Head and neck squamous cell carcinomas (HNSCC) preferentially spread to regional cervical tissues and lymph nodes. Here, we hypothesized that lymphotoxin-β (LTβ), receptor LTβR, and NF-κB-inducing kinase (NIK), promote the aberrant activation of alternative NF-κB2/RELB pathway and genes, that enhance migration and invasion of HNSCC. Genomic and expression alterations of the alternative NF-kB pathway were examined in 279 HNSCC tumors from The Cancer Genome Atlas (TCGA) and a panel of HNSCC lines. LTβR is amplified or overexpressed in HNSCC of the larynx or oral cavity, while LTβ, NIK, and RELB are overexpressed in cancers arising within lymphoid oropharyngeal and tonsillar sites. Similarly, subsets of HNSCC lines displayed overexpression of LTβR, NIK, and RELB proteins. Recombinant LTβ, and siRNA depletion of endogenous LTβR and NIK, modulated expression of LTβR, NIK, and nuclear translocation of NF-κB2(p52)/RELB as well as functional NF-κB promoter reporter activity. Treatment with a NIK inhibitor (1,3[2H,4H]-Iso-Quinoline Dione) reduced the protein expression of NIK and NF-κB2(p52)/RELB, and blocked LTβ induced nuclear translocation of RELB. NIK and RELB siRNA knockdown or NIK inhibitor slowed HNSCC migration or invation in vitro. LTβ-induces expression of migration and metastasis related genes, including hepatocyte growth/scatter factor receptor MET. Knockdown of NIK or MET similarly inhibited the migration of HNSCC cell lines. This may help explain why HNSCC preferentially migrate to local lymph nodes, where LTβ is expressed. Our findings show that LTβ/LTβR promotes activation of the alternative NIK-NF-κB2/RELB pathway to enhance MET-mediated cell migration in HNSCC, which could be potential therapeutic targets in HNSCC.

BACKGROUND: Genome-wide association studies are widely used to map genomic regions contributing to lung cancer (LC) susceptibility, but they typically do not identify the precise disease-causing genes/variants. To unveil the inherited genetic variants that cause LC, we performed focused exome-sequencing analyses on genes located in 121 genome-wide association study-identified loci previously implicated in the risk of LC, chronic obstructive pulmonary disease, pulmonary function level, and smoking behavior.
METHODS: Germline DNA from 260 case patients with LC and 318 controls were sequenced by utilizing VCRome 2.1 exome capture. Filtering was based on enrichment of rare and potential deleterious variants in cases (risk alleles) or controls (protective alleles). Allelic association analyses of single-variant and gene-based burden tests of multiple variants were performed. Promising candidates were tested in two independent validation studies with a total of 1773 case patients and 1123 controls.
RESULTS: We identified 48 rare variants with deleterious effects in the discovery analysis and validated 12 of the 43 candidates that were covered in the validation platforms. The top validated candidates included one well-established truncating variant, namely, BRCA2, DNA repair associated gene (BRCA2) K3326X (OR = 2.36, 95% confidence interval [CI]: 1.38-3.99), and three newly identified variations, namely, lymphotoxin beta gene (LTB) p.Leu87Phe (OR = 7.52, 95% CI: 1.01-16.56), prolyl 3-hydroxylase 2 gene (P3H2) p.Gln185His (OR = 5.39, 95% CI: 0.75-15.43), and dishevelled associated activator of morphogenesis 2 gene (DAAM2) p.Asp762Gly (OR = 0.25, 95% CI: 0.10-0.79). Burden tests revealed strong associations between zinc finger protein 93 gene (ZNF93), DAAM2, bromodomain containing 9 gene (BRD9), and the gene LTB and LC susceptibility.
CONCLUSION: Our results extend the catalogue of regions associated with LC and highlight the importance of germline rare coding variants in LC susceptibility.

