GINS2 facilitates epithelial-to-mesenchymal transition in non-small-cell lung cancer through modulating PI3K/Akt and MEK/ERK signaling

Non-small-cell lung cancer (NSCLC) is a cancer with high morbidity and mortality. We aimed to define the effect of Go-Ichi-Ni-San complex subuint 2 (GINS2) acting on NSCLC. The expressions of GINS2 in NSCLC tissues and cells were detected using real-time quantitative polymerase chain reaction, western blot, and immunohistochemistry (IHC). The relationship between GINS2 expression and NSCLC prognosis or clinicopathologic features was analyzed through statistical analysis. The overexpressed or downexpressed plasmids of GINS2 were transfected into NSCLC cell lines, and then cell proliferation, invasion, and migration viability were, respectively, determined by Cell Counting Kit-8 assay, transwell, and wound healing assay. The epithelial–mesenchymal transition (EMT) was observed and the EMT-related proteins were measured using IHC and western blot. The function of GINS2 in vivo was assessed by mice model. The related proteins of mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) pathways were evaluated using western blot. GINS2 expression was upregulated in NSCLC tissues and cell lines, and its high expression was correlated with the poor prognosis and several clinicopathologic features, such as TMN stages (tumor size, lymph node, and metastasis) and clinical stages. GINS2 enhanced NSCLC cell proliferation, migration, and invasion viability in vivo and in vitro. GINS2 also promoted NSCLC cells EMT. In addition, GINS2 could regulate phosphorylated proteins of PI3K p85, Akt, MEK, and ERK expressions, it revealed that GINS2 effected on PI3K/Akt and MEK/ERK pathways. GINS2 promoted cell proliferation, migration, invasion, and EMT via modulating PI3K/Akt and MEK/ERK signaling pathways. It might be a target in NSCLC treatment.     

Estrogen‐related receptor alpha triggers the proliferation and migration of human non‐small cell lung cancer via interleukin‐6

Human non‐small cell lung cancer (NSCLC) is one of the leading causes of cancer deaths worldwide. Estrogenic signals have been suggested to be important for the growth and metastasis of NSCLC cells. Our present data showed that estrogen‐related receptor alpha (ERRα), while not ERRβ or ERRγ, was significantly elevated in NSCLC cell lines as compared with that in normal bronchial epithelial cell line BEAS‐2B. The expression of ERRα in clinical NSCLC tissues was significantly greater than that in their matched normal adjacent tissues. Over expression of ERRα can trigger the proliferation, migration, and invasion of NSCLC cells, while si‐ERRα or ERRα inhibitor showed opposite effects. ERRα can increase the mRNA and protein expression of IL‐6, while not IL‐8, IL‐10, IL‐22, VEGF, TGF‐β, or TNF‐α, in NSCLC cells. Silence of IL‐6 attenuated ERRα induced proliferation and cell invasion. Furthermore, our data revealed the inhibition of NF‐κB, while not ERK1/2 or PI3K/Akt, abolished ERRα induced production of IL‐6. This might be due to that overexpression of ERRα can increase the expression and nuclear translocation of p65 in NSCLC cells. Collectively, our data showed that activation of NF‐κB/IL‐6 is involved in ERRα induced migration and invasion of NSCLC cells. It suggested that ERRα might be a potential target for NSCLC treatment.     

CCL21/CCR7 promotes G2/M phase progression via the ERK pathway in human non-small cell lung cancer cells

