Restoration of miR-101 suppresses lung tumorigenesis through inhibition of DNMT3a-dependent DNA methylation

The deregulation of miR-101 and DNMT3a has been implicated in the pathogenesis of multiple tumor types, but whether and how miR-101 silencing and DNMT3a overexpression contribute to lung tumorigenesis remain elusive. Here we show that miR-101 downregulation associates with DNMT3a overexpression in lung cancer cell lines and patient tissues. Ectopic miR-101 expression remarkably abrogated the DNMT3a 3′-UTR luciferase activity corresponding to the miR-101 binding site and caused an attenuated expression of endogenous DNMT3a, which led to a reduction of global DNA methylation and the re-expression of tumor suppressor CDH1 via its promoter DNA hypomethylation. Functionally, restoration of miR-101 expression suppressed lung cancer cell clonability and migration, which recapitulated the DNMT3a knockdown effects. Interestingly, miR-101 synergized with decitabine to downregulate DNMT3a and to reduce DNA methylation. Importantly, ectopic miR-101 expression was sufficient to trigger in vivo lung tumor regression and the blockage of metastasis. Consistent with these phenotypes, examination of xenograft tumors disclosed an increase of miR-101, a decrease of DNMT3a and the subsequent DNA demethylation. These findings support that the loss or suppression of miR-101 function accelerates lung tumorigenesis through DNMT3a-dependent DNA methylation, and suggest that miR-101-DNMT3a axis may have therapeutic value in treating refractory lung cancer.Owing to a high propensity for recurrence and a high rate of metastasis at the advanced stages,^{1, 2, 3} lung cancer remains the leading cause of cancer-related mortality. DNA methylation is a major epigenetic rule controlling chromosomal stability and gene expression.^{4, 5} It is under control of DNA methyltransferases (DNMTs), whose overexpression in lung cancer cells predicts worse outcomes.^{6, 7} It is postulated that DNMT overexpression induces DNA hypermethylation and silencing of tumor suppressor genes (TSGs), leading to an aggressive lung cancer. Indeed, enforced expression of DNMT1 or DNMT3a increases DNA methylation, while the abolition of DNMT expression by genetic depletion, microRNAs (miRs) or small molecules reduces genome-wide and gene-specific DNA methylation and restores TSG expression.^{8, 9, 10, 11, 12, 13} As TSGs are the master controllers for cell multiplicity and their silencing predicts poor prognosis,^{14, 15} TSG re-expression via promoter DNA hypomethylation inhibits cell proliferation and induces cell differentiation.^{13, 16} Thus, DNMT gene abundance could serve as a target for anticancer therapy, but how DNMT upregulation occurs in lung cancer is incompletely understood.MiRs are small non-coding RNAs that crucially regulate target gene expression. Up to 30% of all protein-coding genes are predicted to be targeted by miRs,^{17, 18} supporting the key roles of miRs in controlling cell fate.^{19, 20, 21, 22} Research is showing that certain miRs are frequently dysregulated in cancers, including lung cancer.^{7, 23, 24} As miR targets can promote or inhibit cancer cell expansion, miRs have huge potential for acting as bona fide oncogenes (i.e., miR-21) or TSGs (i.e., miR-29b).^{7, 25} We and others demonstrated that the levels of DNMT1 or DNMT3a or DNMT3b are regulated by miR-29b, miR-148, miR-152 or miR-30c,^{7, 13, 26, 27} and overexpression of these miRs results in DNA hypomethylation and TSG reactivation with the concurrent blockage of cancer cell proliferation.^{7, 13} These findings underscore the importance of miRs as epigenetic modulators and highlight their therapeutic applications.MiR-101 is frequently silenced in human cancers^{28, 29, 30, 31} and, importantly, exhibits antitumorigenic properties when overexpressed. Mechanistically, miR-101 inactivation by genomic loss causes the overexpression of EZH2, a histone methyltransferase, via 3′-UTR targeting, which is followed by histone hypermethylation and aggressive tumorigenesis.^{29, 30, 32} However, whether and how miR-101 silencing contributes to DNA hypermethylation patterning in lung cancer is unclear. In this study, we explore the role of miR-101 in regulating DNMT3a expression and the impacts of miR-101-DNMT3a nexus on lung cancer pathogenesis. We showed that the expression of miR-101 and DNMT3a was negatively correlated in lung cancer. We presented evidence that ectopic miR-101 expression decreased DNMT3a levels, reduced global DNA methylation and upregulated CDH1 via its promoter DNA demethylation. The biological significance of miR-101-mediated DNA hypomethylation and CDH1 re-expression was evident by its inhibition of lung tumor cell growth in vitro and in vivo. Thus, our findings mechanistically and functionally link miR-101 silencing to DNA hypermethylation in lung cancer cells.     

