Cholangiocarcinoma (CCA) is characterized by an abundant stromal reaction. Cancer-associated fibroblasts (CAFs) are pivotal in tumor growth and invasiveness and represent a potential therapeutic target. To understand the mechanisms leading to CAF recruitment in CCA, we studied (1) expression of epithelial-mesenchymal transition (EMT) in surgical CCA specimens and CCA cells, (2) lineage tracking of an enhanced green fluorescent protein (EGFP)-expressing human male CCA cell line (EGI-1) after xenotransplantation into severe-combined-immunodeficient mice, (3) expression of platelet-derived growth factors (PDGFs) and their receptors in vivo and in vitro, (4) secretion of PDGFs by CCA cells, (5) the role of PDGF-D in fibroblast recruitment in vitro, and (6) downstream effectors of PDGF-D signaling. CCA cells expressed several EMT biomarkers, but not alpha smooth muscle actin (α-SMA). Xenotransplanted CCA masses were surrounded and infiltrated by α-SMA-expressing CAFs, which were negative for EGFP and the human Y-probe, but positive for the murine Y-probe. CCA cells were strongly immunoreactive for PDGF-A and -D, whereas CAFs expressed PDGF receptor (PDGFR)β. PDGF-D, a PDGFRβ agonist, was exclusively secreted by cultured CCA cells. Fibroblast migration was potently induced by PDGF-D and CCA conditioned medium and was significantly inhibited by PDGFRβ blockade with Imatinib and by silencing PDGF-D expression in CCA cells. In fibroblasts, PDGF-D activated the Rac1 and Cdc42 Rho GTPases and c-Jun N-terminal kinase (JNK). Selective inhibition of Rho GTPases (particularly Rac1) and of JNK strongly reduced PDGF-D-induced fibroblast migration. Conclusion: CCA cells express several mesenchymal markers, but do not transdifferentiate into CAFs. Instead, CCA cells recruit CAFs by secreting PDGF-D, which stimulates fibroblast migration through PDGFRβ and Rho GTPase and JNK activation. Targeting tumor or stroma interactions with inhibitors of the PDGF-D pathway may offer a novel therapeutic approach. (Hepatology 2013;53:1042–1053)
An extensive desmoplastic reaction is a distinctive feature of cholangiocarcinoma (CCA), a highly aggressive cancer originating from the biliary epithelium, characterized by strong invasiveness with limited opportunities of curative treatment. The “tumor reactive stroma” is the site of complex functional interactions between cancer cells and the host microenvironment, and it plays a pivotal role in tumor growth and invasiveness.
Cancer-associated fibroblasts (CAFs) provide tumor cells with proliferative and antiapoptotic signals that ultimately promote cancer growth. On one hand, cancer cells produce a range of signals able to instruct the stromal microenvironment to become permissive and supportive for tumor progression. On the other hand, CAFs communicate with other cell types (endothelial cells [ECs], pericytes, and inflammatory cells) inducing angiogenesis and remodeling of the extracellular matrix (ECM), ultimately favoring tumor invasiveness. In CCA, overexpression of proinflammatory cytokines in the tumor stroma is associated with a more malignant tumor phenotype. Paracrine signals from CAFs protect CCA cells from proapoptotic stimuli.
The origin of CAFs is still uncertain. It has been proposed that CAFs undergo an epithelial-mesenchymal transition (EMT) of carcinoma cells, during which cancer cells lose their epithelial properties and acquire a mesenchymal phenotype that consequently favors increased invasive and migratory capabilities. Alternatively, CAFs may be recruited by cancer cells from resident fibroblasts or from circulating mesenchymal progenitor cells of bone marrow origin.
Members of the platelet-derived growth factor (PDGF) family are of interest because of their ability to promote fibroblast and hepatic stellate cell (HSC) migration and proliferation. Furthermore, PDGF expression has been shown to correlate with cancer progression in colon carcinoma as well as to protect CCA cells from apoptosis.[5, 7] The PDGF family encompasses five dimeric ligand isoforms (PDGF-AA, -BB, -AB, -CC, and -DD), which signal through two structurally related tyrosine kinase receptors, PDGF receptor (PDGFR)α and PDGFRβ. Although PDGFRα binds all PDGF isoforms except for PDGF-DD, PDGFRβ has a preferential and high affinity for PDGF-BB and PDGF–DD. The possible role of PDGF-D in tumor development and progression is only starting to be recognized.
