Autophagy limits the cytotoxic effects of the AKT inhibitor AZ7328 in human bladder cancer cells.
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.
Department of Urology, University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Rian J
RJ
Dickstein
Giovanni
G
Nitti
Colin P
CP
Dinney
Barry R
BR
Davies
Ashish M
AM
Kamat
David J
DJ
McConkey
D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D001343:Autophagy / Q000187:drug effects* / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D058534:Class I Phosphatidylinositol 3-Kinases; D006801:Humans; D009154:Mutation / Q000187:drug effects / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors*; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000235:genetics; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology
D051017:Apoptosis Regulatory Proteins; D058534:Class I Phosphatidylinositol 3-Kinases; D019869:Phosphatidylinositol 3-Kinases; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; C546842:MTOR protein, human; C484760:PIK3CA protein, human