MiR‐376a suppresses the proliferation and invasion of non‐small‐cell lung cancer by targeting c‐Myc

It has been reported that miR‐376a is involved in the formation and progression of several types of cancer. However, the expression and function of miR‐376a is still unknown in non‐small cell lung carcinomas (NSCLC). In this study, the expression of miR‐376a in NSCLC tissues and cell lines were examined by real‐time PCR, the effects of miR‐376a on cell proliferation, apoptosis and invasion were evaluated in vitro. Luciferase reporter assay was performed to identify the targets of miR‐376a. The results showed that miR‐376a was significantly downregulated in NSCLC tissues and cell lines. Restoration of miR‐376a in NSCLC cell line A549 significantly inhibited cell proliferation, increased cell apoptosis and suppressed cell invasion, compared with control‐transfected A549 cells. Luciferase reporter assay showed that c‐Myc, an oncogene that regulating cell survival, angiogenesis and metastasis, was a direct target of miR‐376a. Over‐expression of miR‐376a decreased the mRNA and protein levels of c‐Myc in A549 cells. In addition, upregulation of c‐Myc inhibited miR‐376a‐induced inhibition of cell proliferation and invasion in A549 cells. Therefore, our results indicate a tumor suppressor role of miR‐376a in NSCLC by targeting c‐Myc. miR‐376a may be a promising therapeutic target for NSCLC.     

Circular RNA hsa_circ_0085131 is involved in cisplatin‐resistance of non‐small‐cell lung cancer cells by regulating autophagy

Non‐small‐cell lung carcinoma (NSCLC) continues to top the list of cancer mortalities worldwide. The role of circular RNAs (circRNAs) in tumorigenesis has been increasingly appreciated, although it is relatively unexplored in NSCLC. Herein, we reported the role of hsa_circ_0085131 in NSCLC. In the present study, NSCLC tumor specimens exhibited a higher hsa_circ_0085131 level in comparison to para‐tumor samples. And the higher level of hsa_circ_0085131 was associated with recurrence and poorer survival of NSCLC. Moreover, hsa_circ_0085131 promoted cell proliferation and cisplatin (DDP)‐resistance. Furthermore, hsa_circ_0085131 regulated cell DDP‐resistance by modulating autophagy. Hsa_circ_0085131 acted as a competing endogenous RNA of miR‐654‐5p to release autophagy‐associated factor ATG7 expression, thereby promoting cell chemoresistance. In conclusion, hsa_circ_0085131 enhances DDP‐resistance of NSCLC cells through sequestering miR‐654‐5p to upregulate ATG7, leading to cell autophagy. Therefore, these findings advocate targeting the hsa_circ_0085131/miR‐654‐5p/ATG7 axis as a potential therapeutic option for patients with NSCLC who are resistant to DDP.     

Knockdown GTSE1 enhances radiosensitivity in non–small‐cell lung cancer through DNA damage repair pathway

Radiotherapy is an important strategy for NSCLC. However, although a variety of comprehensive radiotherapy‐based treatments have dominated the treatment of NSCLC, it cannot be avoided to overcome the growing radioresistance during radiotherapy. The purpose of this study was to elucidate the radiosensitizing effects of NSCLC via knockdown GTSE1 expression and its mechanism. Experiments were performed by using multiple NSCLC cells such as A549, H460 and H1299. Firstly, we found GTSE1 conferred to radioresistance via clonogenic assay and apoptosis assay. Then, we detected the level of DNA damage through comet assay and γH2AX foci, which we could clearly observe knockdown GTSE1 enhance DNA damage after IR. Furthermore, through using laser assay and detecting DNA damage repair early protein expression, we found radiation could induce GTSE1 recruited to DSB site and initiate DNA damage response. Our finding demonstrated that knockdown GTSE1 enhances radiosensitivity in NSCLC through DNA damage repair pathway. This novel observation may have therapeutic implications to improve therapeutic efficacy of radiation.     

