Estrogen receptor β signaling regulates the progression of Chinese non-small cell lung cancer

Prospective studies have found that the risk of non-small cell lung cancer (NSCLC) has close relationship with estrogen. The effects of estrogens are mediated via two estrogen receptor (ER) isoforms, that is, ER alpha (ERα) and ER beta (ERβ). ERα in NSCLC has been evaluated mostly by immunohistochemistry. However, our previous study showed that ERβ was also highly expressed in Chinese NSCLC. But the roles of ERβ in Chinese NSCLC have not been clarified as yet. So in the present study, two Chinese lung adenocarcinoma cell lines, SPC-A1 and LTEP-a2, were used and the role of ERβ in lung tumorigenesis was focused to be investigated by in vitro and in vivo experiments. The results showed that over-expressed ERβ can promote the development of NSCLC, while siRNAs targeting ERβ gene can inhibit growth of NSCLC cells and induce apoptosis of these cells via mitochondrial depolarization and caspase-3 activation. These results indicated that ERβ plays an important role in development of Chinese NSCLC. This suggests that ERβ deactivation or down-regulation may possess potential therapeutic utility for the treatment of lung cancer.     

Activation of PKR/eIF2α signaling cascade is associated with dihydrotestosterone-induced cell cycle arrest and apoptosis in human liver cells

Androgen receptor (AR) signaling plays an important role in the development and progression of several liver diseases, including hepatocellular carcinoma (HCC) and non-alcoholic fatty liver disease (NAFLD). Dihydrotestosterone (DHT) is the active metabolite of the major circulating androgen, testosterone. In this study, we investigated the effect of DHT on human liver cells. We found that DHT not only induces cell cycle arrest but also initiates apoptosis in androgen-sensitive liver cells, such as SMMC-7721 and L02. Importantly, DHT/AR induces the activation of RNA-dependent protein kinase (PKR)/eukaryotic initiation factor-2 alpha (eIF2α) cascades in androgen-sensitive liver cells. PKR/eIF2α activation-induced growth arrest and DNA damage-inducible gene 153 (GADD153) and heat shock protein 27 (Hsp27) expression contribute to cell cycle arrest in response to DHT. It is notable that DHT administration results in androgen-sensitive liver cells apoptosis, at least in part, through PKR/eIF2α/GADD153 cascades. These results suggest that the androgen/AR pathway plays a pivotal role in liver cell growth and apoptosis regulating, whose deregulation might be involved in the pathogenesis of liver diseases.     

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.     

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.

     

Curcumin suppresses proliferation and invasion in non-small cell lung cancer by modulation of MTA1-mediated Wnt/β-catenin pathway

Curcumin, a naturally occurring phenolic compound, has a diversity of antitumor activities. It has been previously demonstrated that curcumin can inhibit the invasion and metastasis of tumors through activation of the tumor suppressor DnaJ-like heat shock protein 40 (HLJ1). However, the specific roles and mechanisms of curcumin in regulating the malignant behaviors of non-small cell lung cancer (NSCLC) cells still remain unclear. In this study, we found that curcumin could inhibit the proliferation and invasion of NSCLC cells and induce G0/G1 phase arrest. Metastasis-associated protein 1 (MTA1) overexpression has been detected in a wide variety of aggressive tumors and plays an important role on cell invasion and metastasis. Our results showed that curcumin could effectively inhibit the MTA1 expression of NSCLC cells. Further research on the subsequent mechanism showed that curcumin inhibited the proliferation and invasion of NSCLC cells through MTA1-mediated inactivation of Wnt/β-catenin pathway. Wnt/β-catenin signaling was reported to play a critical cooperative role on promoting lung tumorigenesis. Thus, these investigations provided novel insights into the mechanisms of curcumin on inhibition of NSCLC cell growth and invasion and showed potential therapeutic strategies for NSCLC.     