Mir-660 is downregulated in lung cancer patients and its replacement inhibits lung tumorigenesis by targeting MDM2-p53 interaction

Lung cancer represents the leading cause of cancer-related death in developed countries. Despite the advances in diagnostic and therapeutic techniques, the 5-year survival rate remains low. The research for novel therapies directed to biological targets has modified the therapeutic approach, but the frequent engagement of resistance mechanisms and the substantial costs, limit the ability to reduce lung cancer mortality. MicroRNAs (miRNAs) are small noncoding RNAs with known regulatory functions in cancer initiation and progression. In this study we found that mir-660 expression is downregulated in lung tumors compared with adjacent normal tissues and in plasma samples of lung cancer patients with poor prognosis, suggesting a potential functional role of this miRNA in lung tumorigenesis. Transient and stable overexpression of mir-660 using miRNA mimics reduced migration, invasion, and proliferation properties and increased apoptosis in p53 wild-type lung cancer cells (NCI-H460, LT73, and A549). Furthermore, stable overexpression using lentiviral vectors in NCI-H460 and A549 cells inhibited tumor xenograft growth in immunodeficient mice (95 and 50% reduction compared with control, respectively), whereas the effects of mir-660 overexpression were absent in H1299, a lung cancer cell line lacking p53 locus, both in in vitro and in vivo assays. We identified and validated mouse double minute 2 (MDM2) gene, a key regulator of the expression and function of p53, as a new direct target of mir-660. In addition, mir-660 expression reduced both mRNA and protein expression of MDM2 in all cell lines and stabilized p53 protein levels resulting in an upregulation of p21^WAF1/CIP1 in p53 wild-type cells. Our finding supports that mir-660 acts as a tumor suppressor miRNA and we suggest the replacement of mir-660 as a new therapeutic approach for p53 wild-type lung cancer treatment.Lung cancer is the leading cause of cancer death worldwide, resulting in >1.4 million deaths/year.¹ Lung tumors are often discovered as locally advanced or metastatic disease, and despite improvements in molecular diagnosis and targeted therapies, the overall 5-year survival rate remains in the 10–20% range. Indeed, nonsmall cell lung cancer (NSCLC) is poorly chemosensitive to most of the available agents with response rates ranging from 10 to 25%.² The discovery of recurrent mutations in the epidermal growth factor receptor (EGFR) kinase,³ as well as gene fusion products involving the anaplastic lymphoma kinase (ALK),⁴ has led to a marked change in the treatment of patients with lung adenocarcinoma, the most common type of lung cancer.^{5, 6} To date, patients with mutations in the EGFR gene, suitable for targeting by EGFR tyrosine kinase inhibitors, represent roughly 10%, whereas the subgroup of tumors with ALK rearrangements, targeted by ALK inhibitors, is only ~5%.⁷ Thus, the majority of lung tumors lack effective treatment and novel therapeutic strategies are still needed.MicroRNAs (miRNAs) are short noncoding RNAs, 20–24 nucleotides long, that have important roles in almost all biological pathways,^{8, 9, 10, 11} and influence cancer-relevant processes, such as proliferation,¹² cell cycle,¹³ apoptosis,¹⁴ and migration.¹⁵ Many studies have reported the critical role of miRNAs in lung cancer pathogenesis and their potential as biomarkers for lung cancer risk stratification,¹⁶ outcome prediction,¹⁷ and classification of histological subtypes.^{18, 19} miRNAs are actively released by various cell types and can be detected in biological fluids, such as plasma and serum, making them suitable as circulating biomarkers in NSCLC.^{20, 21}There is limited evidence of mir-660 deregulation in cancer and little is known about its role in lung tumorigenesis and its putative target genes. Mir-660 has been reported to be upregulated in chronic lymphocytic leukemia^{22, 23} and in leukemic cells after treatment with 4-hydroxynonenal, a compound that induces differentiation and blocks proliferation of leukemic cells.²⁴ In a previous study we demonstrated that mir-660 was one of the 24 miRNAs deregulated in plasma samples of NSCLC patients identified in a low-dose computed tomography (LDCT) screening trial.²⁰The p53 tumor suppressor protein is a key regulator of cell cycle G0/G1 checkpoint, senescence, and apoptosis in response to cellular stress signals.^{25, 26} Mouse double minute 2 (MDM2), a p53–E3 ubiquitin ligase,²⁷ is the principal negative regulator of the expression level and function of p53.^{28, 29} Several studies have illustrated different mechanisms of p53 regulation by MDM2,^{30, 31} such as binding transactivation region of p53,^{32, 33} promoting nuclear export and cytoplasmic accumulation of p53 by monoubiquitination,^{34, 35} and inducing p53 proteosomal degradation by polyubiquitination.³⁶ In addition, MDM2 gene has been reported to be amplified or overexpressed in a variety of human cancers, such as sarcoma,³⁷ lymphoma,³⁸ breast cancer,³⁹ lung cancer,⁴⁰ and testicular germ cell tumor.⁴¹ Several miRNAs targeting MDM2 have been identified, such as the mir-143/mir-145 cluster that can be induced by p53,⁴² as well as mir-25 and mir-32, known to inhibit tumor glioblastoma growth in mouse brain.⁴³In this study, we report that mir-660 is downregulated in tissue and plasma samples of lung cancer patients and demonstrate that mir-660 replacement impairs the functionality of p53 wild-type (wt) lung cancer cells and inhibits in vitro and in vivo tumor growth. We showed that all the effects observed after mir-660 overexpression were absent in p53 ko cells, identified MDM2 as mir-660 direct target gene and indicate impairment of the MDM2/p53 interaction as the mechanism underlying tumor growth inhibition.     