Endogenous thrombopoietin promotes non‐small‐cell lung carcinoma cell proliferation and migration by regulating EGFR signalling

Thrombopoietin (TPO) is a haematopoietic cytokine mainly produced by the liver and kidneys, which stimulates the production and maturation of megakaryocytes. In the past decade, numerous studies have investigated the effects of TPO outside the haematopoietic system; however, the role of TPO in the progression of solid cancer, particularly lung cancer, has not been well studied. Exogenous TPO does not affect non‐small‐cell lung cancer (NSCLC) cells as these cells show no or extremely low TPO receptor expression; therefore, in this study, we focused on endogenous TPO produced by NSCLC cells. Immunohistochemical analysis of 150 paired NSCLC and adjacent normal tissues indicated that TPO was highly expressed in NSCLC tissues and correlated with clinicopathological parameters including differentiation, P‐TNM stage, lymph node metastasis and tumour size. Suppressing endogenous TPO by small interfering RNA inhibited the proliferation and migration of NSCLC cells. Moreover, TPO interacted with the EGFR protein and delayed ligand‐induced EGFR degradation, thus enhancing EGFR signalling. Notably, overexpressing TPO in EGF‐stimulated NSCLC cells facilitated cell proliferation and migration, whereas no obvious changes were observed without EGF stimulation. Our results suggest that endogenous TPO promotes tumorigenicity of NSCLC via regulating EGFR signalling and thus could be a therapeutic target for treating NSCLC.     

Dieckol inhibits non‐small–cell lung cancer cell proliferation and migration by regulating the PI3K/AKT signaling pathway

Non‐small–cell lung cancer (NSCLC) is one of the most prevalent type of lung cancers with an increased mortality rate in both developed and developing countries worldwide. Dieckol is one such polyphenolic drug extracted from brown algae which has proven antioxidant and anti‐inflammatory properties. In the present study, we evaluated the anticancer property of dieckol against NSCLC cell line A549. The LC₅₀ value of dieckol was found to be 25 µg/mL by performing 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and the antiapoptotic property of dieckol was analyzed by dual staining technique with acridine orange/propidium iodide (AO/PI) stains. It was further confirmed with flow cytometry analysis with Annexin FITC and JC‐1 staining and the anti‐invasive property was assessed by Transwell assay. The molecular mechanism of dieckol anticancer activity was confirmed by estimating the levels of caspases and by estimating the signaling proteins of Pi3K/AKT/mTOR signaling pathway using the immunoblotting technique. Our data suggest that dieckol is potent anticancer agent, it effectively inhibits the invasive and migratory property A549 cells and it also induces apoptosis via inhibiting Pi3K/AKT/mTOR signaling, activating the tumor suppressor protein E‐cadherin signifying that dieckol is potent natural anticancer drug to treat NSCLC.     

The deubiquitinase USP10 restores PTEN activity and inhibits non–small cell lung cancer cell proliferation

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein is a key player in tumorigenesis of non–small cell lung cancer (NSCLC) and was recently found to be inactivated by tripartite motif containing 25 (TRIM25)–mediated K63-linked polyubiquitination. However, the deubiquitinase (Dub) coordinate TRIM25 in PTEN ubiquitination is still elusive. In the present study, we found that this K63-linked polyubiquitination could be ablated by the ubiquitin-specific protease 10 (USP10) in a screen against a panel of Dubs. We found using coimmununoprecipitation/immunoblotting that USP10 interacted with PTEN and reduced the K63-linked polyubiquitination of PTEN mediated by TRIM25 in NSCLC cells. Moreover, USP10, but not its inactive C424A deubiquitinating mutant or other Dubs, abolished PTEN from K63-linked polyubiquitination mediated by TRIM25. In contrast to TRIM25, USP10 restored PTEN phosphatase activity and reduced the production of the secondary messenger phosphatidylinositol-3,4,5-trisphosphate, thereby inhibiting AKT/mammalian target of rapamycin progrowth signaling transduction in NSCLC cells. Moreover, USP10 was downregulated in NSCLC cell lines and primary tissues, whereas TRIM25 was upregulated. Consistent with its molecular activity, re-expression of USP10 suppressed NSCLC cell proliferation and migration, whereas knockout of USP10 promoted NSCLC cell proliferation and migration. In conclusion, the present study demonstrates that USP10 coordinates TRIM25 to modulate PTEN activity. Specifically, USP10 activates PTEN by preventing its K63-linked polyubiquitination mediated by TRIM25 and suppresses the AKT/mammalian target of rapamycin signaling pathway, thereby inhibiting NSCLC proliferation, indicating that it may be a potential drug target for cancer treatment.     

