Autophagy limits the cytotoxic effects of the AKT inhibitor AZ7328 in human bladder cancer cells

Abstract

Background: Mutations that activate the PI3K/AKT/mTOR pathway are relatively common in urothelial (bladder) cancers, but how these pathway mutations affect AKT dependency is not known. We characterized the relationship between AKT pathway mutational status and sensitivity to the effects of the selective AKT kinase inhibitor AZ7328 using a panel of 12 well-characterized human bladder cancer cell lines.

Methods: Sequenome DNA sequencing was performed to identify mutations in a panel of 12 urothelial cancer cell lines. Drug-induced proliferative inhibition and apoptosis were quantified using MTT assays and propidium iodide staining with FACS analyses. Protein activation via phosphorylation was measured by immunoblotting. Autophagy was measured by LC3 immunofluorescence and immunoblotting.

Results: AZ7328 inhibited proliferation and AKT substrate phosphorylation in a concentration-dependent manner but had minimal effects on apoptosis. Proliferative inhibition correlated loosely with the presence of activating PIK3CA mutations and was strengthened in combination with the mTOR inhibitor rapamycin. AZ7328 induced autophagy in some of the lines, and in the cells exposed to a combination of AZ7328 and chemical autophagy inhibitors apoptosis was induced.

Conclusions: The cytostatic effects of AZ7328 correlate with PIK3CA mutations and are greatly enhanced by dual pathway inhibition using an mTOR inhibitor. Furthermore, AZ7328 can interact with autophagy inhibitors to induce apoptosis in some cell lines. Overall, our results support the further evaluation of combinations of PI3K/AKT/mTOR pathway and autophagy inhibitors in pre-clinical in vivo models and ultimately in patients with PIK3CA mutant bladder cancers.

Keywords: :

• AKT/PKB
• PI3K
• mTOR
• rapamycin
• autophagy
• urothelial cancer

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We would like to thank Goodwin Jinesh G, Aditi Das, Eswaran Devarajan, I-ling Lee, Neema Navai and Eugene K Lee for providing technical support. We also thank Katherine Stemke Hale and Michael Davies for help with the gene sequencing analyses. STR DNA fingerprinting was done by the Cancer Center Support grant funded Characterized Cell Line core, NCI # CA16672.

This research was supported by the MD Anderson Genitourinary SPORE Grant (P50 CA091846), a sponsored research agreement from AstraZeneca and MD Anderson’s Cancer Center Support Grant (P30 CA016672).

Supplemental Materials

Supplemental material may be found here:

http://www.landesbioscience.com/journals/cbt/article/21793/