Transforming growth factor-beta signaling network regulates plasticity and lineage commitment of lung cancer cells

Identification of target cells in lung tumorigenesis and characterization of the signals that control their behavior is an important step toward improving early cancer diagnosis and predicting tumor behavior. We identified a population of cells in the adult lung that bear the EpCAM+CD104+CD49f+CD44+CD24loSCA1+ phenotype and can be clonally expanded in culture, consistent with the properties of early progenitor cells. We show that these cells, rather than being restricted to one tumor type, can give rise to several different types of cancer, including adenocarcinoma and squamous cell carcinoma. We further demonstrate that these cells can be converted from one cancer type to the other, and this plasticity is determined by their responsiveness to transforming growth factor (TGF)-beta signaling. Our data establish a mechanistic link between TGF-beta signaling and SOX2 expression, and identify the TGF-beta/SMAD/SOX2 signaling network as a key regulator of lineage commitment and differentiation of lung cancer cells.Lung cancer is the leading cause of cancer-related mortality in both men and women worldwide. Lung cancers are divided into two major categories: non-small-cell lung cancer (NSCLC) and small-cell lung cancer. NSCLC accounts for ∼80% of all lung cancers and is divided further into adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large-cell lung carcinoma. Of the four major types of lung cancer, Kras mutations are present in about 30–50% of ADC, a smaller percentage of SCC (5–7%) and <1% of SCLC.^{1, 2} Mutations of the p53 gene are common in all types of lung cancer and range from ∼30% in ADC to more than 70% in SCC and SCLC.³ Other alterations occur at lower frequencies in NSCLC, including mutations in EGFR (15%), EML4-ALK (4%), ERBB2 (2%), AKT1, BRAF, MAP2K1 and MET.^{2, 4} Previous efforts in comprehensive characterization of lung cancer include copy number and gene expression profiling, targeted sequencing of candidate genes and large-scale genome sequencing of tumor samples.^{5, 6, 7, 8, 9} Significant progress has also been made in developing mouse models of lung carcinogenesis.^{10, 11} The unifying theme underlying these studies is that there exists a permissive cellular context for each specific oncogenic lesion, and that only certain types of cells are capable of cancer initiation.^{12, 13, 14}The lung consists of three anatomically distinct regions such as trachea, bronchioles and alveoli, each maintained by a distinct population of progenitor cells, that is, basal, Clara and alveolar type 2 (AT2) cells, respectively.^{15, 16} Previous work has focused upon AT2 cells, Clara cells (or variant Clara cells with low CC10 expression) and the putative bronchioalveolar stem cells (BASCs) as potential cells of origin for lung ADC.^{12, 14, 17} However, to date, only AT2 cells have been conclusively identified as having the potential to be the cells of origin for lung ADC.^{14, 17} This raises the question of whether Clara cells, their restricted subpopulations or the newly identified candidate stem cells, termed distal airway stem cells,¹⁸ alveolar epithelial progenitor cells (AECs)^{19, 20} and BASCs,¹² also have the capacity to give rise to ADC. Current knowledge on the cellular origins of SCC, the second most common type of lung cancer, lags behind that of ADC, partly owing to the fact that squamous cells are not normally present in the respiratory epithelium, and therefore arise through either metaplasia (conversions between stem cell states) or trans-differentiation (conversions between differentiated cells).^{21, 22} Whether the mechanisms of SCC causation vary by cell type, their responses to various cells signaling cascades (e.g., transforming growth factor (TGF)-beta, WNT, etc.), or other tumor characteristics is unknown at present.To address the questions of cell type of origin and signal cascades that control their behavior, we developed in vitro culture conditions that favor the growth of lung epithelial cells with stem cell-like properties. We describe a population of cells isolated from the adult lung that, rather than being restricted to one tumor type, can give rise to several different types of cancer, including ADC and SCC. We also show that these cells can be converted from one cancer type to the other, and this plasticity is largely, if not solely, determined by TGF-beta signaling.     