MiR‐34b‐3p represses cell proliferation,cell cycle progression and cell apoptosis in non‐small‐cell lung cancer (NSCLC) by targeting CDK4

Lung cancer is the most common incident cancer, with a high mortality worldwide, and non‐small‐cell lung cancer (NSCLC) accounts for approximately 85% of cases. Numerous studies have shown that the aberrant expression of microRNAs (miRNAs) is associated with the development and progression of cancers. However, the clinical significance and biological roles of most miRNAs in NSCLC remain elusive. In this study, we identified a novel miRNA, miR‐34b‐3p, that suppressed NSCLC cell growth and investigated the underlying mechanism. miR‐34b‐3p was down‐regulated in both NSCLC tumour tissues and lung cancer cell lines (H1299 and A549). The overexpression of miR‐34b‐3p suppressed lung cancer cell (H1299 and A549) growth, including proliferation inhibition, cell cycle arrest and increased apoptosis. Furthermore, luciferase reporter assays confirmed that miR‐34b‐3p could bind to the cyclin‐dependent kinase 4 (CDK4) mRNA 3′‐untranslated region (3′‐UTR) to suppress the expression of CDK4 in NSCLC cells. H1299 and A549 cell proliferation inhibition is mediated by cell cycle arrest and apoptosis with CDK4 interference. Moreover, CDK4 overexpression effectively reversed miR‐34‐3p‐repressed NSCLC cell growth. In conclusion, our findings reveal that miR‐34b‐3p might function as a tumour suppressor in NSCLC by targeting CDK4 and that miR‐34b‐3p may, therefore, serve as a biomarker for the diagnosis and treatment of NSCLC.     

Aspirin boosts the synergistic effect of EGFR/p53 inhibitors on lung cancer cells by regulating AKT/mTOR and p53 pathways

The regimen of afatinib and vinorelbine has been used to treat breast or lung cancer cells with some limitations. Aspirin alone or in combination with other agents has shown unique efficacy in the treatment of cancer. We designed a preclinical study to investigate whether the triple therapy of aspirin, afatinib, and vinorelbine could synergistically inhibit the growth of p53 wild-type nonsmall cell lung cancer (NSCLC) cells. Three NSCLC cells A549, H460, and H1975 were selected to study the effect of triple therapy on cell proliferation and apoptosis. Compared to single agents, triple therapy synergistically inhibited the proliferation of lung cancer cells with combination index <1. Meanwhile, the therapeutic index of triple therapy was superior to that of single agents, indicating a balance between efficacy and safety in the combination of three agents. Mechanistic studies showed that triple therapy significantly induced apoptosis by decreasing mitochondrial membrane potential, increasing reactive oxygen species, and regulating mitochondria-related proteins. Moreover, epidermal growth factor receptor (EGFR) downstream signaling proteins including JNK, AKT, and mTOR were dramatically suppressed and p53 was substantially increased after NSCLC cells were exposed to the triple therapy. We provided evidence that the triple therapy of aspirin, afatinib and vinorelbine synergistically inhibited lung cancer cell growth through inactivation of the EGFR/AKT/mTOR pathway and accumulation of p53, providing a new treatment strategy for patients with p53 wild-type NSCLC.     

