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Dual Inhibition of PI3K-AKT-mTOR- and RAF-MEK-ERK-signaling is synergistic in cholangiocarcinoma and reverses acquired resistance to MEK-inhibitors

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Summary

Until today, there is no systemic treatment available for advanced cholangiocarcinoma (CCA). Recent studies have shown a frequent upregulation of the PI3K-AKT-mTOR and RAF-MEK-ERK pathways in this type of cancer. However, considering their high extend of redundancy and cross-talk, targeting only one pathway is likely to result in therapy failure and emergence of resistances. To provide a rationale for treatment of CCA with inhibitors of these respective pathways, we analyzed the effects of AKT inhibitor MK-2206, MEK inhibitor AZD6244 (ARRY-142886) and mTOR kinase inhibitor AZD8055 on three CCA cell lines in vitro, concerning proliferation, cell signaling and apoptosis. Furthermore, AZD6244 resistant cell lines have been generated to investigate, how their response may be affected by prolonged treatment with only a single inhibitor. Our data demonstrates that co-targeting of both, the PI3K/AKT/mTOR and RAF-MEK-ERK pathway, as well as vertical targeting of AKT and mTOR results in strong synergistic effects on proliferation and cell survival with combination indices below 0.3. Mechanistically, the combinatorial treatment with MK-2206 in addition to AZD8055 is necessary because AKT kinase activity was quickly restored after mTOR kinase inhibition. Interestingly, acquired MEK inhibitor resistance to AZD6244 was reversed by combined treatment with AZD6244 and either MK-2206 or AZD8055. Our data suggest that a combination of inhibitors targeting those respective pathways may be a viable approach for future application in patients with cholangiocarcinoma. Implications: AKT, mTOR and MEK are promising targets for a combinatorial treatment of cholangiocarcinoma cells even after acquisition of MEK inhibitor resistance.

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Abbreviations

mTOR:

mammalian target of rapamycin

PI3K:

phosphatidylinositol 3-kinase

PI3KCA:

catalytic subunit of phosphatidylinositol 3-kinase

IRS-1:

insulin receptor substrate 1

BrdU:

5-bromo-2’-deoxyuridine

DMSO:

dimethyl sulfoxide

PBS:

phosphate buffered saline

CCA:

cholangiocarcinoma

CI:

combination index

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Acknowledgements

We are grateful to Dr. Knuth from the University Hospital Zürich, Department of Oncology for providing SK-ChA-1 cells. Florian Ewald is a fellow of the University Cancer Center Hamburg (UCCH).

Conflict of interest statement

There is no conflict of interest.

Authors’ contributions

FE, DN, BN and MJ designed the study. FE, DN, AG and BH performed the experiments and interpreted the experimental findings. FE, DN and MJ drafted the manuscript and wrote the final version of the manuscript. All authors read and approved the final manuscript.

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Authors and Affiliations

  1. Department of Hepatobiliary and Transplant Surgery, University Medical Center Hamburg-Eppendorf, Martinistr. 52, Hamburg, 20246, Germany

    Florian Ewald & Björn Nashan

  2. Center of Experimental Medicine Institute of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246, Hamburg, Germany

    Dominik Nörz, Astrid Grottke, Bianca T. Hofmann & Manfred Jücker

  3. Department of General, Visceral and Thoracic Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    Bianca T. Hofmann

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  1. Florian Ewald
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  2. Dominik Nörz
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  3. Astrid Grottke
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  4. Bianca T. Hofmann
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  5. Björn Nashan
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  6. Manfred Jücker
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Corresponding author

Correspondence to Manfred Jücker.

Additional information

Florian Ewald and Dominik Nörz  contributed equally to this work

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Online Resource 1

Cells were treated with the indicated concentrations of AZD6244 or AZD8055 for 24h. PI3K-AKT-mTOR and RAF-MEK-ERK signaling pathway activity was analyzed by western blot with antibodies directed against the indicated targets. HSC70 served as loading control. (PDF 246 kb)

Online Resource 2

CCA cell lines were treated with increasing concentrations of AZD8055 (a) or AZD6244 (b), controls were treated with DMSO. Proliferation of CCA cell lines was analyzed after 72h by BrdU incorporation. Each data point represents mean of three independent experiments, normalized to controls. The respective concentrations of AZD6244 and AZD8055 resulting in 50% inhibition of proliferation are given in (c). (PDF 311 kb)

Online Resource 3

Fractional Effect blots were computed based on the experiment as described in Fig. 1. CI values from 0.3 to 0.7 are considered to indicate synergism, and CI values below 0.3 are considered to represent strong synergism. (PDF 138 kb)

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Ewald, F., Nörz, D., Grottke, A. et al. Dual Inhibition of PI3K-AKT-mTOR- and RAF-MEK-ERK-signaling is synergistic in cholangiocarcinoma and reverses acquired resistance to MEK-inhibitors. Invest New Drugs 32, 1144–1154 (2014). https://doi.org/10.1007/s10637-014-0149-7

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