Platelet-derived growth factor-D and Rho GTPases regulate recruitment of cancer-associated fibroblasts in cholangiocarcinoma

Abstract

Cholangiocarcinoma (CCA) is characterized by an abundant stromal reaction. Cancer-associated fibroblasts (CAF) are pivotal players in tumor growth and invasiveness and represent a potential therapeutic target. To understand the mechanisms leading to CAF recruitment in CCA, we studied: 1) the expression of epithelial-mesenchymal transition (EMT) in surgical CCA specimens and CCA cells; 2) the lineage tracking of an EGFP-expressing human male CCA cell line (EGI-1) after xenotransplantation into severe-combined-immunodeficient mice; 3) the expression of platelet-derived growth factors (PDGFs) and their receptors in vivo and in vitro; 4) the secretion of PDGFs by CCA cells; 5) the role of PDGF-D in fibroblast recruitment in vitro; 6) the downstream effectors of PDGF-D signaling. CCA cells expressed several EMT biomarkers but not α-SMA. Xenotransplanted CCA masses were surrounded and infiltrated by α-SMA-expressing CAF, 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, whilst CAF expressed PDGFRβ. 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 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 CAF. Instead, CCA cells recruit CAF by secreting PDGF-D, which stimulates fibroblast migration via PDGFRβ and Rho GTPase and JNK activation. Targeting tumor/stroma interactions with inhibitors of PDGF-D pathway may offer a novel therapeutic approach.

Impact of microenvironment and stem-like plasticity in cholangiocarcinoma: Molecular networks and biological concepts

“…The MF-to-CCA crosstalk furthermore involves several signalling pathways, such as PLK [91], PDGF [92,93], Hh [56], and Notch [56,57]. In particular, Cadamuro et al indicated that CCA cells recruit CAFs by secreting PDGF-D, which stimulates fibroblast migration through PDGFRB, Rho GTPase and JNK activation [94]. Recently, a key role of PLK2 was identified that links PLK and Hedgehog signalling in TRAIL-induced cell death, by demonstrating the ability of Hh to regulate PLK2 expression [91].…”

The Role of Cancer-Associated Fibroblasts and Fibrosis in Liver Cancer

“…Potential sources of CAFs include HSCs, portal or periductular fibroblasts, BM-derived fibroblasts, and tumor-derived fibroblasts via the EMT. Although the expression of EMT biomarkers such as vimentin, fibronectin, and snail CCA cells supports a pivotal role for EMT in this tumor (206), recent studies have not provided evidence for a transdifferentiation of cholangiocytes into CAFs in CCA (207). Given that HSCs represent a major source of myofibroblasts in multiple models of biliary liver fibrosis (44), it is likely that this cell type also makes a major contribution to the CAF population in CCA.…”

Cholangiocarcinoma: current knowledge and future perspectives consensus statement from the European Network for the Study of Cholangiocarcinoma (ENS-CCA)

“…CAFs, which express α-smooth muscle actin, are able to modulate several processes (that is, prolifer ation, migration, invasion and EMT) in CCA tumour cells 139,[175][176][177][178][179][180] . Accordingly, patients with high levels of α-smooth muscle actin expression in CCA tissue samples exhibit worse prognosis 181,182 .…”