C-C chemokine receptor 7 (CCR7) contributes to the survival of certain cancer cell lines, but its role in the proliferation of human non-small cell lung cancer (NSCLC) cells remains vague. Proliferation assays performed on A549 and H460 NSCLC cells using Cell Counting Kit-8 indicated that activation of CCR7 by its specific ligand, exogenous chemokine ligand 21 (CCL21), was associated with a significant linear increase in cell proliferation with duration of exposure to CCL21. The CCL21/CCR7 interaction significantly increased the fraction of cells in the G(2)/M phase of the cell cycle as measured by flow cytometry. In contrast, CCL21/CCR7 had no significant influence on the G(0)/G(1) and S phases. Western blot and real-time PCR indicated that CCL21/CCR7 significantly upregulated expression of cyclin A, cyclin B1, and cyclin-dependent kinase 1 (CDK1), which are related to the G(2)/M phase transition. The expression of cyclin D1 and cyclin E, which are related to the G(0)/G(1) and G(1)/S transitions, was not altered. The CCL21/CCR7 interaction significantly enhanced phosphorylation of extracellular signal-regulated kinase (P-ERK) but not Akt, as measured by Western blot. LY294002, a selective inhibitor of PI3K that prevents activation of the downstream Akt, did not weaken the effect of CCL21/CCR7 on P-ERK. Coimmunoprecipitation further confirmed that there was an interaction between P-ERK and cyclin A, cyclin B1, or CDK1, particularly in the presence of CCL21. CCR7 small interfering RNA or PD98059, a selective inhibitor of MEK that disrupts the activation of downstream ERK, significantly abolished the effects of exogenous CCL21. These results suggest that CCL21/CCR7 contributes to the time-dependent proliferation of human NSCLC cells by upregulating cyclin A, cyclin B1, and CDK1 potentially via the ERK pathway.     

Baicalein inhibits cell growth and increases cisplatin sensitivity of A549 and H460 cells via miR‐424‐3p and targeting PTEN/PI3K/Akt pathway

Lung cancer is the leading cause of death in individuals with malignant disease. Non‐small‐cell lung cancer (NSCLC) is the most common type of lung cancer, and chemotherapy drugs such as cisplatin are the most widely used treatment for this disease. Baicalein is a purified flavonoid compound that has been reported to inhibit cancer cell growth and metastasis and increase sensitization to chemotherapeutic drugs via different pathways. Therefore, we assessed the effects of baicalein on the proliferation, apoptosis and cisplatin sensitivity in the NSCLC A549 and H460 cell lines and determined the pathways through which baicalein exerts its effects. Baicalein was slightly toxic to normal human bronchial NHBE cells but inhibited growth, induced apoptosis and increased cisplatin sensitivity in A549 and H460 cells. Baicalein down‐regulated miR‐424‐3p, up‐regulated PTEN expression and down‐regulated expression of PI3K and p‐Akt in A549 and H460 cells. Dual‐luciferase reporter assay demonstrated that PTEN is a target gene of miR‐424‐3p, and overexpression of miR‐424‐3p or silencing of PTEN partially attenuated the effects of baicalein on A549 and H460 cells. Taken together, we concluded that baicalein inhibits cell growth and increases cisplatin sensitivity to A549 and H460 cells via down‐regulation of miR‐424‐3p and targeting the PTEN/PI3K/Akt pathway.     

Potential mechanism of interleukin-8 production from lung cancer cells: an involvement of EGF-EGFR-PI3K-Akt-Erk pathway

Tumor inflammatory microenvironment is considered to play the role in the sensitivity of tumor cells to therapies and prognosis of lung cancer patients. Interleukin-8 (IL-8) is one of critical chemo-attractants responsible for leukocyte recruitment, cancer proliferation, and angiogenesis. The present study aimed at investigating potential mechanism of IL-8 production from human non-small cell lung cancer (NSCLC) SPC-A1 cells. We initially found that EGF could directly stimulate IL-8 production, proliferation, and bio-behaviors of lung cancer cells through the activation of EGFR, PI3K, Akt, and Erk signal pathway. EGF-stimulated IL-8 production, phosphorylation of Akt and Erk, and cell proliferation and movement could be inhibited by EGFR inhibitor (Erlotinib), PI3K inhibitor (GDC-0941 BEZ-235 and SHBM1009), and ERK1/2 inhibitor (PD98059). Our data indicate that IL-8 production from lung cancer cells could be initiated by their own produced factors, leading to the recruitment of inflammatory cells in the cancer tissue, and the formation of inflammatory microenvironment. Thus, it seems that the signal pathway of EGFR-PI3K-Akt-Erk can be the potential target of therapies for inflammatory microenvironment in lung cancer.     