Metastatic melanoma is an aggressive skin cancer and is one of the global malignancies with high mortality and morbidity. It is essential to identify and verify diagnostic biomarkers of early metastatic melanoma. Previous studies have systematically assessed protein biomarkers and mRNA-based expression characteristics. However, molecular markers for the early diagnosis of metastatic melanoma have not been identified. To explore potential regulatory targets, we have analyzed the gene microarray expression profiles of malignant melanoma samples by co-expression analysis based on the network approach. The differentially expressed genes (DEGs) were screened by the EdgeR package of R software. A weighted gene co-expression network analysis (WGCNA) was used for the identification of DEGs in the special gene modules and hub genes. Subsequently, a protein-protein interaction network was constructed to extract hub genes associated with gene modules. Finally, twenty-four important hub genes (RASGRP2, IKZF1, CXCR5, LTB, BLK, LINGO3, CCR6, P2RY10, RHOH, JUP, KRT14, PLA2G3, SPRR1A, KRT78, SFN, CLDN4, IL1RN, PKP3, CBLC, KRT16, TMEM79, KLK8, LYPD3 and LYPD5) were treated as valuable factors involved in the immune response and tumor cell development in tumorigenesis. In addition, a transcriptional regulatory network was constructed for these specific modules or hub genes, and a few core transcriptional regulators were found to be mostly associated with our hub genes, including GATA1, STAT1, SP1, and PSG1. In summary, our findings enhance our understanding of the biological process of malignant melanoma metastasis, enabling us to identify specific genes to use for diagnostic and prognostic markers and possibly for targeted therapy.

Acute myeloid leukemia (AML) is the most common and severe form of acute leukemia diagnosed in adults. Owing to its heterogeneity, AML is divided into classes associated with different treatment outcomes and specific gene expression profiles. Based on previous studies on AML, in this study, we designed and generated an AML-array containing 900 oligonucleotide probes complementary to human genes implicated in hematopoietic cell differentiation and maturation, proliferation, apoptosis and leukemic transformation. The AML-array was used to hybridize 118 samples from 33 patients with AML of the M1 and M2 subtypes of the French-American‑British (FAB) classification and 15 healthy volunteers (HV). Rigorous analysis of the microarray data revealed that 83 genes were differentially expressed between the patients with AML and the HV, including genes not yet discussed in the context of AML pathogenesis. The most overexpressed genes in AML were STMN1, KITLG, CDK6, MCM5, KRAS, CEBPA, MYC, ANGPT1, SRGN, RPLP0, ENO1 and SET, whereas the most underexpressed genes were IFITM1, LTB, FCN1, BIRC3, LYZ, ADD3, S100A9, FCER1G, PTRPE, CD74 and TMSB4X. The overexpression of the CPA3 gene was specific for AML with mutated NPM1 and FLT3. Although the microarray-based method was insufficient to differentiate between any other AML subgroups, quantitative PCR approaches enabled us to identify 3 genes (ANXA3, S100A9 and WT1) whose expression can be used to discriminate between the 2 studied AML FAB subtypes. The expression levels of the ANXA3 and S100A9 genes were increased, whereas those of WT1 were decreased in the AML-M2 compared to the AML-M1 group. We also examined the association between the STMN1, CAT and ABL1 genes, and the FLT3 and NPM1 mutation status. FLT3+/NPM1- AML was associated with the highest expression of STMN1, and ABL1 was upregulated in FLT3+ AML and CAT in FLT3- AML, irrespectively of the NPM1 mutation status. Moreover, our results indicated that CAT and WT1 gene expression levels correlated with the response to therapy. CAT expression was highest in patients who remained longer under complete remission, whereas WT1 expression increased with treatment resistance. On the whole, this study demonstrates that the AML-array can potentially serve as a first-line screening tool, and may be helpful for the diagnosis of AML, whereas the differentiation between AML subgroups can be more successfully performed with PCR-based analysis of a few marker genes.

In solid tumors, the presence of lymph node-like structures called tertiary lymphoid structures (TLS) is associated with improved patient survival. However, little is known about how TLS develop in cancer, how their function affects survival, and whether they are affected by cancer therapy. In this study, we used multispectral microscopy, quantitative pathology, and gene expression profiling to analyze TLS formation in human lung squamous cell carcinoma (LSCC) and in an experimental model of lung TLS induction. We identified a niche of CXCL13

Monoclonal gammopathy of undetermined significance is a pre-malignant precursor of multiple myeloma with a 1% risk of progression per year. Although targeted analyses have shown the presence of specific genetic abnormalities such as

