK7-SMO plasmid to generate a 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 polymerase chain reaction artifacts were introduced.

SMO trafficking

HuCCT-1 cells were cultured on coverslips, treated as indicated, and fixed with PBS containing 4% paraformaldehyde for 20 min at 37°C. After being washed with PBS, cells were incubated with 0.5% Triton X-100 in PBS for 15 min at RT and then blocked with PBS containing 5% BSA for 60 min at 37°C. Cells were subsequently incubated with anti-SMO antiserum (1:250; Santa Cruz, Santa Cruz, CA; H-300) at 4°C overnight. After being washed, coverslips were incubated with Texas Red®-X goat anti-rabbit IgG (1:1000; Invitrogen, Camarillo, CA; T6391) for 1 hr 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; Zeiss, Jena, Germany). In additional experiments, SMO trafficking was examined by total internal reflection microscopy (TIRF).³¹ KMCH-1 cells cultured on coverslips were transfected with GFP-SMO plasmid 48 hours prior to study. Cells were treated as indicated, and fixed with ddH₂O containing 2.5% formaldehyde, 0.1 M PIPES, 1.0mM EGTA, and 3.0 mM MgSO₄ for 20 min at 37°C. Cells were then washed three times in PBS, one time in water and mounted using Prolong Antifade (Invitrogen). The slides were analyzed with a TIRF microscop (Zeiss AxioObserver.Z1, Munich, Germany). GFP-SMO localized to the plasma membrane was quantified using image analysis software (Carl Zeiss AxioVision 4.8.2.0, Munich, Germany). Data were 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 (8x-GLI) downstream of the luciferase gene was employed (pδ51LucII plasmid; δ-crystalline promoter). ³² The 8x-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 shSMO KMCH-1 cells (0.5 μg/well) using FuGene HD (Roche Diagnosis, Basel, Switzerland). Cells were co-transfected with 50 ng of a plasmid expressing Renilla luciferase (pRL-CMV; Promega, Madison, WI). 24 hours after transfection, cells were treated as indicated, cell lysates prepared, and both firefly and Renilla luciferase activities 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 8x-GLI/pRL-CMV reporter constructs.

Animal experiments

All animal studies were performed in accordance with and approved by the Institutional Animal Care and Use Committee. In vivo intrahepatic cell implantation (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 BW; 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 one week (1^st injection: 7^th post-operative day; 7^th injection: 13^th post-operative day). Twenty-four hours after receiving the last injection, the rats were euthanized and the livers removed for further analysis including histopathology and mRNA extraction. To assess the numbers of metastases-free and metastases-bearing rats, the abdominal cavities, the retroperitoneal spaces and the thoracic cavities were thoroughly examined as previously described.²⁹

Supplemental methods

Materials, generation of shSMO KMCH-1 cells, quantitation of PDGF-BB and cAMP, co-culture experiments, quantitation of apoptosis, immunoblot analysis, immunohistochemistry for α-SMA, PDGFR-β, PDGF-BB, and cytokeratin 7 as well as RT-PCR, the genome-wide mRNA expression assay, and statistical analysis are described in the online supplementary material.

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 ELISA (monoculture conditiones, 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). Immunoblot analysis confirmed protein expression of PDGFR-β in KMCH-1 cells (Fig. 1B, lower), while 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 mRNA expression of PDGFR-β in KMCH-1 cells and 4 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; Suppl. 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 expressed by CCA cells.

MFB-derived PDGF-BB promotes resistance to TRAIL cytotoxicity

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

PDGF-BB cytoprotection is dependent on Hh signaling

Given that PDGF-BB modulates antiapoptotic Hh signaling in immature cholangiocytes ¹⁶ 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 following 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.

PDGF-BB induces translocation of SMO to the plasma membrane

We next sought to explore how PDGF-BB stimulates Hh signaling in order 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 (Supplemental 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 if PDGF-BB promotes Hh signaling by downregulation of known negative regulators of this pathway, we also measured the 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 (Supplemental 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 translocation of SMO from intracellular compartments to the plasma membrane (Arrows; Fig 4A, middle). This process appears to be PKA-dependent as 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 microscopy ²⁶ (Fig. 4B). Moreover, PDGF-BB derived from co-cultured LX-2 cells also induced SMO trafficking as assessed by TIRF microscopy (Suppl. 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 immunoblot 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).

Since PKA function is cAMP-dependent,³⁷ we measured the effect of PDGF-BB on intracellular cAMP levels (Suppl. 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- (50 genes) or downregulated (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).

Hh signaling inhibition is tumor suppressive in vivo

To determine if the proapoptotic in vitro-effect of Hh signaling inhibition by cyclopamine observed in co-cultures is 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 (Supplemental 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 non-tumor 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.

Next, we examined the potential therapeutic effects of the hedgehog 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 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 and 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.