RHCG基因在非小细胞肺癌中的表达及对细胞增殖的影响

为了探讨Rh type C glycoprotein (RHCG)对非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞增殖的影响及可能的作用机制,本研究使用荧光定量PCR法检测12对NSCLC及癌旁组织样本中RHCG mRNA的表达水平及pcDNA3.1-RHCG质粒对A549细胞RHCG m RNA的表达;采用CCK-8法检测细胞增殖能力;运用PI染色法检测细胞周期;使用免疫印迹法检p-PI3K、PI3K、p-AKT以及AKT蛋白表达水平。本研究发现,与癌旁组织比较,NSCLC中RHCG m RNA表达水平明显降低。RHCG过表达能抑制NSCLC细胞系A549细胞增殖能力。此外,RHCG过表达使A549细胞周期G1/S期转化发生阻滞。本研究还发现,RHCG过表达可下调A549细胞p-PI3K/PI3K和p-AKT/AKT水平。本研究表明,RHCG抑制NSCLC细胞增殖的作用与其抑制PI3K/AKT信号通路有关。     

CCL21/CCR7 prevents apoptosis via the ERK pathway in human non-small cell lung cancer cells

Previously, we confirmed that C-C chemokine receptor 7 (CCR7) promotes cell proliferation via the extracellular signal-regulated kinase (ERK) pathway, but its role in apoptosis of non-small cell lung cancer (NSCLC) cell lines remains unknown. A549 and H460 cells of NSCLC were used to examine the effect of CCL21/CCR7 on apoptosis using flow cytometry. The results showed that activation of CCR7 by its specific ligand, exogenous chemokine ligand 21 (CCL21), was associated with a significant decline in the percent of apoptosis. Western blot and real-time PCR assays indicated that activation of CCR7 significantly caused upregulation of anti-apoptotic bcl-2 and downregulation of pro-apoptotic bax and caspase-3, but not p53, at both protein and mRNA levels. CCR7 small interfering RNA significantly attenuated these effects of exogenous CCL21. Besides, PD98059, a selective inhibitor of MEK that disrupts the activation of downstream ERK, significantly abolished these effects of CCL21/CCR7. Coimmunoprecipitation further confirmed that there was an interaction between p-ERK and bcl-2, bax, or caspase-3, particularly in the presence of CCL21. These results strongly suggest that CCL21/CCR7 prevents apoptosis by upregulating the expression of bcl-2 and by downregulating the expression of bax and caspase-3 potentially via the ERK pathway in A549 and H460 cells of NSCLC.     

Mitochondrial aldehyde dehydrogenase attenuates hyperoxia-induced cell death through activation of ERK/MAPK and PI3K-Akt pathways in lung epithelial cells

