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Theranostics 2022; 12(9):4348-4373. doi:10.7150/thno.68299 This issue Cite

Research Paper

RNA interference screens discover proteases as synthetic lethal partners of PI3K inhibition in breast cancer cells

Lena Hölzen^1,2,3,4, Jan Mitschke⁵, Claudia Schönichen¹, Maria Elena Hess^4,6, Sophia Ehrenfeld⁵, Melanie Boerries^2,3,6,8, Cornelius Miething^2,3,5,8, Tilman Brummer^1,2,3,7,8, Thomas Reinheckel^1,2,3,7,✉

1. Institute of Molecular Medicine and Cell Research, Faculty of Medicine, University of Freiburg, 79104 Freiburg, Germany
2. German Cancer Consortium (DKTK) Partner Site Freiburg, DKFZ, 79104 Freiburg, Germany
3. German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
4. Faculty of Biology, Albert-Ludwigs-University of Freiburg, 79104 Freiburg, Germany
5. Center for Translational Cell Research, Department of Internal Medicine I, Department of Hematology, Oncology and Stem Cell Transplantation, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany
6. Institute of Medical Bioinformatics and Systems Medicine, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany
7. Center for Biological Signaling Studies BIOSS, University of Freiburg, 79104 Freiburg, Germany
8. Comprehensive Cancer Center Freiburg (CCCF), University Medical Center, University of Freiburg, 79106 Freiburg, Germany

✉ Corresponding author: Univ. Prof. Dr. Thomas Reinheckel: Institute of Molecular Medicine and Cell Research, Faculty of Medicine, Albert-Ludwigs-University of Freiburg, Stefan Meier Str. 17, 79104 Freiburg, Germany. Phone: +49 761 203-9606. Email: thomas.reinheckeluni-freiburg.de. More

Abstract

Rationale: PI3K/mTOR signaling is frequently upregulated in breast cancer making inhibitors of this pathway highly promising anticancer drugs. However, PI3K-inhibitors have a low therapeutic index. Therefore, finding novel combinatory treatment options represents an important step towards clinical implementation of PI3K pathway inhibition in breast cancer therapy. Here, we propose proteases as potential synergistic partners with simultaneous PI3K inhibition in breast cancer cells.

Methods: We performed mRNA expression studies and unbiased functional genetic synthetic lethality screens by a miR-E based knockdown system targeting all genome-encoded proteases, i.e. the degradome of breast cancer cells. Importantly theses RNA interference screens were done in combination with two PI3K pathway inhibitors. Protease hits were validated in human and murine breast cancer cell lines as well as in non-cancerous cells by viability and growth assays.

Results: The degradome-wide genetic screens identified 181 proteases that influenced susceptibility of murine breast cancer cells to low dose PI3K inhibition. Employing independently generated inducible knockdown cell lines we validated 12 protease hits in breast cancer cells. In line with the known tumor promoting function of these proteases we demonstrated Usp7 and Metap2 to be important for murine and human breast cancer cell growth and discovered a role for Metap1 in this context. Most importantly, we demonstrated that Usp7, Metap1 or Metap2 knockdown combined with simultaneous PI3K inhibition resulted in synergistic impairment of murine and human breast cancer cell growth

Conclusion: We successfully established proteases as combinatory targets with PI3K inhibition in human and murine breast cancer cells. Usp7, Metap1 and Metap2 are synthetic lethal partners of simultaneous protease/PI3K inhibition, which may refine future breast cancer therapy.

Keywords: Proteases, Cancer Degradome, Breast Cancer, RNA Interference, Phosphatidylinositol-3-kinase (PI3K)-Pathway

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