To better understand the mechanisms underlying the formation of tumor reactive stroma in CCA, we investigated whether CAFs are generated from cancer cells or are recruited by cancer cells through a PDGF-dependent mechanism. Specifically, this study sought (1) expression of several EMT markers in human CCA specimens and cells (2) the fate of human CCA cells xenotransplanted in severe combined immunodeficient (SCID) mice after transfection with enhanced green fluorescent protein (EGFP), (3) expression of PDGF ligands and receptors in CCA specimens and cells, (4) the ability of cultured human CCA cells to secrete PDGF isoforms, (5) the role of PDGF-D in tumor epithelial/mesenchymal cross-talk in vitro, and (6) the intracellular signaling pathways involved.
The incidence of CCA is increasing in Western countries and accounts for 10%-20% of deaths from primary hepatobiliary malignancies. CCA is characterized by the presence of an abundant tumor reactive stroma, a feature common to other aggressive malignancies of ductal origin, such as pancreatic and breast carcinomas.
The tumor reactive stroma is the microanatomical site of multiple functional interactions between cancer cells and several kinds of host cells and thus it behaves as an important determinant of cancer invasiveness. CAFs, the main cellular component of the tumoral stroma, produce tumoral matrix and release a variety of growth factors and chemokines, which modulate tumor cell survival, migration, and invasion. For example, it has been shown that CAF-derived PDGF protects CCA cells from death induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a Hedgehog-signaling-dependent manner. CAFs are also an important source of matrix metalloproteinases, cathepsins, and plasminogen activators that enable cancer cells to escape from the primary site of growth. Some researchers propose that factors originating from the stroma (transforming growth factor beta [TGF-β] and hepatocyte growth factor [HGF]) signal to cancer cells to undergo an EMT and to become endowed with functional properties that favor the metastatic process, such as the ability to detach from the neoplastic cluster and to migrate to and invade lymphatic or blood vessels. The role of EMT in liver diseases and tumors remains unclear and controversial.
In this study, CCA cells expressed several phenotypic features, known to correlate with increased motility and invasiveness, including down-regulation of E-cadherin and β-catenin and up-regulation of Snail1, Twist, and S100A4. However, there was no evidence of EMT. This conclusion is based on the lack of coexpression of K7 and α-SMA in CCA tissue sections as well as on the lack of coincidence between CCA cholangiocyte lineage markers (EGFP and human Y chromosome [Y Chr]) and an activated myofibroblast marker (α-SMA) after intraportal injection of the highly invasive EGI-1 cells into SCID mice. EGFP-positive CCA cholangiocytes expressed the human Y-probe, but did not express α-SMA, whereas α-SMA-positive CAFs expressed the murine Y-probe, rather than the human Y-probe (Fig. 1). After xenotransplantation, in spite of the immunotolerant environment, an abundant stroma formed around the CCA cholangiocytes, suggesting a direct effect of factors secreted by tumoral cells.
Several factors can regulate epithelial-mesenchymal cross-talk, including Hedgehog, Wnt, and PDGF. We present IHC and in vitro evidences suggesting that PDGF secreted by tumoral cells plays a key role on migratory properties of CAFs.
We demonstrate that PDGF-D is secreted by neoplastic, but not by control, cholangiocytes. PDGF-D is one of the players responsible for the increased migration of fibroblasts when exposed to CCA conditioned medium. In contrast with the other members of the PDGF family, PDGF-D binds only to the PDGFRβ. Mechanisms leading to the up-regulation of PDGF-D in neoplastic cholangiocytes are uncertain. However, our data suggest that hypoxia may behave as a critical inducer of PDGF-D secretion, as shown by the potent stimulation exerted on CCA cells by DMOG, an agent that prevents HIF-1α degradation. This effect is in line with the typical hypovascularization featured in CCA. Our IF studies show that a subset of inflammatory cells may represent an additional source of PDGF-D released in the tumor microenvironment, albeit their PDGF-D expression is less relevant than CCA cells.
The importance of PDGF-D in cancer biology is just beginning to be understood.[23, 24] Our findings strongly suggest that PDGF-D plays a major role in promoting CAF recruitment in CCA. In fact, siRNA of PDGF-D significantly impaired the ability of CCA cholangiocytes to promote fibroblast migration. In addition, rhPDGF-D induced a clear dose-dependent effect on fibroblast migration, whereas the effect on proliferation was milder and evident only at the highest dosages. PDGFRα, which binds all isoforms except for PDGF-D, may theoretically contribute to CAF recruitment in CCA, because PDGFRα was also expressed by CAF, and EGI-1 cells were able to secrete PDGF-A. However, administration of conditioned medium from control cholangiocytes, which contained amounts of PDGF-A comparable to those produced by CCA cells, exerted only a weak effect on fibroblast transwell migration. Interestingly, whereas PDGFRα signaling plays a pivotal role in embryonic development and in fibrosis of nonhepatic conditions, PDGFRβ seems to be more relevant in activating HSCs and in stimulating the production of profibrogenic growth factors and ECM components by liver myofibroblasts.