Oxygen toxicity is one of the major risk factors in the development of the chronic lung disease or bronchopulmonary dysplasia in premature infants. Using proteomic analysis, we discovered that mitochondrial aldehyde dehydrogenase (mtALDH or ALDH2) was downregulated in neonatal rat lung after hyperoxic exposure. To study the role of mtALDH in hyperoxic lung injury, we overexpressed mtALDH in human lung epithelial cells (A549) and found that mtALDH significantly reduced hyperoxia-induced cell death. Compared with control cells (Neo-A549), the necrotic cell death in mtALDH-overexpressing cells (mtALDH-A549) decreased from 25.3 to 6.5%, 50.5 to 9.1%, and 52.4 to 15.1% after 24-, 48-, and 72-h hyperoxic exposure, respectively. The levels of intracellular and mitochondria-derived reactive oxygen species (ROS) in mtALDH-A549 cells after hyperoxic exposure were significantly lowered compared with Neo-A549 cells. mtALDH overexpression significantly stimulated extracellular signal-regulated kinase (ERK) phosphorylation under normoxic and hyperoxic conditions. Inhibition of ERK phosphorylation partially eliminated the protective effect of mtALDH in hyperoxia-induced cell death, suggesting ERK activation by mtALDH conferred cellular resistance to hyperoxia. mtALDH overexpression augmented Akt phosphorylation and maintained the total Akt level in mtALDH-A549 cells under normoxic and hyperoxic conditions. Inhibition of phosphatidylinositol 3-kinase (PI3K) activation by LY294002 in mtALDH-A549 cells significantly increased necrotic cell death after hyperoxic exposure, indicating that PI3K-Akt activation by mtALDH played an important role in cell survival after hyperoxia. Taken together, these data demonstrate that mtALDH overexpression attenuates hyperoxia-induced cell death in lung epithelial cells through reduction of ROS, activation of ERK/MAPK, and PI3K-Akt cell survival signaling pathways.     

CK2 inhibitor CX4945 induces sequential inactivation of proteins in the signaling pathways related with cell migration and suppresses metastasis of A549 human lung cancer cells

Casein kinase 2 (CK2) is known to be involved in various cellular processes such as cell cycle, apoptosis and proliferation. It has been reported that the inhibition of CK2 induced by recently developed small molecule CX4945 shows anti-cancer effects including anti-proliferation and anti-angiogenesis in several different cancers including prostate cancer. Here we report that migration and invasion of A549 human lung cancer cells are suppressed by the inhibition of CK2 induced by CX4945. We found that CX4945 sequentially attenuates the proteins in PI3K/Akt and MAPK pathways, two signaling pathways related with cell migration. This sequential control of signal pathways inhibits the expression of membrane type 1-matrix metalloproteinase and this leads to the selective attenuation of one of the gelatinases, MMP-2, which can degrade components of extracellular matrix, and metastasis of A549 human lung cancer cell.     

Insulin-like growth factor I-stimulated melanoma cell migration requires phosphoinositide 3-kinase but not extracellular-regulated kinase activation

Dysregulated signaling contributes to altered cellular growth, motility, and survival during cancer progression. We have evaluated the ability of several factors to stimulate migration in WM1341D, a cell line derived from an invasive human vertical growth phase melanoma. Basic fibroblast growth factor, hepatocyte growth factor, interleukin-8, and CCL27 each slightly increased migration. Insulin-like growth factor I (IGF-I), however, stimulated a 15-fold increase in migration. This response required the IGF-I receptor, which activates phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways. Both pathways have been implicated in migration in a variety of cell types, but the signaling required for IGF-I-induced melanoma cell migration is not well defined. IGF-I-stimulated activation of MAPK/ERK signaling in WM1341D cells was inhibited by U0126, but a 33-fold higher dose of U0126 was needed to inhibit IGF-I-stimulated cellular migration. In contrast, similar concentrations of either wortmannin or LY294002 were required to inhibit both IGF-I-induced PI3K activation and migration. These results indicate that IGF-I-stimulated migration of WM1341D cells requires PI3K activation but is independent of MAPK/ERK signaling. Determining the contributions of IGF-I signaling pathways to migration will help us to understand melanoma progression and may lead to new therapeutic targets of this highly metastatic cancer.     