The clinical and prognostic significance of YWHAZ in non-small–cell lung cancer patients: Immunohistochemical analysis

YWHAZ has been suggested to as an oncogene in various human malignancies, including non-small–cell lung cancer (NSCLC). Our study presents more evidence to confirm the clinical significance and biological function of YWHAZ in NSCLC. In our results, YWHAZ was upregulated in lung squamous cell carcinoma tissues and lung adenocarcinoma tissues through analyzing The Cancer Genome Atlas (TCGA) database, and confirmed high levels of YWHAZ messenger RNA and protein in lung squamous cell carcinoma tissues and lung adenocarcinoma tissues through quantitative real-time polymerase chain reaction and immunohistochemistry. Moreover, YWHAZ overexpression was correlated with advanced clinical stage, more lymph node metastasis and present distant metastasis in NSCLC patients. Survival analysis indicated that high level of YWHAZ protein expression was associated with short overall survival time in NSCLC patients, and YWHAZ expression was independent prognostic factors for overall survival in NSCLC patients. Moreover, Silencing of YWHAZ expression represses NSCLC cell migration and invasion. In conclusion, YWHAZ is a credible prognostic biomarker, and may be a therapeutic target in NSCLC.     

Transcriptional regulation of livin by β-catenin/TCF signaling in human lung cancer cell lines

Heregulin can regulate the survival of cardiomyocytes, epithelial cells, neuron, glial cells, and other cell types through binding with the ErbB receptors. The aim of this study is to investigate the effects of heregulin (HRG) on the apoptosis of Bone marrow Mesenchymal stem cells (MSCs). We used the MSCs from adult Sprague–Dawley rats and the model of serum deprivation (SD) and hypoxia-induced apoptosis. The apoptosis was detected by TUNEL method. The apoptosis of MSCs significantly increased 12 h or 18 h after SD and hypoxia, but treatment with HRG significantly decreased the apoptosis induced by SD and hypoxia. Tyrphostin AG1478 (ErbB3/4 inhibitor) or Tyrphostin AG825 (ErbB2 inhibitor) could block this effects of HRG. Akt and ERK were activated by HRG under SD and hypoxia conditions, but HRG had no effects on the activation of JNK and p38. HRG also increased the ratio of Bcl-2/Bax and decreased the activation of caspase3 induced by SD and hypoxia. These results suggested HRG could decrease the apoptosis of MSCs induced by SD and hypoxia through the activation of Akt and ERK, the increase of Bcl-2/Bax ratio and the inhibition of caspase3 activation.     

microRNA-383 regulates cell viability and apoptosis by mediating Wnt/β-catenin signaling pathway in non–small cell lung cancer

The aim of this study was to investigate the roles of microRNA-383 (miRNA-383) in progression of non–small cell lung cancer (NSCLC) and the potential mechanism. The expressions of miR-383 and Wnt1 protein were detected in lung cancer tissues and cells by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. After the transfection of miR-383 mimics, si-Wnt1 or miR-383+Wnt1, the viability and apoptosis of NSCLC cells were detected by cell counting kit-8 and terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling, respectively. The interaction between miR-383 and Wnt1 was investigated by luciferase activity and Western blot analysis. Cells stably transfected with miR-383 mimics were inoculated into the right axillary of nude mice by subcutaneous injection. The tumor volume and weight were measured, and the expressions of miR-383, Wnt1, β-catenin, and cyclin D1 were detected by qRT-PCR and Western blot analysis. The expression of miR-383 was significantly decreased, and the level of Wnt1 was significantly increased (P < 0.05) in lung cancer tissues and cells. Upregulation of miR-383 or inhibition of Wnt1 expression inhibited the cell viability and induce apoptosis in NSCLC cells. Moreover, Wnt1 was the target gene of miR-383, and its overexpression weakened the regulatory effect of miR-383 on cell viability and apoptosis in NSCLC cells. Besides, the addition of miR-383 decreased the tumor volume and size and inhibited the expressions of Wnt1, β-catenin, and cyclin D1 at the protein level in nude mice. Collectively, miR-383 induced apoptosis and inhibited cell viability as well as tumorigenic capacity in nude mice via regulating the Wnt/β-catenin signaling pathway.     

