Akt1 and Akt3 but not Akt2 through interaction with DNA-PKcs stimulate proliferation and post-irradiation cell survival of K-RAS-mutated cancer cells

Mahmoud Toulany; Julia Maier; Mari Iida; Simone Rebholz; Marina Holler; Astrid Grottke; Manfred Jüker; Deric L Wheeler; Ulrich Rothbauer; H Peter Rodemann

doi:10.1038/cddiscovery.2017.72

Akt1 and Akt3 but not Akt2 through interaction with DNA-PKcs stimulate proliferation and post-irradiation cell survival of K-RAS-mutated cancer cells

Cell Death Discov. 2017 Oct 30:3:17072. doi: 10.1038/cddiscovery.2017.72. eCollection 2017.

Authors

Mahmoud Toulany^{1

2}, Julia Maier^{3

4}, Mari Iida⁵, Simone Rebholz^{1

2}, Marina Holler¹, Astrid Grottke⁶, Manfred Jüker⁶, Deric L Wheeler⁵, Ulrich Rothbauer^{3

4}, H Peter Rodemann^{1

2}

Affiliations

¹ Division of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Tuebingen, Germany.
² German Cancer Consortium (DKTK), Partner Site Tuebingen, and German Cancer Research Center (DKFZ), Heidelberg, Germany.
³ Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany.
⁴ Pharmaceutical Biotechnology, Eberhard Karls University Tuebingen, Tuebingen, Germany.
⁵ Department of Human Oncology, University of Wisconsin, Madison, WI, USA.
⁶ Institute of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Abstract

Akt1 through the C-terminal domain interacts with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and stimulates the repair of DNA double-strand breaks (DSBs) in K-RAS-mutated (K-RASmut) cells. We investigated the interactions of distinct domain(s) of DNA-PKcs in binding to full-length Akt1. Similarly, we analyzed potential interactions of DNA-PKcs with Akt2 and Akt3. Finally the effect of Akt isoforms in cell proliferation and tumor growth was tested. We demonstrated that Akt1 preferentially binds to the N-terminal domain of DNA-PKcs using pull-down studies with distinct eGFP-tagged DNA-PKcs fragments that were expressed by plasmids in combination with mCherry-tagged full-length Akt isoforms. These binding studies also indicated an interaction with the intermediate and C-terminal domains of DNA-PKcs. In contrast, Akt3 interacted with all four DNA-PKcs fragments without a marked preference for any specific domain. Notably, we could not see binding of Akt2 to any of the tested DNA-PKcs fragments. In subsequent studies, we demonstrated that Akt inhibition interferes with binding of Akt1 to the N-terminal domain of DNA-PKcs. This indicated a correlation between Akt1 activity and the Akt1/DNA-PKcs complex formation. Finally, knockdown studies revealed that the depletion of endogenous Akt1 and Akt3, but not Akt2, inhibit clonogenic activity and repair of ionizing radiation (IR)-induced DNA DSBs, leading to radiosensitization. Furthermore, in a xenograft study the expression of shAkt1 or shAkt3, but not shAkt2 in K-RASmut breast cancer cell line MDA-MB-231 showed major tumor growth delay. Together, these data indicate that Akt1 and Akt3, but not Akt2, physically interact with DNA-PKcs, thus stimulating the repair of DSBs and therefore protecting K-RASmut cells against IR. Likewise, interaction of Akt isoforms with DNA-PKcs could be crucial for their role in regulating tumor growth.