Tyr1068-phosphorylated epidermal growth factor receptor (EGFR) predicts cancer stem cell targeting by erlotinib in preclinical models of wild-type EGFR lung cancer

Tyrosine kinase inhibitors (TKIs) have shown strong activity against non-small-cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations. However, a fraction of EGFR wild-type (WT) patients may have an improvement in terms of response rate and progression-free survival when treated with erlotinib, suggesting that factors other than EGFR mutation may lead to TKI sensitivity. However, at present, no sufficiently robust clinical or biological parameters have been defined to identify WT-EGFR patients with greater chances of response. Therapeutics validation has necessarily to focus on lung cancer stem cells (LCSCs) as they are more difficult to eradicate and represent the tumor-maintaining cell population. Here, we investigated erlotinib response of lung CSCs with WT-EGFR and identified EGFR phosphorylation at tyrosine1068 (EGFR^tyr1068) as a powerful biomarker associated with erlotinib sensitivity both in vitro and in preclinical CSC-generated xenografts. In contrast to the preferential cytotoxicity of chemotherapy against the more differentiated cells, in EGFR^tyr1068 cells, erlotinib was even more active against the LCSCs compared with their differentiated counterpart, acquiring potential value as CSC-directed therapeutics in the context of WT-EGFR lung cancer. Although tumor growth was inhibited to a similar extent during erlotinib or chemotherapy administration to responsive tumors, erlotinib proved superior to chemotherapy in terms of higher tolerability and reduced tumor aggressiveness after treatment suspension, substantiating the possibility of preferential LCSC targeting, both in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) tumors. We conclude that EGFR^tyr1068 may represent a potential candidate biomarker predicting erlotinib response at CSC-level in EGFR-WT lung cancer patients. Finally, besides its invariable association with erlotinib sensitivity in EGFR-WT lung CSCs, EGFR^tyr1068 was associated with EGFR-sensitizing mutations in cell lines and patient tumors, with relevant diagnostic, clinical and therapeutic implications.Non-small-cell lung cancer (NSCLC) accounts for ∼80% of lung cancer subtypes and is the leading cause of cancer-related death worldwide.¹ In recent years, molecular characterization of NSCLC has reached an unprecedented detail and has allowed segregating NSCLC into discrete molecular subgroups, characterized by specific oncogenic drivers, such as epidermal growth factor receptor (EGFR), BRAF, KRAS, epidermal growth factor receptor 2 (HER2) mutations, MET amplification and anaplastic lymphoma kinase gene rearrangements (ALK).^{2, 3} Consequently, the understanding of NSCLC biology has brought two new classes of targeted agents into the clinical setting: EGFR tyrosine kinase inhibitors (TKIs) and ALK inhibitors.^{4, 5} In particular, clinical trials have shown that NSCLC patients whose tumors harbor sensitizing EGFR mutations significantly benefit from the upfront use of an EGFR TKI, rather than conventional chemotherapy.^{6, 7, 8, 9, 10, 11} Although licensed for clinical use in chemotherapy-pretreated patients, regardless of EGFR mutational status, the EGFR TKI erlotinib has limited efficacy when compared with standard chemotherapy in patients with WT-EGFR NSCLC.^{12, 13, 14}However, a fraction of patients on erlotinib treatment may achieve clinically significant objective responses and prolonged disease control, despite the lack of detectable EGFR mutations.¹⁵ Nevertheless, no biomarker investigated so far was felt sufficiently robust to select for the use of erlotinib in the maintenance or refractory setting.¹⁶ Thus, it would be crucial to identify molecular predictors of TKI sensitivity in EGFR wild-type (WT) tumors in order to prospectively select the subgroup of patients who may benefit from erlotinib therapy. Moreover, EGFR TKIs have also shown a modest therapeutic effect in lung squamous cell carcinoma (SCC), where EGFR mutations are very rare and patients have limited therapeutic options in the maintenance and relapsed settings.^{16, 17, 18, 19, 20}Even more importantly, in order to obtain meaningful clinical responses it is crucial to effectively target the population of cells that are able to escape treatment and maintain the growth of a resistant tumor.²¹ Cancer stem cells (CSCs) have been in fact identified within most solid tumors, including lung tumors, and are associated with increased resistance to therapies.^{22, 23, 24, 25, 26, 27, 28, 29, 30} Thus, the efficacy of innovative therapeutic strategies should be validated against these more aggressive, tumor-maintaining cells.^{23, 27, 31} Importantly, TKI response has never been determined at the level of the tumor-maintaining CSCs. Thus, we investigated erlotinib response of EGFR mutation-negative lung cancer stem cells (LCSCs) and LCSC-based xenografts with the attempt to evaluate their sensitivity to the drug and correlate it with their molecular pattern in order to identify potential biomarkers predictive of erlotinib response in a WT-EGFR context at the CSC level.     