MICAL1 facilitates breast cancer cell proliferation via ROS‐sensitive ERK/cyclin D pathway

Molecule interacting with CasL 1 (MICAL1) is a multidomain flavoprotein mono‐oxygenase that strongly involves in cytoskeleton dynamics and cell oxidoreduction metabolism. Recently, results from our laboratory have shown that MICAL1 modulates reactive oxygen species (ROS) production, and the latter then activates phosphatidyl inositol 3‐kinase (PI3K)/protein kinase B (Akt) signalling pathway which regulates breast cancer cell invasion. Herein, we performed this study to assess the involvement of MICAL1 in breast cancer cell proliferation and to explore the potential molecular mechanism. We noticed that depletion of MICAL1 markedly reduced cell proliferation in breast cancer cell line MCF‐7 and T47D. This effect of MICAL1 on proliferation was independent of wnt/β‐catenin and NF‐κB pathways. Interestingly, depletion of MICAL1 significantly inhibited ROS production, decreased p‐ERK expression and unfavourable for proliferative phenotype of breast cancer cells. Likewise, MICAL1 overexpression increased p‐ERK level as well as p‐ERK nucleus translocation. Moreover, we investigated the effect of MICAL1 on cell cycle‐related proteins. MICAL1 positively regulated CDK4 and cyclin D expression, but not CDK2, CDK6, cyclin A and cyclin E. In addition, more expression of CDK4 and cyclin D by MICAL1 overexpression was blocked by PI3K/Akt inhibitor LY294002. LY294002 treatment also attenuated the increase in the p‐ERK level in MICAL1‐overexpressed breast cancer cells. Together, our results suggest that MICAL1 exhibits its effect on proliferation via maintaining cyclin D expression through ROS‐sensitive PI3K/Akt/ERK signalling in breast cancer cells.     

NUSAP1 knockdown inhibits cell growth and metastasis of non-small-cell lung cancer through regulating BTG2/PI3K/Akt signaling

Non-small-cell lung cancer (NSCLC) is the most common malignancy along with high mortality rate worldwide. Recently, nucleolar and spindle-associated protein 1 (NUSAP1) has been reported to be involved in the malignant progression of several cancers. However, in NSCLC, the biological function of NUSAP1 and its molecular mechanism have not been reported. Here, our findings indicated that the NUSAP1 messenger RNA expression level was remarkably upregulated in NSCLC tissues compared with that of adjacent normal tissues. We also found that NUSAP1 gene expression was notably upregulated in NSCLC cell lines (A549, 95-D, H358, and H1299) compared with that of normal human bronchial epithelial cell line (16HBE). Subsequently, the biological function of NUSAP1 was investigated in A549 and H358 cells transfected with NUSAP1 small interfering RNA (siRNA), respectively. Results showed that NUSAP1 knockdown inhibited NSCLC cell proliferation, and promoted cell apoptosis. Furthermore, the number of cell migration and invasion was significantly suppressed by NUSAP1 knockdown. In addition, our results indicated that NUSAP1 knockdown increased the gene expression of B-cell translocation gene 2 (BTG2), but decreased the expression levels of phosphoinositide 3-kinase (PI3K) and phosphorylated serine/threonine kinase (p-AKT). BTG2 siRNA partly abrogates the effect of NUSAP1 knockdown on BTG2 gene expression. Fumonisin B1 (FB1), a AKT activator, reversed the effect of NUSAP1 knockdown on the biological function in NSCLC. Taken together, NUSAP1 knockdown promotes NSCLC cell apoptosis, and inhibits cell proliferation, cell migration, and invasion, which is associated with regulating BTG2/PI3K/Akt signal pathway. Our findings suggest that NUSAP1 is a promising molecular target for NSCLC treatment.     

CXCL5 regulation of proliferation and migration in human non-small cell lung cancer cells

Epithelial neutrophil-activating peptide-78 (CXCL5), a member of the subgroup of CXC-type chemokine family, is an inflammatory factor involved in the progression of lung cancer, but the underlying mechanism remains unclear. In this study, we investigated the effects of CXCL5 on proliferation and migration in non-small cell lung cancer (NSCLC) using tissue microarrays from NSCLC patients and H460 cells transfected with a CXCL5-interfered lentivirus vector or stimulated with recombinant CXCL5. We observed that the expression of CXCL5 was significantly higher in lung cancer cell lines, and high CXCL5 was associated with high chemokine (C-X-C motif) receptor 2 expression and was significantly associated with poor differentiation. The high expression of CXCL5 was associated with poor NSCLC prognosis and was an independent predictive factor. Furthermore, downregulation of CXCL5 in H460 cells significantly reduced proliferation and migration. Recombinant CXCL5 promoted H460 cell proliferation and movement by activating MAPK/ERK1/2 and PI3K/AKT signaling. Our study elucidates the important role of CXCL5 in the progression and prognosis of NSCLC. These findings suggested that CXCL5 might be a potential biomarker and novel therapeutic target for lung cancer.     

