Cyclic AMP/PKA-dependent paradoxical activation of Raf/MEK/ERK signaling in polycystin-2 defective mice treated with sorafenib.

Polycystic Liver Disease (PLD) is characterized by multiple liver cysts that originate from the biliary epithelium and progressively enlarge, eventually causing complications related to mass effects, hemorrhages, infection or rupture(1, 2). Some patients may require cyst fenestration, liver resection and even liver transplantation(3). Most cases of PLD are associated to Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease caused by mutations in PKD1 or PKD2. These genes encode polycystin-1 (PC1) and polycystin-2 (PC2) respectively, two proteins expressed by cholangiocytes(2). PC1 is localized in the primary cilium; its main function is to serve as chemosensor and a mechanosensor for the apical flow and pressure. PC1 physically interacts with PC2 (or TRPP2), a member of the transient receptor potential channels (TRP) family, that functions as a non-selective Ca2+ channel(1). PC2 is expressed in the cilium and the endoplasmic reticulum (ER), and is able to regulate cytoplasmic and ER Ca2+ concentrations. Defective PC2 function affects the resting cell [Ca2+] by reducing extracellular Ca2+ entry and altering ER Ca2+ homeostasis. In the absence of PC2, cells are unable to activate the store-operated increase Ca2+ entry mechanisms (SOCE) and respond to the subsequent reduction in ER Ca2+ levels by stimulating the activity of adenylyl cyclase 6 (AC6), a Ca2+-inhibitable AC that is not active at resting Ca2+ concentrations. This mechanism generates increased levels of cAMP(4). Inappropriate production of cAMP, the main signaling abnormality of cystic cholangiocytes, is responsible for the brisk proliferative activity of cystic cholangiocytes(5). Cyclic-AMP activates the PKA/Ras/Raf/MEK/ERK1/2 cascade, resulting in stimulation of cholangiocytes proliferation(6, 7). In addition, studies in PC2-defective cholangiocytes, have shown that the over-activation of this pathway, causes the downstream activation of the mTOR pathway and that both ERK1/2 and mTOR converge in stimulating cyclins and HIF1α-dependent VEGF-A secretion(8). Mice deficient in PC2 show a severe liver phenotype, high proliferation rate of the cystic epithelium and high expression of pERK1/2, p-mTOR, HIF1α, VEGF and VEGFR-2(7–9). The pathophysiological relevance of this model is demonstrated by the reduction of cyst growth in vivo, after administration of SU5418 (inhibition of VEGFR2 signaling)(7, 9) rapamycin (inhibition of mTOR and of VEGF production)(8) or somatostatin, that inhibits cAMP production through its receptor SSTR2(10). Clinical trials of somatostatin-analogues in PLD patients have shown only a modest reduction in cyst growth(11–13), and thus a medical treatment for patients with symptomatic PLD is still not available. Because of its role in the PKA/Ras/Raf/MEK/ERK cascade, the key signaling pathway altered in PLD, and the availability of chemical inhibitors approved for clinical use, we considered Raf as a potential new target molecule for the treatment of PLD and sought to generate experimental proof of this concept. Sorafenib is an oral Raf inhibitor used in the treatment of kidney and liver cancer that was shown to increase apoptosis and to block cell proliferation and neo-angiogenesis in a wide range of tumor models by targeting Raf/MEK/ERK signaling(14, 15). In this study, we performed in vivo and in vitro experiments to test the hypothesis that sorafenib inhibits liver cyst growth in PC2-defective mice. Contrary to our hypothesis, we found that sorafenib caused an increase in liver cyst growth in vivo and stimulated pERK, cell proliferation and Raf-1 kinase activity in Pkd2cKO cells in vitro. Inhibition of PKA restored the expected inhibitory effect of sorafenib in PC2-defective cells. Consistent with this observation, a significant reduction in liver cyst growth in vivo was achieved when sorafenib was given in combination with octreotide, an analogue of somatostatin known to inhibit cAMP production(10). These data are consistent with a model in which sorafenib inhibits B-Raf, but paradoxically activates Raf-1 in the context of PKA-dependent, Ras-induced B-Raf/Raf-1 heterodimerization. These results also suggest that the potential consequence of paradoxical activation of Raf-1 should be carefully considered when treating conditions characterized by activation of non-mutated Raf.