Poster Presentation 2nd Australian Cancer and Metabolism Meeting 2017

PI3K/Akt signalling coordinates LAT3-mediated leucine uptake for mTORC1 pathway activation (#36)

Blake Zhang 1 2 3 , Anne Moran 1 2 3 , Chuck Bailey 2 3 , John EJ Rasko 2 3 4 , Jeff Holst 1 2 3 , Qian Wang 1 2 3
  1. Origins of Cancer, ACRF Centenary Institute, The University of Sydney, Camperdown, NSW, Australia
  2. Gene & Stem Cell Therapy, Centenary Institute, The University of Sydney, Camperdown, NSW, Australia
  3. Sydney Medical School, The University of Sydney, Camperdown, NSW, Australia
  4. Cell and Molecular Therapies, Royal Prince Alfred Hospital, Camperdown, NSW, Australia

Objective: PI3K/Akt and leucine-activated signalling are essential mediators of mTORC1 activation, thereby regulating protein synthesis and cell growth. In primary prostate cancer, leucine is provided to the cells through L-type amino acid transporter 3 (LAT3; SLC43A1), and LAT3 is therefore essential for mTORC1 pathway activation. PhosphoSitePlus database showed that several residues of LAT3 are phosphorylated, suggesting that LAT3 can be regulated by signalling pathways. Therefore, we aim to investigate the crosstalk between the PI3K/Akt signalling and leucine transport in prostate cancer.

Methods: LNCaP human prostate cancer cell line was used for the investigation. We conducted leucine uptake assays and analysed Akt phosphorylation, in the presence and absence of EGF and various kinase inhibitors of PI3K (LY294002) and Akt (MK2206), to examine the effect on cellular uptake of leucine and Akt activation. We used the proximity ligation assay (PLA) to explore the in situ interaction between phosphorylated Akt and LAT3.

Results: Our results showed that inhibition of PI3K/Akt signalling using LY294002 or MK2206 decreased leucine transport in LNCaP cell line. EGF treatment induced PI3K/Akt activation, leading to a significant increase in leucine uptake. PLA analysis showed an increased signal in EGF-treated LNCaP cells compared to untreated control cells, indicating that phosphorylated Akt might interact with LAT3 to regulate leucine transport. With the stimulation of EGF, LAT3 protein level increased in 30 min, but decreased in the presence of MK2206, indicating Akt regulates the LAT3 protein level.

Conclusion: Growth factor-activated PI3K/Akt signalling regulates leucine transport through amino acid transporter LAT3 in prostate cancer cell lines. Therefore, combination of therapeutics targeting Akt and LAT3 may provide an increased benefit for prostate cancer patients.