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

Carlo Spirli, Carola M. Morell, Luigi Locatelli, Stefano Okolicsanyi, Cecilia Ferrero, Amy Kim, Luca Fabris, Romina Fiorotto, Mario Strazzabosco

Research output: Contribution to journalArticle

Abstract

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., Pkd2flox/-:pCxCreERTM [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. Conclusion: 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.

Original languageEnglish (US)
Pages (from-to)2363-2374
Number of pages12
JournalHepatology
Volume56
Issue number6
DOIs
StatePublished - Dec 2012
Externally publishedYes

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Adenylate Kinase
Fibrosarcoma
Mitogen-Activated Protein Kinase Kinases
Extracellular Signal-Regulated MAP Kinases
Cyclic AMP-Dependent Protein Kinases
Cyclic AMP
Cysts
Cell Proliferation
Octreotide
Liver
Liver Neoplasms
Growth
Sarcoma
polycystic kidney disease 2 protein
sorafenib
TRPP Cation Channels
Mitogen-Activated Protein Kinase 3
Kidney Neoplasms
Mitogen-Activated Protein Kinase 1
Bromodeoxyuridine

ASJC Scopus subject areas

  • Hepatology

Cite this

Spirli, C., Morell, C. M., Locatelli, L., Okolicsanyi, S., Ferrero, C., Kim, A., ... Strazzabosco, M. (2012). Cyclic AMP/PKA-dependent paradoxical activation of Raf/MEK/ERK signaling in polycystin-2 defective mice treated with sorafenib. Hepatology, 56(6), 2363-2374. https://doi.org/10.1002/hep.25872

Cyclic AMP/PKA-dependent paradoxical activation of Raf/MEK/ERK signaling in polycystin-2 defective mice treated with sorafenib. / Spirli, Carlo; Morell, Carola M.; Locatelli, Luigi; Okolicsanyi, Stefano; Ferrero, Cecilia; Kim, Amy; Fabris, Luca; Fiorotto, Romina; Strazzabosco, Mario.

In: Hepatology, Vol. 56, No. 6, 12.2012, p. 2363-2374.

Research output: Contribution to journalArticle

Spirli, C, Morell, CM, Locatelli, L, Okolicsanyi, S, Ferrero, C, Kim, A, Fabris, L, Fiorotto, R & Strazzabosco, M 2012, 'Cyclic AMP/PKA-dependent paradoxical activation of Raf/MEK/ERK signaling in polycystin-2 defective mice treated with sorafenib', Hepatology, vol. 56, no. 6, pp. 2363-2374. https://doi.org/10.1002/hep.25872
Spirli, Carlo ; Morell, Carola M. ; Locatelli, Luigi ; Okolicsanyi, Stefano ; Ferrero, Cecilia ; Kim, Amy ; Fabris, Luca ; Fiorotto, Romina ; Strazzabosco, Mario. / Cyclic AMP/PKA-dependent paradoxical activation of Raf/MEK/ERK signaling in polycystin-2 defective mice treated with sorafenib. In: Hepatology. 2012 ; Vol. 56, No. 6. pp. 2363-2374.
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abstract = "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., Pkd2flox/-:pCxCreERTM [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. Conclusion: 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.",
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AU - Spirli, Carlo

AU - Morell, Carola M.

AU - Locatelli, Luigi

AU - Okolicsanyi, Stefano

AU - Ferrero, Cecilia

AU - Kim, Amy

AU - Fabris, Luca

AU - Fiorotto, Romina

AU - Strazzabosco, Mario

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N2 - 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., Pkd2flox/-:pCxCreERTM [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. Conclusion: 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.

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