Id-1 promotes migration and invasion of non-small cell lung cancer cells through activating NF-κB signaling pathway

Background

Numerous studies have shown that Id-1 (Inhibitor of differentiation 1) is upregulated in several cancers and associated with tumor malignant characters. However, the clinical significance and biological role of Id-1 in non-small cell lung cancer (NSCLC) remains unclear.

Methods

We used RT-PCR, Western blot and Immunohistochemistry to measure Id-1 expression in NSCLC tissues and matched adjacent noncancerous tissues. The expression pattern of Id-1 in NSCLC tissues was determined by scoring system of immunohistochemical analysis. The Kaplan-Meier method was used to calculate the survival curve, and log-rank test to determine statistical significance. The Id-1 gene was overexpressed or downreuglated with Lentiviral vectors in NSCLC cells. And, the migration ability of NSCLC cells was tested in a Transwell Boyden Chamber.

Results

We found that Id-1 is generally expressed higher in NSCLC tissues compared with matched adjacent noncancerous tissues. We also found that high Id-1 expression in tumor tissues is significantly correlated with tumor progression and poor survival in NSCLC patients. Furthermore, our experimental data revealed that knockdown of Id-1 significantly suppressed the proliferation, migration and invasion of NSCLC cells, whereas ectopic expression of Id-1 promoted the malignant phenotype of NSCLC cells. Mechanistic study showed that NF-κB signaling pathway contributed to the effects of Id-1 in NSCLC cells. Moreover, blocking the NF-κB pathway significantly inhibited the tumor-promoting actions of Id-1 in NSCLC cells.

Conclusions

We identified a tumorigenic role of Id-1 in NSCLC and provided a novel therapeutic target for NSCLC patients.

     

MARK2/4 promotes Warburg effect and cell growth in non-small cell lung carcinoma through the AMPKα1/mTOR/HIF-1α signaling pathway

MARKs kinase belongs to an AMPK-related family kinase plays a critical role in tumor progression, but its exact role and contribution of four different isoforms remain largely ambiguous. In this study, we used a clinical dataset compiled by The Cancer Genome Atlas (TCGA) and GEO revealed that MARK2 and MARK4 expressions were significantly upregulated in non-small cell lung cancer (NSCLC) compared with normal tissues. Furthermore, expressions of MARK2/4 were highly appeared in advanced stages and associated with the low survival rate of NSCLC patients. Functional assays demonstrated that MARK2/4 deletion or MARKs inhibition significantly suppressed aerobic glycolysis and cell growth in NSCLC cells. Mechanistically, MARK2/4 stimulates the mTOR/HIF-1α pathway and subsequently alleviates AMPK activity via physically associate with Raptor and AMPKα1, thereby facilitating aerobic glycolysis and cell growth in NSCLC cells. However, these effects were markedly reversed by MARKs inhibitor 39621, or MARK2/4 deletion, mTOR inhibitor rapamycin, or AMPK activator AICAR. Together, the data demonstrated that MARK2/4 exerts its oncogenic effects by facilitating metabolic reprogramming in NSCLC cells. Therefore, MARK2/4 might be a potential therapeutic target for lung cancer.     

Essential roles of the nitric oxide (no)/cGMP/protein kinase G type-Iα (PKG-Iα) signaling pathway and the atrial natriuretic peptide (ANP)/cGMP/PKG-Iα autocrine loop in promoting proliferation and cell survival of OP9 bone marrow stromal cells

