CIP2A regulates cell proliferation via the AKT signaling pathway in human lung cancer

Abstract

Cancerous inhibitor of PP2A (CIP2A) is an intracellular endogenous protein phosphatase 2A (PP2A) inhibitor with oncogenic activities. Initially identified as a tumor-associated antigen (TAA) in gastric and liver cancer patients, CIP2A was overexpressed in a variety of cancer types. The overexpression of CIP2A in cancer cells is associated with increased cell proliferation. However, the mechanism of CIP2A in cancer cell proliferation remains poorly understood. In the present study, we reported that CIP2A can regulate AKT phosphorylation at S473 under growth factor stimulation and our results also showed that CIP2A may promote cell proliferation through the AKT signaling pathway. Notably, depletion of CIP2A did not induce a global change of AKT phosphatase activity, which indicated that CIP2A may recognize specific AKT targets and play certain roles in the signaling pathway. In addition, we detected that CIP2A expression was associated with mTOR phosphorylation. Our further analysis corroborated the relationship between CIP2A and AKT-mTOR signaling pathway. Therefore, our study addressed a novel role of CIP2A in mediating cancer progression through interacting with the AKT-mTOR signaling pathway.

Figure 1

CIP2A expression and cell proliferation assay. (A) CIP2A expression in NSCLC was examined in four cell lines, A549, H838, H1299 and H460, by using mouse anti-CIP2A monoclonal antibody (1:2,000). (B) Cells transduced with shRNA control or shRNA CIP2A or pLOX or pLOX-CIP2A were evaluated in three cell lines (H838, H1299 and H460). (C) Cell proliferation assay was performed in H1299 cell line transduced with shRNA control or shRNA CIP2A and H460 cell line transduced with pLOX or pLOX-CIP2A. Five thousand cells of each group were plated sextuplet in 96-well plates and grew for the indicated times (day 1, 2, 3 and 4). Cell proliferation assay was performed by incubation of cell culture with MTT for 4 h and the absorbance was measured at 570 nm in a colorimetric meter.

Figure 2

CIP2A regulates AKT phosphorylation in lung cancer cells. Phosphorylation of AKT between cells transduced with shRNA control or shRNA CIP2A or pLOX or pLOX-CIP2A was evaluated in three cell lines (H838, H1299 and H460). Equal number of cells was starved for 24 h and then stimulated with EGF for 30 min. Cells were harvested, lysed in 1X Laemmli sample buffer, resolved on 10% SDS-PAGE, transferred to nitrocellouse membrane and probed with rabbit anti-phospho-AKT (1:1,000). CIP2A to actin ratio was analyzed with densitometry.

Figure 3

CIP2A modulates AKT-associated PP2A phosphatase activity. AKT-associated PP2A phosphatase activity was evaluated in two lung cancer cell lines, H838 and H460, with either CIP2A depletion (left panel) or overexpression (right panel). Western blot results showed the amount of AKT in the 5% of the immunoprecipitates in both of H1299 and H460, which is used to normalize the PP2A phosphatase activity.

Figure 4

CIP2A promotes cell proliferation through AKT signaling. Cell proliferation was measured in cell lines transfected or co-transfected with either shRNA control, CIP2A shRNA (shRNA1), shRNA1 and AKT CA, pCDNA3.1+CIP2A or pcDNA3.1-CIP2A and AKT KD. Five thousand transfected cells were plated sextuplet in 96-well plates and cell proliferation was measured 48 h post-transfection. After 4 h incubation with MTT, cell proliferation was stopped with Stop solution and absorption was measured at 570 nm in a colorimetric meter.

Figure 5

CIP2A modulates mTOR phosphorylation and the expression level of mTOR downstream substrates. Phosphorylation of mTOR phosphorylation and its downstream effectors, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and P70 ribosomal S6 kinase 1 (P70S6K1) were analyzed in H838 and H460 cancer cell lines with either CIP2A depletion or CIP2A overexpression. Cells were starved, stimulated with EGF (100 ng/ml) for 30 min and lysed with 1X Laemmli buffer. Proteins were resolved on 10% SDS-PAGE gel. Phosphorylation of mTOR was analyzed with rabbit anti-phospho-mTOR (S2441) (1:1,000) and normalized to the expression level of actin.