001, 0.01, 0.1, 1, and 10 μM). In WT cholangiocytes, sorafenib significantly decreased

pERK1/2 at a concentration of 10 μM, but sorafenib had a dose-dependent biphasic effect: in Pkd2cKO cells receiving doses of 0.001 or 1 μM sorafenib, there was a statistically significant increase of pERK1/2 compared with baseline, already at a dose of 0.01 μM) (see Fig. 3); similar to the control cells, pERK1/2 was significantly inhibited at a dose of 10 μM sorafenib MG-132 clinical trial but had no significant effect on ERK1/2 phosphorylation at lower doses (Fig 3). In Pkd2cKO cells, we previously reported that baseline pERK1/2 was significantly increased with respect to WT.7, 8 The effects of sorafenib on cell proliferation were studied using MTS and BrdU assays. Our results (Fig. 4A,B) confirmed a significant increase in cell proliferation with doses up to 1 μM and this website a significant inhibition when cells were exposed to 10 μM sorafenib. Sorafenib was shown to induce apoptosis

in malignant cells24, 25 by a ERK1/2-independent decrease in the expression of Mcl1, a major antiapoptotic protein in cholangiocytes.26 To evaluate the effects of sorafenib on apoptosis, we measured the expression of CC3 in WT and Pkd2cKO cholangiocytes exposed to the above range of sorafenib concentrations. As shown in Fig. 4C, significant stimulation of apoptosis was found after 10 μM sorafenib, both in WT and in Pkd2cKO cholangiocytes, whereas at lower concentrations, CC3 expressions were slightly decreased, with statistical significance. As shown in Supporting Fig 3, higher doses of sorafenib (100 μM) caused cell toxicity and a dramatic increase in apoptosis. ERK phosphorylation is dependent on the upstream activation of Raf. Cholangiocytes express two isoforms of Raf, B-Raf, and Raf-1 (or C-Raf) (Supporting Fig 4), that may be differentially regulated by sorafenib. The effects of sorafenib on Ras kinases activity were measured in vitro

after immunoprecipitation of B-Raf or Raf-1 from whole lysates of WT or Pkd2cKO cells, using exogenous mouse MEK as a substrate for phosphorylation.20 As shown in Fig. 5, B-Raf activity was inhibited in both WT and Pkd2cKO treated with sorafenib in a dose-dependent way. On the contrary, in Pkd2cKO cells but not WT cells, Raf-1 activity showed the same biphasic effect described above for pERK1/2 and BCKDHB cell proliferation. In fact, Raf-1 was significantly stimulated at doses between 0.001 and 1 μM, followed by a significant inhibition at 10 μM. Similar results were found using the more potent Raf inhibitor RAF265 (Supporting Fig 5). Pkd2cKO cells are characterized by PKA-mediated, Ras-dependent activation of Raf/MEK/ERK signaling.7 The inhibition of B-Raf with paradoxical activation of Raf-1 caused by sorafenib in Pkd2cKO cells is consistent with the concept that PKA-activated Ras induces a heterodimerization of B-Raf and Raf-1. If so, sorafenib-stimulated Raf-1 activation should be blocked by inhibition of PKA.