Cyclic AMP/PKA-dependent paradoxical activation of Raf/MEK/ERK signaling in polycystin-2 defective mice treated with sorafenib.
Mutations in polycystins are a cause of polycystic liver disease. In polycystin-2 (PC2)-defective mice, cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-dependent activation of the Rat Sarcoma (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen signal-regulated kinase-extracellular signal-regulated kinase (ERK) 1/2 pathway stimulates the growth of liver cysts. To test the hypothesis that sorafenib, a Raf inhibitor used for the treatment of liver and kidney cancers, inhibits liver cyst growth in PC2-defective mice, we treated PC2 (i.e., Pkd2(flox/-) :pCxCreER(TM) [Pkd2cKO]) mice with sorafenib-tosylate for 8 weeks (20-60 mg/kg/day). Sorafenib caused an unexpected increase in liver cyst area, cell proliferation (Ki67), and expression of phosphorylated ERK (pERK) compared with Pkd2cKO mice treated with vehicle. When given to epithelial cells isolated from liver cysts of Pkd2cKO mice (Pkd2cKO-cells), sorafenib progressively stimulated pERK1/2 and cell proliferation [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and bromodeoxyuridine assay (MTS)] at doses between 0.001 and 1 μM; however, both pERK1/2 and cell proliferation significantly decreased at the dose of 10 μM. Raf kinase activity assay showed that whereas B-Raf is inhibited by sorafenib in both wild-type (WT) and Pkd2cKO cells, Raf-1 is inhibited in WT cells but is significantly stimulated in Pkd2cKO cells. In Pkd2cKO cells pretreated with the PKA inhibitor 14-22 amide, myristolated (1 μM) and in mice treated with octreotide in combination with sorafenib, the paradoxical activation of Raf/ERK1/2 was abolished, and cyst growth was inhibited.


CONCLUSIONS
In PC2-defective cells, sorafenib inhibits B-Raf but paradoxically activates Raf-1, resulting in increased ERK1/2 phosphorylation, cell proliferation, and cyst growth in vivo. These effects are consistent with the ability of Raf inhibitors to transactivate Raf-1 when a PKA-activated Ras promotes Raf-1/B-Raf heterodimerization, and are inhibited by interfering with cAMP/PKA signaling both in vitro and in vivo, as shown by the reduction of liver cysts in mice treated with combined octreotide and sorafenib.
Liver Center & Section of Digestive Diseases, Department of Internal Medicine, Yale University, New Haven, CT 06520, USA.
Carlo
C
Spirli
Carola M
CM
Morell
Luigi
L
Locatelli
Stefano
S
Okolicsanyi
Cecilia
C
Ferrero
Amy K
AK
Kim
Luca
L
Fabris
Romina
R
Fiorotto
Mario
M
Strazzabosco
D000818:Animals; D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology / Q000627:therapeutic use; D001557:Benzenesulfonates / Q000494:pharmacology* / Q000627:therapeutic use; D001652:Bile Ducts / Q000166:cytology / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003560:Cysts / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology; D004359:Drug Therapy, Combination; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology; D019394:Ki-67 Antigen / Q000378:metabolism; D008107:Liver Diseases / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D051379:Mice; D018345:Mice, Knockout; D009536:Niacinamide / Q000031:analogs & derivatives; D015282:Octreotide / Q000494:pharmacology / Q000627:therapeutic use; D010671:Phenylurea Compounds; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D048493:Proto-Oncogene Proteins B-raf / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology* / Q000627:therapeutic use; D000077157:Sorafenib; D050396:TRPP Cation Channels / Q000172:deficiency* / Q000235:genetics
D018931:Antineoplastic Agents, Hormonal; D001557:Benzenesulfonates; D053148:Caspase 3; D017868:Cyclic AMP-Dependent Protein Kinases; D019394:Ki-67 Antigen; D009536:Niacinamide; D015282:Octreotide; D010671:Phenylurea Compounds; D047428:Protein Kinase Inhibitors; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf; D011725:Pyridines; D000077157:Sorafenib; D050396:TRPP Cation Channels