Cholesterol oxidase from Bordetella species promotes irreversible cell apoptosis in lung adenocarcinoma by cholesterol oxidation

Cholesterol oxidase (COD), an enzyme catalyzing the oxidation of cholesterol, has been applied to track the distribution of membrane cholesterol. Little investigations about the effect of COD on tumor cells have been performed. In the present study, we provided evidence that COD from Bordetella species (COD-B), induced apoptosis of lung cancer cells in vitro and in vivo. COD-B treatment inhibited Akt and ERK1/2 phosphorylation in dose- and time-dependent manner, which was not reversed and was even aggravated by cholesterol addition. Further investigation indicated that COD-B treatment promoted the generation of reactive oxygen species (ROS) and that cholesterol addition further elevated ROS levels. Moreover, COD-B treatment resulted in JNK and p38 phosphorylation, downregulation of Bcl-2, upregulation of Bax, activated caspase-3 and cytochrome C release, which likely responded to freshly produced hydrogen peroxide that accompanied cholesterol oxidation. Catalase pretreatment could only partially prevent COD-B-induced events, suggesting that catalase inhibited H₂O₂-induced signal transduction but had little effect on signal pathways involved in cholesterol depletion. Our results demonstrated that COD-B led to irreversible cell apoptosis by decreasing cholesterol content and increasing ROS level. In addition, COD-B may be a promising candidate for a novel anti-tumor therapy.Cholesterol is an essential component of the plasma membrane in eukaryotic cells and has an important role in maintaining structure integrity, receptor function, dynamics and ion channels in the plasma membrane.^{1, 2, 3} Cholesterol is a critical constituent for the formation of lipid rafts.⁴ Lipid rafts are plasma membrane microdomains locating abundant signaling molecules, such as caveolin-1 protein and epidermal growth factor receptor (EGFR).⁵ These molecules conduct a series of cellular functions, including cell proliferation and apoptosis.⁶ Because cholesterol has a bridging role in liquid-ordered rafts by binding tightly to the sphingolipids with saturated hydrocarbon chains, modification or depletion of membrane cholesterol is speculated to perturb the properties of lipid rafts.^{7, 8} Several studies have demonstrated that the depletion of membrane cholesterol led to the disruption of lipid rafts and dissociation of signaling molecules from lipid rafts, which generated aberrant signal transductions and disturbed cellular functions.⁹ Therefore, membrane cholesterol concentration is accurately regulated.¹⁰Cholesterol metabolism is disorganized in various tumors, such as prostate, lung, acute myeloid leukemia and breast cancer and especially in chemoresistant tumors.^{11, 12, 13, 14, 15} Solid tumors accumulate more cholesterol compared with normal tissue, which contributes to the proliferation, differentiation and migration of tumor cell.^{16, 17} The elevated content of membrane cholesterol modulates the activation of cellular surface receptors, such as EGFR.^{18, 19} Several reports have demonstrated that EGFR is upregulated in most malignant cells and stimulates the proliferation of cells by promoting the downstream activation of protein kinase B (Akt).^{20, 21, 22} Depletion of cholesterol from plasma membrane induces Akt inactivation and cell death.^{23, 24} Furthermore, the alteration of membrane cholesterol also affects the expression of the B-cell lymphoma/leukemia-2 (BCL-2) family members.^{21, 25} Therefore, it has been proposed that membrane cholesterol could potentially be a therapeutic target for tumors.The modification of membrane cholesterol can be mainly performed by methyl-beta-cyclodextrin (MβCD), statin and filipin.^{9, 21} Previous reports have indicated that MβCD could take up cholesterol into the internal hydrophobic pocket to form a MβCD–cholesterol complex and that the hydrophilic surface promoted the complex to dissolve in the aqueous phase.²⁶ Statins serve as competitive inhibitors to 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA) to block synthesis of cholesterol. Filipin, as a macrolide polyene antibiotic, binds specifically to cholesterol and causes the disruption of lipid rafts.²⁷ A common factor shared by the above three agents is that the cholesterol alteration caused by each of them can be reversed by the supplement of exogenous cholesterol. In view of high cholesterol in the modern diet, the effect of MβCD, statin and filipin on tumors would be attenuated, although prolonged suppression of cholesterol synthesis reduces the risk of advanced prostate cancer.²⁸Cholesterol oxidase (COD) is a flavoprotein from microorganisms that catalyzes the oxidation of cholesterol to 4-cholesten-3-one with the reduction of oxygen to hydrogen peroxide.²⁹ COD can convert membrane cholesterol to 4-cholesten-3-one and can inhibit the formation of lipid rafts.³⁰ Different from other cholesterol-depleting agents, COD disrupts lipid rafts by displacing cholesterol with 4-cholesten-3-one. However, because the action of COD is dependent on the microenvironment of membrane cholesterol, such as the phospholipid composition, cholesterol content and ionic strength, COD does not attack all cell types. So far, despite the fact that COD has been well known as a lipid raft destroyer, few investigations have been conducted to directly determine the impact of COD on signal transduction in cancer.In this study, we investigated whether COD purified from Bordetella species (COD-B) could suppress the growth of a lung adenocarcinoma cell line by its effect on membrane cholesterol. We provide the evidence that COD-B induces apoptosis in lung adenocarcinoma cells by catalyzing oxidation of membrane cholesterol and elevating reactive oxygen species (ROS) levels. Moreover, COD-B-induced apoptosis is not reversed and is aggravated by cholesterol supplementation.     