The RNA-binding protein HuR stabilizes cytosolic phospholipase A2α mRNA under interleukin-1β treatment in non-small cell lung cancer A549 Cells

The activation of cytosolic phospholipase A(2)α (cPLA(2)α) plays an important role in initiating the inflammatory response. The regulation of cPLA(2)α mRNA turnover has been proposed to control cPLA(2)α gene expression under cytokine and growth factor stimulation. However, the detailed mechanism is still unknown. In this report, we have demonstrated that the cPLA(2)α mRNA stability was increased under IL-1β treatment in A549 cells. By using EMSAs, HuR was identified as binding with the cPLA(2)α mRNA 3'-UTR, and the binding region was located at nucleotides 2716-2807, a fragment containing AUUUA flanked by U-rich sequences. IL-1β treatment enhanced the association of cPLA(2)α mRNA with cytosolic HuR. The reduction of HuR expression by RNA interference technology inhibited IL-1β-induced cPLA(2)α mRNA and protein expression. Furthermore, blocking the p38 MAPK signaling pathway with SB203580 abolished the effect of IL-1β-induced cPLA(2)α gene expression. Phosphorylation at residue Thr-118 of HuR is crucial in regulating the interaction between HuR and its target mRNAs. Mutation of HuR Thr-118 reduced the association between HuR and cPLA(2)α mRNA under IL-1β treatment. This inhibitory effect was also observed in binding with COX-2 mRNA. This result indicated that p38 MAPK-mediated Thr-118 phosphorylation may play a key role in regulating the interaction of HuR with its target mRNAs in inflammation.     

lncRNA SNHG11 promotes lung cancer cell proliferation and migration via activation of Wnt/β-catenin signaling pathway

Lung cancer ranks topmost among the most frequently diagnosed cancers. Despite increasing research, there are still unresolved mysteries in the molecular mechanism of lung cancer. Long noncoding RNA small nucleolar RNA host gene 11 (SNHG11) was found to be upregulated in lung cancer and facilitated lung cancer cell proliferation, migration, invasion, and epithelial–mesenchymal transition progression while suppressed cell apoptosis. Moreover, the high expression of SNHG11 was correlated with poor prognosis of lung cancer patients, TNM stage, and tumor size. Further assays demonstrated that SNHG11 functioned in lung cancer cells via Wnt/β-catenin signaling pathway. Subsequently, Wnt/β-catenin pathway was found to be activated through SNHG11/miR-4436a/CTNNB1 ceRNA axis. As inhibiting miR-4436 could only partly rescue the suppression of cell function induced by silencing SNHG11, it was suspected that β-catenin might enter cell nucleus through other pathways. Mechanism investigation proved that SNHG11 would directly bind with β-catenin to activate classic Wnt pathway. Subsequently, in vivo tumorigenesis was also demonstrated to be enhanced by SNHG11. Hence, SNHG11 was found to promote lung cancer progression by activating Wnt/β-catenin pathway in two different patterns, implying that SNHG11 might contribute to lung cancer treatment by acting as a therapeutic target.     

Mast cell exosomes promote lung adenocarcinoma cell proliferation – role of KIT-stem cell factor signaling

Background

Human cells release nano-sized vesicles called exosomes, containing mRNA, miRNA and specific proteins. Exosomes from one cell can be taken up by another cell, which is a recently discovered cell-to-cell communication mechanism. Also, exosomes can be taken up by different types of cancer cells, but the potential functional effects of mast cell exosomes on tumor cells remain unknown.

Methods and results

Exosomes were isolated from the human mast cell line, HMC-1, and uptake of PKH67-labelled exosomes by the lung epithelial cell line, A549, was examined using flow cytometry and fluorescence microscopy. The RNA cargo of the exosomes was analyzed with a Bioanalyzer and absence or presence of the c-KIT mRNA was determined by RT-PCR. The cell proliferation was determined in a BrdU incorporation assay, and proteins in the KIT-SCF signaling pathway were detected by Western blot. Our result demonstrates that exosomes from mast cells can be taken up by lung cancer cells. Furthermore, HMC-1 exosomes contain and transfer KIT protein, but not the c-KIT mRNA to A549 cells and subsequently activate KIT-SCF signal transduction, which increase cyclin D1 expression and accelerate the proliferation in the human lung adenocarcinoma cells.

