The Study Of The Regulatory Mechanisms Of PI3K/Akt Signaling On Skp2 And Its Downstream Proteins In Lung Cancers

ObjectiveLung cancer is a malignant tumor with high mortality and morbidity. Accumulating data suggested that the incidence of lung cancer increased dramatically since 2000. And currently the death toll caused by lung cancer was up to 60 million each year in China, ranking first among all malignancies. The pathogenesis of cancer is complicated, a variety of signaling pathways could regulate the process of tumorigenesis. Among them Phosphatidylinositol-3-kinase (PI3K) signaling pathway is of great concern, which regulates proliferation, differentiation, apoptosis and migration of tumor cells by phosphorylating Akt, then activating or inhibiting a number of its downstream target proteins, such as Bad, Caspase9, NF-κB, p21 and p27. It had been reported previously that PI3K signaling was abnormally activated in lung tumor cells; and when it was blocked, the tumor cells showed growth inhibition, partly due to the blockage of cell cycles.S-phase kinase-associated protein 2 (Skp2) is an important regulator in cell cycles, which affects cell proliferation, apoptosis, tumorigenesis and development of cancers. Skp2 was originally discovered as a component of Cyclin-A-CDK2 complex. Many studies have now shown that Skp2 is also a member of the F-box family and the substrate recognition component of the Skpl-Cullin-Skp2 (SCF)-type ubiquitin ligase. Skp2 could regulate the G1/S phase progression via the ubiquitin-dependent degradation of cell cycle regulators such as p21, p57, E2F1, MYC, Cyclin A, Cyclin E, Cyclin D1, and p27. Cyclin-dependent kinase inhibitor (CKI) p27 is the major degradation substrate of Skp2, in addition, only Thr-187 phosphorylated p27 is degraded by Skp2. Kei-Ichi reported that Skp2 could ubiquitin ligase phosphorylated Cyclin E and promote its degradation, which regulated the cells'entrance into S phase and maintained the stability of chromosome.As shown in previous studies, the expression level of Skp2 was elevated in many cancers, which was related to the tumorigenesis, development and prognosis of some cancers including breast, colon and lung. Skp2 was an oncoprotein, regulating the G1/S transition via ubiquitin-dependent degradation of cell cycle regulators involved in the transition of G1 phase to S phase, therefore, Skp2 could serve as a new target of oncotherapy. However, the effects of PI3K/Akt pathway on the expression of Skp2 and its role in cell proliferation in lung cancers is unclear. In the current study, we used four types of lung cancer cell lines H460, LK2, H446, and A549 to explore the regulatory effects of PI3K/Akt signaling on Skp2. We also investigated the effects of PI3K/Akt signaling on E2F1, p27, Cyclin E.Methods1,LK2, A549 cells were cultured in DMEM medium supplemented with 10% calf serum, H460 and H446 cells in RPMI1640 medium containing 10% calf serum. Cells were maintained at 37℃in a humidified atmosphere of 5% CO2 in air.2,Cells with 70% coverage were divided into two groups:the control group and the treatment group. PI3K/Akt pathway specific inhibitor LY294002 was added to the treatment group at 25μmol/L. The same amount volume of DMSO was added to the control group.3,MTT assay was used to detect the effects of LY294002 on cells growth and proliferation. The growth curves were drawn, and the appropriate length of treated time was optimized.4,Flow cytometer (FCM) was used to analyze the cell cycle progression in H460, LK2, H446 and A549 cell lines treated with DMSO or LY294002.5,Western-blot was used to detect the expression levels of Skp2, p27, CyclinE, E2F1 proteins in H460, LK2, H446 and A549 cell lines treated with DMSO or LY294002.6,Real-time RT-PCR was used to detect the mRNA levels in H460, LK2, H446 and A549 cell lines treated with DMSO or LY294002.7,Immunofluorescence assay was used to detect the subcellular localization of Skp2 protein in lung cancer cells H460, LK2, H446, A549 and lung normal cells HBE treated with DMSO or LY294002.Results1,Inhibition of cell proliferation by LY294002 in lung cancer cellsMTT assays showed that the growth rates of H446, A549, LK2, H460 cell lines were decreased after the treatment of LY294002. The inhibition rates were 54.7±4.97%, 55.7±3.24%,36.24%±4.56%and 38.96%±3.17%respectively when cells were treated for 24 hours.2,G1/S cell cycle arrest by LY294002 in lung cancer cellsAs shown in FCM, the cells in G1 phase were dramatically increased, while the cells in S phase were decreased in H446, A549, LK2, H460 cells treated with LY294002 when compared those treated with DMSO. The differences were statistically significant, indicating that the cells treated with LY294002 were arrested at Gl/S phase.3,The effects of LY294002 on expression of Skp2, p27, CyclinE, E2F1 proteinsWestern blot showed that compared to DMSO treated control group, the expression levels of Skp2 protein were significantly decreased in H460, LK2, H446 and A549 cells treated with LY294002 (P<0.01); the expression levels of p27 were increased in H446, A549 cell lines, while unaltered in H460, LK2 cell lines; the expression levels of Cyclin E were decreased in these four cell lines; the expression levels of E2F1 were decreased in H460, LK2, H446 cell lines, especially in H446 cell lines; on the other hand, E2F1 was not expressed in A549 cell lines.4,The effects of LY294002 on the expression of Skp2, p27 mRNAAs shown in Real-time RT-PCR, the expression levels of Skp2 mRNA were decreased and the expression levels of p27 mRNA were increased in these four cell lines.5,The effects of LY294002 on subcellular localization of Skp2 proteinImmunofluorescence showed that Skp2 protein was mainly expressed in nucleus in HBE and H460 cell lines, while partly located in cytoplasm in LK2, H446 and A549 cell lines. LY294002 treated LK2, H446 and A549 cells demonstrated increased nuclear localization of Skp2 protein, in H460 cells Skp2 expression was increased in cytoplasm and nucleus membrane area after exposure to LY294002.Conclusion1,In H446 and A549 cell lines, Skp2 and p27 are negatively regulated, indicating that p27 was degraded via Skp2-mediated ubiquitin-proteasome system (UPS); while in H460 and LK2 cell lines, increase in p27 proteins were not observed upon downregulation of Skp2, indicating that p27 wasn't degraded through the Skp2-mediated UPS in these cells. E2F1, CyclinE weren't degraded through the Skp2 mediated UPS in these four cell lines either.2,PI3K/Akt pathway possibly regulated the expression of Skp2 through transcriptional factor E2F1 in H460, H446 and LK2 cells, but not in lung adenocarcinoma A549 cells; E2F1 might also regulate the expression of Cyclin E.3,Skp2 protein was mainly located in cytoplasm and inhibition of PI3K/Akt resulted in nuclear translocation of Skp2 in LK2, H446 and A549 cell lines. On the contrary, Skp2 protein was mainly presented in nucleus and inhibition of PI3K/Akt resulted in its cytoplasmic distribution.4,PI3K/Akt pathway could regulate the G1/S phase and cell proliferation through E2F1, CyclinE, p27 in lung cancers.