The novel miR-9500 regulates the proliferation and migration of human lung cancer cells by targeting Akt1

MicroRNAs have crucial roles in lung cancer cell development. They regulate cell growth, proliferation and migration by mediating the expression of tumor suppressor genes and oncogenes. We identified and characterized the novel miR-9500 in human lung cancer cells. The miR-9500 forms a stem-loop structure and is conserved in other mammals. The expression levels of miR-9500 were reduced in lung cancer cells and lung cancer tissues compared with normal tissues, as verified by TaqMan miRNA assays. It was confirmed that the putative target gene, Akt1, was directly suppressed by miR-9500, as demonstrated by a luciferase reporter assay. The miR-9500 significantly repressed the protein expression levels of Akt1, as demonstrated via western blot, but did not affect the corresponding mRNA levels. Akt1 has an important role in lung carcinogenesis, and depletion of Akt1 has been shown to have antiproliferative and anti-migratory effects in previous studies. In the current study, the overexpression of miR-9500 inhibited cell proliferation and the expression of cell cycle-related proteins. Likewise, the overexpression of miR-9500 impeded cell migration in human lung cancer cells. In an in vivo assay, miR-9500 significantly suppressed Fluc expression compared with NC and ASO-miR-9500, suggesting that cell proliferation was inhibited in nude mice. Likewise, miR-9500 repressed tumorigenesis and metastasis by targeting Akt1. These data indicate that miR-9500 might be applicable for lung cancer therapy.MicroRNAs (miRNAs) are small, non-coding RNAs, 18–25 nucleotides (nt) in length that regulate gene expression by binding to the 3′-untranslated region (UTR) of their target genes,^{1, 2} and these RNAs are processed from introns, exons or intergenic regions.³ First, miRNAs are transcribed by RNA polymerase II into primary miRNA (pri-miRNA) molecules that contain several thousand nucleotides. The pri-miRNAs are then sequentially processed by a microprocessor, such as Drosha RNase III endonuclease and DiGeorge syndrome region gene 8 protein (DGCR8), to form ∼70 nt-stem-loop intermediates known as miRNA precursors (pre-miRNAs).^{4, 5} The pre-miRNAs are then exported from the nucleus into the cytoplasm via Exportin-5 (EXP5), with its cofactor Ran-GTP; in the cytoplasm, these pre-miRNAs are processed into 18–25 nt mature miRNA duplexes by the RNase III endonuclease Dicer.^{6, 7} The mature miRNA duplexes, along with the Argonaute proteins, are integrated as single-stranded RNAs into an RNA-induced silencing complex, which induces either the cleavage or the translational inhibition of the targeted mRNAs.^{8, 9, 10} miRNAs have been implicated in a variety of biological processes associated with cancer development, including cell proliferation and invasion,¹¹ and miRNA expression is deregulated in many forms of cancer.¹²Cancer is a major public health problem worldwide. Lung cancer represents one of the most predominant types of cancer, with high mortality rates in both men and women. Epithelial lung cancer can be categorized into one of two types: small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). NSCLC accounts for ∼80% of lung cancer cases, and these cases can be further categorized as adenocarcinoma (40%), squamous cell carcinoma (30–35%), and large cell carcinoma (5–15%). NSCLC has a 5-year survival rate of only 16%.^{13, 14, 15} Current studies have shown that miRNAs are deregulated in various cancers, including NSCLC, and may act as oncogenes or tumor suppressor genes.¹⁶ For example, the Let-7 family,¹⁷ miR-15a/16,¹⁸ miR-17-92,¹⁹ miR-107 and miR-185,²⁰ are deregulated in lung cancer.Some studies have reported that phosphatidylinositol 3-kinase (PI3K) signaling is activated in human cancers^{21, 22} and has an important role in the progression of NSCLC. The PI3K pathway modulates several cellular mechanisms, such as cell survival, proliferation, migration and motility, and thereby significantly affects the growth of tumors.^{23, 24} The primary regulator of the PI3K pathway is Akt, a protein kinase B that mediates cell survival, cell death,²⁵ cell growth, cell migration and angiogenesis.^{26, 27, 28} The silencing of the Akt1 gene has been shown to inhibit the proliferation of gastric cancer cells both in vitro and in vivo.²⁹ Other studies have shown that aberrant AKT activation has a critical role in tumorigenesis.³⁰In this study, we identified small RNAs in lung cancer cells. To analyze a novel miRNA signature, we examined the structure and sequence of the small RNAs, analyzed the expression patterns of the novel miRNAs in lung cancer tissues and assessed the miRNA target genes. Our data revealed that miR-9500 regulates certain human lung cancer cell functions, including cell growth, proliferation, and migration.     