Conclusions

Our results indicate that exosomes can transfer KIT as a protein to tumor cells, which can affect recipient cell signaling events through receptor-ligand interactions.

     

microRNA-448 inhibits the progression of non-small–cell lung cancer through regulating IRS2

Recently, microRNA-448 (miR-448) has been reported to be a tumor-associated miRNA in many human cancers. In this study, we investigated the function of miR-448 in non-small–cell lung cancer (NSCLC) progression and confirmed the relationship between miR-448 and insulin receptor substrates 2 (IRS2). First, downregulation of miR-448 and upregulation of IRS2 were detected in NSCLC using the quantitative real-time polymerase chain reaction (qRT-PCR) assay. Furthermore, the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay showed that miR-448 inhibited cell viability in NSCLC. Transwell and Western blot assays indicated that the upregulation of miR-448 inhibited cell metastasis and epithelial-to-mesenchymal transition (EMT) in NSCLC. And it was found that overexpression of miR-448 reduced the adhesion of A549 cells to HUVEC cells using the adhesion assay. Furthermore, the dual luciferase assay indicated that miR-448 directly targeted IRS2 in NSCLC. In addition, it was found that IRS2 silencing had an inhibitory effect on the progression of NSCLC, and the upregulation of IRS2 partially impaired the inhibitory effect of miR-448 in NSCLC. Briefly, overexpression of miR-448 inhibited cell proliferation, metastasis, and EMT by suppressing IRS2 expression in NSCLC.     

Mitochondrial reactive oxygen species mediates nicotine-induced hypoxia-inducible factor-1α expression in human non-small cell lung cancer cells

Cigarette smoking is not only a documented risk for lung carcinogenesis but also promotes lung cancer development. Nicotine, a major component of cigarette smoke but not a carcinogen by itself, has been found to induce proliferation, invasion and metastasis of non-small cell lung cancer (NSCLC). Here we reported that proinvasive effect of nicotine is analogous to that of hypoxia and involves stabilization and activation of hypoxia-inducible factor (HIF)-1α, a key factor in determining the presence of HIF-1 and expression of its downstream metastasis-associated genes. Furthermore, nicotine-induced upregulation of HIF-1α was dependent on mitochondria-derived reactive oxygen species (ROS). Ecotopic expression of mitochondrial targeted catalase effectively prevented nicotine-induced accumulation of HIF-1α protein. In addition, we demonstrated that the effect of nicotine in upregulation of HIF-1α was mediated by Dihydro-β-erythroidine (DhβE)-sensitive nicotine acetylcholine receptors (nAChRs) and required synergistic cooperation of Akt and mitogen-activated protein kinase (MAPK) pathways. These results suggest that exposure to nicotine could mimic effects of hypoxia to stimulate HIF-1α accumulation and activity that might underlie the high metastatic potential of lung cancer.     

α7-Nicotinic acetylcholine receptor antagonist QND7 suppresses non-small cell lung cancer cell proliferation and migration via inhibition of Akt/mTOR signaling

Lung cancer, one of the most commonly found carcinoma type, has the highest mortality rate in cancer patients worldwide. Therapeutic interventions targeting to lung cancer become remaining the world significant challenge. Recently, the α7-nicotinic acetylcholine receptor (α7-nAChR) was reported to play an important role in the mechanism underlying lung cancer progression, being intriguing drug target for lung cancer therapy. Hence, the top four α7-nAChR antagonists (QND7, PPRD10, PPRD11 and PPRD12) among our previously developed ligands were proceeded to the in vitro anti-cancer evaluations in non-small cell lung cancer (NSCLC) cell lines (H460 and A549). In this study, we found that QND7 exhibited the highest cytotoxic effect and induced cell apoptosis in both cell lines at a level comparable to cisplatin, whereas the PPRD compounds showed much lower cytotoxicity. Low doses of QND7 and PPRD11 were able to suppress H460 and A549 cell proliferation, whereas PPRD10 and PPRD12 were considered ineffective. In an in vitro wound healing assay, QND7-treatment showed the greatest suppression of H460 and A549 cell migration. The variations in the anti-cancer activities of PPRD compounds might be, at least in part of, their non-selective antagonisms to serotonin receptor (5-HT3) and α4β2-nAChR. Further investigation revealed that QND7 was able to minimize protein kinase B/mammalian target of rapamycin (Akt/mTOR) activity, in correlating to its anti-cancer effects. These findings warrant QND7 for further preclinical evaluation and demonstrate the potential of α7-nAChR as cancer drug target.     