Atorvastatin overcomes gefitinib resistance in KRAS mutant human non-small cell lung carcinoma cells

The exact influence of statins on gefitinib resistance in human non-small cell lung cancer (NSCLC) cells with KRAS mutation alone or KRAS/PIK3CA and KRAS/PTEN comutations remains unclear. This work found that transfection of mutant KRAS plasmids significantly suppressed the gefitinib cytotoxicity in Calu3 cells (wild-type KRAS). Gefitinib disrupted the Kras/PI3K and Kras/Raf complexes in Calu3 cells, whereas not in Calu3 KRAS mutant cells. These trends were corresponding to the expression of pAKT and pERK in gefitinib treatment. Atorvastatin (1 μM) plus gefitinib treatment inhibited proliferation, promoted cell apoptosis, and reduced the AKT activity in KRAS mutant NSCLC cells compared with gefitinib alone. Atorvastatin (5 μM) further enhanced the gefitinib cytotoxicity through concomitant inhibition of AKT and ERK activity. Atorvastatin could interrupt Kras/PI3K and Kras/Raf complexes, leading to suppression of AKT and ERK activity. Similar results were also obtained in comutant KRAS/PTEN or KRAS/PIK3CA NSCLC cells. Furthermore, mevalonate administration reversed the effects of atorvastatin on the Kras/Raf and Kras/PI3K complexes, as well as AKT and ERK activity in both A549 and Calu1 cells. The in vivo results were similar to those obtained in vitro. Therefore, mutant KRAS-mediated gefitinib insensitivity is mainly derived from failure to disrupt the Kras/Raf and Kras/PI3K complexes in KRAS mutant NSCLC cells. Atorvastatin overcomes gefitinib resistance in KRAS mutant NSCLC cells irrespective of PIK3CA and PTEN statuses through inhibition of HMG-CoA reductase-dependent disruption of the Kras/Raf and Kras/PI3K complexes.     

miR-126 enhances the sensitivity of non-small cell lung cancer cells to anticancer agents by targeting vascular endothelial growth factor A

Increasing evidence suggests that hsa-miR-126 (miR-126) is down-regulated in non-small cell lung cancer (NSCLC) cell lines and the restoration of miR-126 impairs tumor cell proliferation, migration, invasion, and survival by targeting specific molecules. Here, we reported for the first time that miR-126 was involved in regulating the response of NSCLC cells to cancer chemotherapy. After transfected A549 cells with miR-126 mimic or inhibitor, we found that an elevated level of miR-126 was significantly associated with a decreased half maximal inhibitory concentration of adriamycin (ADM) and vincristine, an increased accumulation of ADM, down-regulation of vascular endothelial growth factor A (VEGFA) and multidrug resistance-associated protein 1 (MRP1), and inactivation of the Akt signaling pathway. Furthermore, enhanced expression of miR-126 suppressed the growth of A549 xenograft and inhibited the expression of VEGFA and MRP1. miR-126 could efficiently down-regulate VEGFA expression through the interaction with the VEGFA 3'-untranslated region, whereas restoration of VEGFA could partially attenuate the suppression of MRP1 by miR-126. However, LY294002, an inhibitor of the PI3K/Akt signaling pathway, diminished this effect, suggesting that enhanced expression of miR-126 increased the sensitivity of NSCLC cells to anticancer agents through negative regulation of a VEGF/PI3K/Akt/MRP1 signaling pathway.