Interleukin-33 enhances programmed oncosis of ST2L-positive low-metastatic cells in the tumour microenvironment of lung cancer

The proinflammatory interleukin-33 (IL-33) binds to its receptor ST2L on the surface of immune cells and stimulates the production of Th2 cytokines; however, the effects of IL-33 on tumour cells are poorly understood. Here we show that ST2 was significantly downregulated in human lung cancer tissues and cells compared with normal lung tissues and cells. IL-33 expression was also inversely correlated with the stages of human lung cancers. In accordance with this finding, low-metastatic cells but not high-metastatic cells derived from Lewis lung carcinoma expressed functional ST2L. IL-33 was abundantly present in the tumours established by the low-metastatic cells compared with those formed by the high-metastatic cells. Although the low-metastatic cells scarcely expressed IL-33 in vitro, these cells did expry 6ess this molecule in vivo, likely due to stimulation by intratumoural IL-1β and IL-33. Importantly, IL-33 enhanced the cell death of ST2L-positive low-metastatic cells, but not of ST2L-negative high-metastatic cells, under glucose-depleted, glutamine-depleted and hypoxic conditions through p38 MAPK and mTOR activation, and in a mitochondria-dependent manner. The cell death was characterised by cytoplasmic blisters and karyolysis, which are unique morphological features of oncosis. Inevitably, the low-metastatic cells, but not of the high-metastatic cells, grew faster in IL-33^−/− mice than in wild-type mice. Furthermore, IL-33 selected for the ST2L-positive, oncosis-resistant high-metastatic cells under conditions mimicking the tumour microenvironment. These data suggest that IL-33 enhances lung cancer progression by selecting for more malignant cells in the tumour microenvironment.Interleukin-33 (IL-33), a member of the IL-1 cytokine family, is a natural ligand for the IL-33 receptor, which is a heterodimer composed of ST2L and the IL-1 receptor accessory protein (IL-1RAcP).^{1, 2, 3} IL-33 is primarily expressed in epithelial cells and endothelial cells as a proinflammatory cytokine.^{4, 5} IL-33 is usually localised in the cell nucleus as an alarmin that signals to local immune cells in response to tissue damage caused by injury, necrosis or exposure to pathogens.^{6, 7, 8} IL-33 polarises naive T cells to produce Th2-associated cytokines, it strongly induces proinflammatory cytokine and chemokine production by mast cells and eosinophils, and it stimulates the polarisation of alternatively activated M2 macrophages.^{9, 10} Thus, IL-33 has an important role in Th2 immunity and Th2-related diseases, such as asthma, atopic dermatitis and anaphylaxis.^{6, 11, 12, 13, 14} ST2L is expressed on the cell surface of Th2 cells, but not of Th1 cells, and on the cell surface of other immune-related cells including NK and NKT cells.^{8, 15, 16, 17} Human bronchial epithelial cells and rat alveolar type-II cells, which can be the cellular origins of bronchoalveolar carcinomas and adenocarcinomas, respectively, human lung microvascular endothelial cells and human intestinal epithelial cells, are also reported to express ST2L.^{18, 19, 20, 21, 22}IL-33 binding to ST2L/IL-1RAcP initiates the recruitment of the myeloid differentiation primary response 88 (MyD88)/IL-1 receptor-associated kinase 4/IRAK1/tumour necrosis factor (TNF) receptor-associated factor 6 module and then activates tumour growth factor-β-activated kinase 1. This activation stimulates the activation of nuclear factor-κB (NF-κB), mitogen-activated protein kinase p38 (p38 MAPK), c-JUN N-terminal kinases (JNK) and, in parallel, extracellularly regulated kinases (p44/42 MAPK), leading to the production of the inflammatory mediators.^{23, 24, 25}A relationship between the IL-33/ST2L axis and cancer is beginning to be recognised. IL-33 enhances murine breast cancer growth and metastasis by increasing the intratumoural accumulation of immunosuppressive and innate lymphoid cells.²⁶ Higher serum levels of IL-33 were found to be a worse prognostic marker in gastric cancer and in non-small-cell lung carcinoma patients.^{27, 28} From these observations, IL-33 appears to promote tumour malignancy by modulating Th2-type immunity.Whether the tumour cells express functional ST2L remains unknown. If these cells do express functional ST2L, then IL-33 in the tumour microenvironment might directly affect their behaviour. In this study, we sought ST2L-positive tumour cells and eventually found that low-metastatic, but not high-metastatic, Lewis lung carcinoma (3LL) cells expressed functional ST2L. We demonstrate that IL-33 induced programmed oncosis of the ST2L-positive low-metastatic cells, under conditions mimicking the tumour microenvironment, thereby allowing the outgrowth of the ST2L-negative high-metastatic cells in equal mixtures, suggesting a role for IL-33 in the malignant progression of lung cancers.     

