Mutant AKT1-E17K is oncogenic in lung epithelial cells.

The hotspot E17K mutation in the pleckstrin homology domain of AKT1 occurs in approximately 0.6–2% of human lung cancers. In this manuscript, we sought to determine whether this AKT1 variant is a bona-fide activating mutation and plays a role in the development of lung cancer. Here we report that in immortalized human bronchial epithelial cells (BEAS-2B cells) mutant AKT1-E17K promotes anchorage-dependent and -independent proliferation, increases the ability to migrate, invade as well as to survive and duplicate in stressful conditions, leading to the emergency of cells endowed with the capability to form aggressive tumours at high efficiency. We provide also evidence that the molecular mechanism whereby AKT1-E17K is oncogenic in lung epithelial cells involves phosphorylation and consequent cytoplasmic delocalization of the cyclin-dependent kinase (cdk) inhibitor p27. In agreement with these results, cytoplasmic p27 is preferentially observed in primary NSCLCs with activated AKT and predicts poor survival.

We also investigated whether AKT1-E17K altered growth potential of NCI-H23 cells. Analysis of cell proliferation by Trypan blue assay demonstrated that H23-AKT1-E17K cells duplicated at an accelerated rate in monolayer compared with H23-pcDNA3 or H23-AKT1-WT cells (Supplemental Fig. S1D). In agreement with the results of AKT activation, the difference in the proliferation rate between H23-AKT1-E17K cells and H23-pcDNA3 or H23-AKT1-WT cells was predominantly observed under conditions of growth factor deprivation.
These results confirm the data obtained in the BEAS-2B cells and indicate that mutant AKT1-E17K, but not wild type AKT1 expressed at similar level, is able to promote anchorage-dependent proliferation of human bronchial epithelial cells, especially under stressful conditions.

AKT1-E17K stimulates migration and invasion in NSCLC NCI-H23 cells
We also evaluated the effects of AKT1-E17K on the capability to migrate of NSCLC cells. All cells were included in 2D Matrigel matrices and followed for 18 hours using time-lapse microscopy. No difference in cell motility was observed in the presence of growth factors (not shown). Conversely, H23-PcDNA3 cells moved with a mean speed of 0.15 ± 0.04 μm/min demonstrating low velocity in serum free conditions. Expression of AKT1-WT did not significantly modify 2D velocity of NCI-H23 cells (0.17 ± 0.04 μm/min). Conversely, AKT1-E17K significantly increased the ability of these cells to move (0.23 ± 0.05 μm/min, p < 0.0001) (Supplemental Fig. S2A). The total path described by each cell was significantly longer in AKT1-E17Kexpressing cells with respect to control or AKT1-WT expressing cells, demonstrating that AKT1-E17K prevents the reduction of cell speed due to serum deprivation (Supplemental Fig. S2B).
These results confirmed the data obtained in the BEAS-2B cells and indicated that mutant AKT1-E17K, but not wild type AKT1 expressed at similar level, is able to stimulate migration, invasion and tumorigenicity in human NSCLC cells.

PIK3CA-E545K and PTEN silencing promote delocalization of p27 in NSCLC cells
Aberrant activation of the PI3K/AKT pathway is a frequent event in NSCLC. It results from gain-offunction mutations of AKT1 itself, of PIK3CA or from PTEN loss. We investigated whether inactivation of p27 by phosphorylation represents a general mechanism whereby abnormally active PI3K/AKT pathway signals in lung epithelial cells. To this aim we generated BEAS-PIK3CA-E545K and BEAS-shPTEN cells after lentiviraltransduction and selection in blasticidin-containing medium (Malanga et al. in preparation). The presence of the exogenous PIK3CA or endogenous PTEN proteins was detected by immunoblot (Malanga et al. in preparation). The status of the PI3K/AKT pathway was determined by analysis of GSK3 phosphorylation (Malanga et al. in preparation). Active PIK3CA (E545K) or PTEN loss are tumorigenic to human lung epithelial cells like mutant AKT1-E17K (Malanga et al., in preparation).
Here we show that, similar to AKT1-E17K, BEAS cells expressing mutant PIK3CA or silenced for PTEN showed a decreased amount of nuclear p27 compared with control BEAS-C cells (Supplemental Fig. S3A) Fig. S3B). Altogether, these results demonstrated that aberrant signalling through the PI3K pathway -induced by mutant AKT1, mutant PIK3CA or by PTEN loss -is associated with T157/T198 phosphorylated p27, which is delocalized in lung epithelial cells. Analysis of cell proliferation by analysis of growth curves indicated that BEAS-PIK3CA and BEAS-shPTEN cells duplicated at an accelerated rate in monolayer compared with BEAS-C (Supplemental Fig. S3C).

Cell culture and transfections
NCI-H23 cells were grown in RPMI supplemented with 10% fetal bovine serum. Transfections were carried out with Fugene 6 reagent (Roche Diagnostics GmbH, Mannheim, Germany). Selection was carried out with 800 mg/ml G418 (Invitrogen, Carlsbad, CA) for 2-3 weeks. Starvation was carried out in RPMI 0.2%. Immunoblot analysis of pAKT and total AKT1 in extracts enriched in cytosolic or membrane proteins. EGFR and Tubulin were used for normalization and to rule out cross-contamination. C. Immunoblot analysis of p27 and FOXO1 in extracts enriched in cytosolic or nuclear proteins. Tubulin and Myc were used for normalization and to rule out cross-contamination. D. Trypan Blue exclusion assay of NCI-H23 cells and derivatives. Data are means ± SD from triplicate samples of a representative experiment. ****p < 0.0001.