Neuropilin 1 expression correlates with the Radio‐resistance of human non‐small‐cell lung cancer cells

The purpose of this study was to determine the correlation between over‐expression of the neuropilin 1 (NRP1) gene and growth, survival, and radio‐sensitivity of non‐small cell lung carcinoma (NSCLC) cells. 3‐[4,5‐dimethylthylthiazol‐2‐yl]‐2,5 diphenyltetrazolium broide (MTT) and colony assays were then performed to determine the effect of NRP1 inhibition on the in vitro growth of NSCLC cells. The Annexin V‐Fluorescein Isothiocyanate (FITC) apoptosis detection assay was performed to analyse the effect of NRP1 enhancement on apoptosis of NSCLC cells. Transwell invasion and migration assays were employed to examine the metastatic ability of A549 cells post X‐ray irradiation. In addition, Western blot assays were carried out to detect the protein level of VEGFR2, PI3K and NF‐κB. Finally, to examine the effect of shNRP1 on proliferation and radio‐sensitivity in vivo, a subcutaneous tumour formation assay in nude mice was performed. Microvessel density in tumour tissues was assessed by immunohistochemistry. The stable transfected cell line (shNRP1‐A549) showed a significant reduction in colony‐forming ability and proliferation not only in vitro, but also in vivo. Moreover, shRNA‐mediated NRP1 inhibition also significantly enhanced the radio‐sensitivity of NSCLC cells both in vitro and in vivo. The over‐expression of NRP1 was correlated with growth, survival and radio‐resistance of NSCLC cells via the VEGF‐PI3K‐ NF‐κB pathway, and NRP1 may be a molecular therapeutic target for gene therapy or radio‐sensitization of NSCLC.     

PTEN‐ and p53‐mediated apoptosis and cell cycle arrest by FTY720 in gastric cancer cells and nude mice

FTY720, a new immunosuppressant, derived from ISP‐1, has been studied for its putative anti‐cancer properties in the recent years. In this study, we have reported that FTY720 greatly inhibited gastric cancer cell proliferation for the first time, and found this effect was associated with G1 phase cell cycle arrest and apoptosis. Results from our Western blotting and Real‐time PCR showed that FTY720 induced obvious PTEN expression in a p53‐independent way, consistent with a substantial decrease in p‐Akt and MDM2. FTY720 dramatically increased the expression of Cip1/p21, p27, and BH3‐only proteins through the accumulation of p53 by PTEN‐mediated inhibition of the PI3K/Akt/MDM2 signaling. Suppression of PTEN expression with siRNA significantly reduced the p53 and p21 levels and activated Akt, resulting in decreased apoptosis and increased cell survival. Furthermore, we have observed an additive effect of FTY720 in killing gastric cancer cells when in combination with Cisplatin, partly through PTEN‐mediated Akt/MDM2 inhibition. In vivo study has also shown that tumor growth was significantly suppressed after FTY720 treatment. In conclusion, our results suggest that FTY720 induces a significant increase of PTEN, which inhibits p‐Akt and MDM2, and then increases the level of p53, thereby inducing G1 phase arrest and apoptosis. We have characterized a novel immunosuppressant, for the first time, which shows potential anti‐tumor effects on gastric cancer by PTEN activation through p53‐independent mechanism, especially in combination with Cisplatin. This PTEN target‐based therapy is worth further investigation and warrants clinical evaluation. J. Cell. Biochem. 111: 218–228, 2010. © 2010 Wiley‐Liss, Inc.     

In non‐small cell lung cancer mitogenic signaling leaves Sprouty1 protein levels unaffected

Sprouty1 protein belongs to a family of receptor tyrosine kinase‐mediated signaling inhibitors, whose members are usually regulated by growth factors to form a negative feedback loop. Correspondingly fluctuations of Sprouty1 mRNA in response to single growth factors have been observed. In this report, we investigate Sprouty1 protein levels and show that in non‐small cell lung carcinoma‐derived cells, the expression levels are unaffected by the serum content in the cellular environment. Although cells harboring K‐Ras mutations express insignificant higher Sprouty1 levels, ectopic expression of constitutive active Ras in normal human lung fibroblasts fails to augment Sprouty1 protein content. Furthermore, serum starvation for three days has no influence on Sprouty1 expression and addition of serum or of singular growth factors leaves Sprouty protein levels unchanged. Cell cycle analysis reveals that Sprouty1 levels remain constant throughout the whole cell cycle. These data demonstrate that Sprouty1 expression is not connected with mitogenic signaling and cell proliferation. Copyright © 2013 John Wiley & Sons, Ltd.