Myofibroblast-derived PDGF-BB promotes hedgehog survival signaling in cholangiocarcinoma cells

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 pro-apoptotic stimuli is incompletely understood. CCAs are highly desmoplastic cancers suggesting 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 α-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 the 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 as they reduce apoptosis of non-malignant cholangiocytes in co-culture 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 process; 18 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 oncogene (GLI) 1, 2, and 3.21 How PTCH1 modulates SMO was enigmatic until quite recently, as 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 (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 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 resistance of CCA cells to TRAIL cytotoxicity. 25, 26 Taken together, these observations suggest MFB-derived PDGF-BB may modulate Hh survival signaling in CCA cells. The objective 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 a Hh signaling-dependent manner. These observations have implications for the treatment of human CCA.