The results indicate that NSCLC cell proliferation is attenuated by reducing PKIB expression. Open in a separate window Figure 3 JX 401 Real-time PCR (a) and western blotting (b) were applied to confirm the efficiency of the PKIB shRNA infection in H1299 cells. that PKIB promotes cell proliferation and tumorigenesis by activating the PI3K/Akt pathway in NSCLC, implying that this is an important underlying mechanism that affects the progression of NSCLC. Keywords: cAMP-dependent protein kinase inhibitor-, proliferation, nonCsmall cell lung cancer, metastasis, invasion Introduction Lung cancer is one of the JX 401 most prevalent malignant tumors and the leading cause of cancer-related death among men and women worldwide.1,2 NonCsmall cell lung cancer (NSCLC) is responsible for approximately 80% of lung cancer diagnoses. Currently, lung cancer therapeutic strategies include surgery, chemotherapy, radiotherapy, and recently established molecular targeted therapies, but the main challenge of targeted therapies is that only a small proportion of patients can benefit from the treatments.3,4 Moreover, the 5-year overall survival rate for patients with NSCLC has not been FJH1 markedly improved by the current therapeutic strategies, which is only 16% for all stages of lung cancer.5 In recent years, although the discovery of targetable driver oncogenes, such as EGFR mutations, ALK fusions, and inhibition of hTERT overexpression, are major treatment strategies for patients with NSCLC,6C8 there is still an urgent need to understand the JX 401 molecular mechanisms of JX 401 lung cancer tumorigenesis and to identify new therapeutic targets to improve the treatment strategies for lung cancer patients. PKIB (cAMP-dependent protein kinase inhibitor-) is a member of the protein kinase inhibitors (PKIs), which are a class of proteins that can inhibit the activity of cAMP-dependent protein kinase (PKA). PKA is a complex composed of two regulatory subunits (R-subunits) and two catalytic subunits (C-subunits)9; the PKIs can bind to the C-subunits of PKA in the nucleus and then transport them to the cytoplasm to reform the inactive PKA complex with the R-subunits.10 To date, studies have found that PKIB has the ability to enhance the constitutive activity of the G-protein-coupled zinc receptor GPR39 and may play important roles in vascular endothelial cells.11,12 Furthermore, there are a few studies about the functions of PKIB in the tumor progression process. In breast cancers, PKIB expression is strongly correlated with the triple-negative breast cancer subtype, and overexpression of PKIB promotes tumor aggressiveness in prostate cancer.13,14 However, it is currently not known whether PKIB is involved in modulating the progression of NSCLC. In the present study, we aim to clarify the roles of PKIB in the proliferation, migration, and invasion of NSCLC cells. We find that the expression of PKIB is significantly up-regulated in NSCLC tissues compared with the normal tissues adjacent to the tumors. Moreover, we have also demonstrated that PKIB serves as an important regulator of the cell proliferation and metastasis of NSCLC cells by activating the PI3K/Akt pathway. All of our results suggest that PKIB may be a novel potential therapeutic target for NSCLC. Materials and methods Materials The antibodies against PCNA and beta-actin were purchased from Santa Cruz Biotechnology Inc., CA, USA, and the PKIB antibody was purchased from Abcam (Cambridge, MA, USA). The BrdU proliferation assay kit was purchased from Millipore Corporation (Billerica, MA). All other reagents were from common commercial sources. Cell line preparation and clinical samples The A549 and H1299 cells, two human NSCLC cell lines, were obtained from the American Type Culture Collection (ATCC, Rockville, Maryland, USA). Both cell lines were routinely cultured in DMEM supplemented with 10% fetal calf serum, penicillin (100?U/mL), and streptomycin (100?g/mL) at 37 in an incubator with 95% air and 5% CO2. The tumor specimens used in this study were obtained from 30 NSCLC patients who underwent curative resection.