The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs

To grant faithful chromosome segregation, the spindle assembly checkpoint (SAC) delays mitosis exit until mitotic spindle assembly. An exceedingly prolonged mitosis, however, promotes cell death and by this means antimicrotubule cancer drugs (AMCDs), that impair spindle assembly, are believed to kill cancer cells. Despite malformed spindles, cancer cells can, however, slip through SAC, exit mitosis prematurely and resist killing. We show here that the Fcp1 phosphatase and Wee1, the cyclin B-dependent kinase (cdk) 1 inhibitory kinase, play a role for this slippage/resistance mechanism. During AMCD-induced prolonged mitosis, Fcp1-dependent Wee1 reactivation lowered cdk1 activity, weakening SAC-dependent mitotic arrest and leading to mitosis exit and survival. Conversely, genetic or chemical Wee1 inhibition strengthened the SAC, further extended mitosis, reduced antiapoptotic protein Mcl-1 to a minimum and potentiated killing in several, AMCD-treated cancer cell lines and primary human adult lymphoblastic leukemia cells. Thus, the Fcp1-Wee1-Cdk1 (FWC) axis affects SAC robustness and AMCDs sensitivity.The spindle assembly checkpoint (SAC) delays mitosis exit to coordinate anaphase onset with spindle assembly. To this end, SAC inhibits the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) to prevent degradation of the anaphase inhibitor securin and cyclin B, the major mitotic cyclin B-dependent kinase 1 (cdk1) activator, until spindle assembly.¹ However, by yet poorly understood mechanisms, exceedingly prolonging mitosis translates into cell death induction.^{2, 3, 4, 5, 6, 7} Although mechanistic details are still missing on how activation of cell death pathways is linked to mitosis duration, prolongation of mitosis appears crucial for the ability of antimicrotubule cancer drugs (AMCDs) to kill cancer cells.^{2, 3, 4, 5, 6, 7} These drugs, targeting microtubules, impede mitotic spindle assembly and delay mitosis exit by chronically activating the SAC. Use of these drugs is limited, however, by toxicity and resistance. A major mechanism for resistance is believed to reside in the ability of cancer cells to slip through the SAC and exit mitosis prematurely despite malformed spindles, thus resisting killing by limiting mitosis duration.^{2, 3, 4, 5, 6, 7} Under the AMCD treatment, cells either die in mitosis or exit mitosis, slipping through the SAC, without or abnormally dividing.^{2, 3, 4} Cells that exit mitosis either die at later stages or survive and stop dividing or proliferate, giving rise to resistance.^{2, 3, 4} Apart from a role for p53, what dictates cell fate is still unknown; however, it appears that the longer mitosis is protracted, the higher the chances for cell death pathway activation are.^{2, 3, 4, 5, 6, 7}Although SAC is not required per se for killing,⁶ preventing SAC adaptation should improve the efficacy of AMCD by increasing mitosis duration.^{2, 3, 4, 5, 6, 7} Therefore, further understanding of the mechanisms by which cells override SAC may help to improve the current AMCD therapy. Several kinases are known to activate and sustain SAC, and cdk1 itself appears to be of primary relevance.^{1, 8, 9} By studying mitosis exit and SAC resolution, we recently reported a role for the Fcp1 phosphatase to bring about cdk1 inactivation.^{10, 11} Among Fcp1 targets, we identified cyclin degradation pathway components, such as Cdc20, an APC/C co-activator, USP44, a deubiquitinating enzyme, and Wee1.^{10, 11} Wee1 is a crucial kinase that controls the G2 phase by performing inhibitory phosphorylation of cdk1 at tyr-15 (Y15-cdk1). Wee1 is also in a feedback relationship with cdk1 itself that, in turn, can phosphorylate and inhibit Wee1 in an autoamplification loop to promote the G2-to-M phase transition.¹² At mitosis exit, Fcp1 dephosphorylated Wee1 at threonine 239, a cdk1-dependent inhibitory phosphorylation, to dampen down the cdk1 autoamplification loop, and Cdc20 and USP44, to promote APC/C-dependent cyclin B degradation.^{10, 11, 12} In this study we analysed the Fcp1 relevance in SAC adaptation and AMCD sensitivity.