Abstract:
The serine-threonine protein kinase, Akt, also known as protein kinase B (PKB), plays a critical role downstream from phosphoinositide 3-kinases (PI3K), which is essential in regulating cell growth, metabolism, and proliferation. Activation of the PI3K/Akt pathway in response to growth factor regulates cell functions by phosphorylating the Forkhead box (Fox) O subfamily of transcription factors. The FOXO family plays a crucial role in many aspects of development and functions of an organism, including tumorigenesis. All four members of the FOXO subfamily identified (FOXO1, FOXO3a, FOXO4, and FOXO6) have overlapping roles in regulating cell death and cell cycle arrest that they function as tumour suppressors. Transcriptional activity of FOXO is regulated by Akt/PKB phosphorylation and nuclear export. Unphosphorylated FOXO in the nucleus FOXO is active for transcription regulation whereas phosphorylated FOXO is inactivated and is subsequently exported from the nucleus for proteasomal degradation. The aim of this project is using FOXO-inducible luciferase reporters to monitor nuclear changes of FOXO levels in response to PI3K inhibitors. Ultimately, this system would be used in vivo for monitoring real-time progression of tumours. Here we showed that sole transduction of the lentiviral FOXO-luciferase reporter or transfection of the DNA-based FOXO-luciferase reporter was not sufficient for detecting the effects of well-known PI3K inhibitors. This is due to the low levels of active FOXO proteins in the nucleus. We also showed that transduction of additional FOXO3a recombinant together with the FOXO-luciferase reporter into certain cancer cell lines, significant changes of luciferase activity in response to PI3K inhibitors and/or insulin could be observed. According to our results, we concluded that with additional stable transduction of exogenous FOXO3a, cell lines that stably expressing FOXO-luciferase may be used in vivo as pharmacodynamic markers for screening cancer drugs targeting the PI3K pathway.
