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

Authors


  • Potential conflict of interest: Dr. Colledan advises Novartis.

  • This work was supported by the Associazione Scientifica Gastroenterologica di Treviso (to L.F. and I.F.), the Telethon (grant no.: GGP09189) and Fondazione Amici Dell' Epatologia (to L.F.), the Progetto di Ricerca Ateneo 2008 (grant no.: CPDA083217) and 2011 (grant no.: CPD113799/11) (to L.F. and M.C.), the National Institutes of Health (grant no.: DK34989: Silvio O. Conte Digestive Diseases Research Core Centers – 5P30DK034989), and a grant from PSC partners for a care (to M.S.). R.F is a recipient of a Liver Scholar Award (American Liver Foundation). The support of Fondazione San Martino (Bergamo, Italy) is gratefully acknowledged.

  • See Editorial on Page 853

Address reprint requests to: Luca Fabris, M.D., Ph.D., Department of Surgery, Oncology, and Gastroenterology, University of Padova, School of Medicine, 2 Via Giustiniani, 35128 Padova, Italy. E-mail: luca.fabris@unipd.it; fax: +39 049 827 8906.

Abstract

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)

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