By interacting with its cognate receptor, PDGFRβ, PDGF-D can activate several signaling cascades to regulate cell survival, cell growth, cell differentiation, cell invasion, and angiogenesis. Because MAPK and PI3K/Akt are two major signal transduction pathways known to be activated by PDGF-D, we studied ERK1/2, JNK, and the small Rho GTPases as downstream effectors, respectively, of MAPK and PI3K/Akt, which are able to control cell proliferation (ERK1/2) and migration (JNK and Rho GTPases).[18, 26] The ability of PDGF-B to induce cytoskeletal remodeling by Rac1 and JNK has recently been reported in NIH3T3 cells,[26, 27] but the effects of PDGF-D on these molecular effectors are hitherto largely unknown. Our findings show that exposure of fibroblasts even to low doses of PDGF-D strongly activates Rho GTPases and JNK, whereas expression levels of p-ERK increased only at the highest doses. These results strongly correlate with the different functional effects on fibroblast migration and proliferation of PDGF-D (as shown in Figs. 3, 5 and Supporting Fig. 9). By regulating the cytoskeleton and adhesion dynamics, the Rho GTPases are key drivers of cell migration. The time-course study of Rho GTPase activation further enforces the role of PDGF-D as a fundamental mediator of CAF recruitment. Rac1 and Cdc42 are two of the members of the family that are most activated by PDGF-D; however, they show different kinetics of activation. Rac1, which induces the assembly of actin-rich surface protrusions (lamellipodia) enabling the start of the mesenchymal cell movement (“random” migration), shows a brisk, but transient, activation by PDGF-D. In contrast, Cdc42, which promotes the formation of actin-rich, finger-like membrane extensions (filopodia) regulating chemotaxis, shows a significantly sustained activation. These data indicate that by activating Rac1 and Cdc42 with different time-dependent patterns, PDGF-D may potentially regulate distinct steps of CAF recruitment, including chemotaxis toward tumoral cells, a critical function in the generation of the tumor stroma. The capability of the small GTPases to orchestrate fibroblast recruitment driven by PDGF-D is confirmed by the observation that fibroblast transwell migration elicited by PDGF-D was completely inhibited by a mix of selective inhibitors (Y-27632, NSC23766, and CASIN). Although the regulation of cell motility by the Rho GTPases has been well documented in cancer cells, their involvement as fundamental molecular determinants of the tumor stromal reaction has not been reported yet. In addition to Rho GTPases, fibroblast migration in response to PDGF-D is also modulated by JNK, as previously shown in murine HSCs and portal myofibroblasts. Notably, our data show that specific JNK inhibition halts fibroblast migration to an extent similar to Rac1, likely indicating that both pathways act in concert to orchestrate the PDGF-D-mediated paracrine fibroblast recruitment by CCA cells.
In addition to Rho GTPase and JNK inhibitors, we found that tyrosine kinase inhibitors were also highly effective in halting fibroblast migration and proliferation induced by PDGF-D. The potential clinical usefulness of tyrosine kinase inhibitors in CCA has recently been outlined by Andersen et al., particularly in those patients where overexpression of inflammatory functions in the microenvironment is a critical signature related to a worse prognosis. Data in this study show that selective blockade of PDGFRβ with imatinib mesylate, a tyrosine kinase inhibitor already in clinical use for other indications, significantly reduces fibroblast recruitment by CCA cholangiocytes in Boyden chambers. The therapeutic relevance of specifically targeting PDGFRβ in CCA is a topic of growing interest. Recently, the ability of PDGFRβ inhibitors to interfere with CAF-to-CCA paracrine signaling mediated by PDGF-BB has been reported on. In fact, PDGFRβ activation promotes Hedgehog survival signaling in CCA cholangiocytes through protection from TRAIL cytotoxicity. Our study further extends the role of PDGFRβ molecular targeting in CCA because it can prevent CAF recruitment induced by CCA cholangiocyte-derived PDGF-D. Notably, overexpression of PDGFRβ in the stromal compartment of CCA was related to the most significant “network connectivity” with the tumoral compartment. Pharmacological targeting of tumor/stroma interactions using PDGF inhibitors may represent a novel molecularly targeted therapeutic approach in CCA.[31, 32]
The authors wish to thank Dr. Scott Swenson (Section of Digestive Disease, Yale University School of Medicine, New Haven, CT) for assistance with FISH experiments.