Inappropriate signaling conditions within bone marrow stromal cells (BMSCs) can lead to loss of BMSC survival, contributing to the loss of a proper micro-environmental niche for hematopoietic stem cells (HSCs), ultimately causing bone marrow failure. In the present study, we investigated the novel role of endogenous atrial natriuretic peptide (ANP) and the nitric oxide (NO)/cGMP/protein kinase G type-Iα (PKG-Iα) signaling pathway in regulating BMSC survival and proliferation, using the OP9 BMSC cell line commonly used for facilitating the differentiation of HSCs. Using an ANP-receptor blocker, endogenously produced ANP was found to promote cell proliferation and prevent apoptosis. NO donor SNAP (S-nitroso-N-acetylpenicillamine) at low concentrations (10 and 50 μM), which would moderately stimulate PKG activity, protected these BMSCs against spontaneous apoptosis. YC-1, a soluble guanylyl cyclase (sGC) activator, decreased the levels of apoptosis, similar to the cytoprotective effects of low-level NO. ODQ (1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one), which blocks endogenous NO-induced activation of sGC and thus lowers endogenous cGMP/PKG activity, significantly elevated apoptotic levels by 2.5- and three-fold. Pre-incubation with 8-Bromo-cGMP or ANP, which bypass the ODQ block, almost completely prevented the ODQ-induced apoptosis. A highly-specific PKG inhibitor, DT-3, at 20, and 30 μM, caused 1.5- and two-fold increases in apoptosis, respectively. ODQ and DT-3 also decreased BMSCs proliferation and colony formation. Small Interfering RNA gene knockdown of PKG-Iα increased apoptosis and decreased proliferation in BMSCs. The data suggest that basal NO/cGMP/PKG-Iα activity and autocrine ANP/cGMP/PKG-Iα are necessary for preserving OP9 cell survival and promoting cell proliferation and migration.     

Effects of ceramide,ceramidase inhibition and expression of ceramide kinase on cytosolic phospholipase A2α; additional role of ceramide-1-phosphate in phosphorylation and Ca2+ signaling

Ceramide and the metabolites including ceramide-1-phosphate (C1P) and sphingosine are reported to regulate the release of arachidonic acid (AA) and/or phospholipase A₂ (PLA₂) activity in many cell types including lymphocytes. Recent studies established that C1P, a product of ceramide kinase, interacts directly with Ca²⁺ binding regions in the C2 domain of α type cytosolic PLA₂ (cPLA₂α), leading to translocation of the enzyme from the cytosol to the perinuclear region in cells. However, a precise mechanism for C1P-induced activation of cPLA₂α has not been well elucidated; such as the phosphorylation signal caused by the extracellular signal-regulated kinases (ERK1/2) pathway, a downstream of the protein kinase C activation with 4β-phorbol myristate acetate (PMA), is required or not. In the present study, we showed that the increase in intracellular ceramide levels (exogenously added cell permeable ceramides and an inhibition of ceramidase by (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and the increase in C1P formation by transfection with the vector for human ceramide kinase significantly enhanced the Ca²⁺ ionophore (A23187) -induced release of AA via cPLA₂α's activation in CHO cells. Ceramides did not show additional effects on the release from the cells treated with the inhibitor of ceramidase. Ceramides and C2-C1P neither had effect on the intracellular mobilization of Ca²⁺ nor the phosphorylation of cPLA₂α in cells. A23187/PMA-induced release of AA was enhanced by ceramides and C2-C1P and by expression of ceramide kinase. Our findings suggest that C1P is a stimulatory factor on cPLA₂α that is independent of the Ca²⁺ signal and the PKC-ERK-mediated phosphorylation signal.     

Huaier shows anti-cancer activities by inhibition of cell growth,migration and energy metabolism in lung cancer through PI3K/AKT/HIF-1α pathway

Huaier has been verified to have anti-cancer effects on many tumours. However, little information is available about the effects of Huaier on non-small cell lung cancer (NSCLC). We sought to probe the anti-cancer effects and related mechanisms of Huaier on lung cancer. A549 cells were pre-treated with 2, 4 and 8 mg/mL Huaier at different time points. Thereafter, cell viability was analysed by CCK-8 and the migration and invasion were detected by Scratch test and Transwell chamber migration assay. Moreover, ELISA, Western blot, shRNA transfection and RT-PCR were conducted to discover the related gene and protein expressions of energy metabolism and phosphatidylinositol 3-kinase (PI3K)/AKT/hypoxia-inducible factor 1α (HIF-1α) pathway. Furthermore, tumour xenografts were accomplished to inspect the anti-cancer effects of Huaier. Our consequences suggested that Huaier considerably repressed cell viability and migration in a dose-dependent way. In addition, Huaier statistically suppressed glycolysis, glucose transport and lactic acid (LA) accumulation. Besides, we detected that Huaier could inactivate the PI3K/AKT/HIF-1α pathway. The in vivo data confirmed that Huaier obviously decreased tumour volume and tumour growth, reduced the glycolysis, glucose transport and HIF-1α expression in the tumour-bearing tissues. Our results suggested Huaier revealed anti-tumour effects in both in vivo and in vitro possibly through PI3K/AKT/HIF-1α pathway.