The non-steroidal anti-inflammatory drug indomethacin activates the eIF2α kinase PKR, causing a translational block in human colorectal cancer cells

The NSAID (non-steroidal anti-inflammatory drug) indomethacin, a cyclo-oxygenase-1 and -2 inhibitor with anti-inflammatory and analgesic properties, is known to possess anticancer activity against CRC (colorectal cancer) and other malignancies in humans; however, the mechanism underlying the anticancer action remains elusive. In the present study we show that indomethacin selectively activates the dsRNA (double-stranded RNA)-dependent protein kinase PKR in a cyclo-oxygenase-independent manner, causing rapid phosphorylation of eIF2α (the α-subunit of eukaryotic translation initiation factor 2) and inhibiting protein synthesis in colorectal carcinoma and other types of cancer cells. The PKR-mediated translational block was followed by inhibition of CRC cell proliferation and apoptosis induction. Indomethacin did not affect the activity of the eIF2α kinases PERK (PKR-like endoplasmic reticulum-resident kinase), GCN2 (general control non-derepressible-2) and HRI (haem-regulated inhibitor kinase), and induced eIF2α phosphorylation in PERK-knockout and GCN2-knockout cells, but not in PKR-knockout cells or in human PKR-silenced CRC cells, identifying PKR as a selective target for indomethacin-induced translational inhibition. The fact that indomethacin induced PKR activity in vitro, an effect reversed by the PKR inhibitor 2-aminopurine, suggests a direct effect of the drug in kinase activation. The results of the present study identify PKR as a novel target of indomethacin, suggesting new scenarios on the molecular mechanisms underlying the pleiotropic activity of this traditional NSAID.     

The RhoGDI-α/JNK signaling pathway plays a significant role in mycophenolic acid-induced apoptosis in an insulin-secreting cell line

Mycophenolic acid (MPA)-induced β-cell toxicity is an important factor for islet graft function. The signal transduction mechanisms underlying this process have not been fully explored. Using a proteomics approach, we examined protein expression patterns in MPA-treated RIN-5 cells and found that RhoGDI-α expression is altered by MPA-treatment. We examined the relationship between RhoGDI-α expression and activated JNK during MPA-induced apoptosis. Cells were treated with N-acetyl-cysteine (NAC), caspase inhibitor, JNK inhibitor, guanosine or GTP for 1 h before being treated with MPA. To investigate the regulatory effects of RhoGDI-α on JNK activity, we examined cells showing either elevated or reduced expression of RhoGDI-α as a result of transfection with cDNA or siRNA constructs, respectively. MPA significantly increased cell death, caspase-3 expression and JNK activation, but it decreased the expression of a protein spot 25 observed by two-dimensional electrophoresis. This protein 25 was identified as RhoGDI-α by mass spectrometry. MPA-induced cell death and down-regulation of RhoGDI-α were prevented by guanosine, GTP or a JNK inhibitor. However, MPA-induced cell death was partially restored by treatment with a caspase inhibitor, but not by NAC treatment. RhoGDI-α expression was not affected by treatment with NAC or caspase inhibitor. Over-expression of RhoGDI-α increased cell viability and decreased activated JNK expression following exposure to MPA, whereas knockdown of RhoGDI-α enhanced MPA-induced cell death and increased the activation of JNK. In conclusion, MPA induces significant apoptosis in insulin-secreting cells via down-regulation of RhoGDI-α linked with increased JNK expression. This RhoGDI-α/JNK pathway might be the focus of therapeutic target for the prevention of MPA-induced islet apoptosis.