LHRH based vaccines are promising candidates for therapy of androgen and estrogen dependent cancers. We report in this communication development of a novel recombinant protein vaccine candidate against LHRH. A synthetic gene was designed in which the codon sequence in the LHRH decapeptide was modified by substituting the codon for 6-glycine with that of l-leucine. Further the LHRH(6leu) gene was linked to heat-labile enterotoxin of E. coli (LTB) as carrier. This LHRH(6leu)-LTB gene was cloned into a prokaryotic expression vector under the control of inducible and strong bacteriophage T7 promoter to over-express LHRH(leu) fused to LTB as recombinant protein in E. coli. Recombinant LHRH(leu)-LTB protein of ∼14 kDa size, was purified from inclusion bodies using in-situ refolding on the column and Ni-NTA based immobilized affinity chromatography. Western blot confirmed the immunoreactivity of purified LHRH(leu)-LTB fusion protein with anti-LHRH monoclonal antibody. The vaccine protein was further characterized by mass spectroscopy, circular dichroism and fluorescence spectroscopy. This communication reports a recombinant LHRH fusion protein with potential for blocking of sex hormones production for eventual therapy of sex hormones dependent neoplasms.

Leukotriene B

OBJECTIVE: To describe the epileptic phenotype of Tsc1(+/-) mice pups in comparison with age-related seizures in human tuberous sclerosis complex (TSC).
METHODS: Tsc1(+/-) and control mice underwent intracranial electroencephalography (EEG) recording at postnatal ages (P)8 to P33, with linear silicon probe implanted in the somatosensory cortex of one or both hemispheres for 8-24 h. Ictal events were classified visually by independent analyzers; distinct EEG patterns were related to age and analyzed to quantify field potential characteristics and signal dynamics between hemispheres. We collected retrospectively 20 infants with prenatally diagnosed TSC and EEG before seizure onset, and analyzed the electroclinical course of epilepsy, taking into account a first-line treatment by vigabatrin.
RESULTS: Spontaneous seizures were disclosed in 55% of Tsc1(+/-) mice at P9-18. Three ictal patterns were identified: from P9 to P12 "spike clusters" consisted of recurring large spikes without clinical correlate; "spasm-like" discharges dominated from P13 to P16 consisting of high amplitude large field potential superimposed with or followed by fast activity repeated every 2-10 s for at least 20 s, accompanied by rhythmic limb contractions; from P14 to P18 a "tonic-clonic like" pattern comprised rhythmic spikes of increasing amplitude with tonic-clonic movements. Early onset "spike clusters" were mainly unilateral, whereas "spasm-like" and "tonic-clonic like" patterns were bilateral. Interhemispheric propagation was significantly faster for "tonic-clonic like" than for "spasm-like" events. In infants diagnosed prenatally with TSC, clusters of sharp waves or spikes preceded the first seizure, and vigabatrin prevented the development of seizures. Patients treated after seizure onset developed spasms or focal seizures that were pharmacoresistant in 66.7% of cases.
SIGNIFICANCE: Tsc1(+/-) mice pups exhibit an age-dependent seizure pattern sequence mimicking early human TSC epilepsy features. Spike clusters before seizure onset in TSC should be considered as a first stage of epilepsy reinforcing the concept of preventive antiepileptic therapy.

Lymphotoxin-mediated activation of the lymphotoxin-β receptor (LTβR; LTBR) has been implicated in cancer, but its role in T-cell acute lymphoblastic leukaemia (T-ALL) has remained elusive. Here we show that the genes encoding lymphotoxin (LT)-α and LTβ (LTA, LTB) are expressed in T-ALL patient samples, mostly of the TAL/LMO molecular subtype, and in the TEL-JAK2 transgenic mouse model of cortical/mature T-ALL (Lta, Ltb). In these mice, expression of Lta and Ltb is elevated in early stage T-ALL. Surface LTα1 β2 protein is expressed in primary mouse T-ALL cells, but only in the absence of microenvironmental LTβR interaction. Indeed, surface LT expression is suppressed in leukaemic cells contacting Ltbr-expressing but not Ltbr-deficient stromal cells, both in vitro and in vivo, thus indicating that dynamic surface LT expression in leukaemic cells depends on interaction with its receptor. Supporting the notion that LT signalling plays a role in T-ALL, inactivation of Ltbr results in a significant delay in TEL-JAK2-induced leukaemia onset. Moreover, young asymptomatic TEL-JAK2;Ltbr(-/-) mice present markedly less leukaemic thymocytes than age-matched TEL-JAK2;Ltbr(+/+) mice and interference with LTβR function at this early stage delayed T-ALL development. We conclude that LT expression by T-ALL cells activates LTβR signalling in thymic stromal cells, thus promoting leukaemogenesis.

PURPOSE: At the molecular level, myeloma is characterized by copy number abnormalities and recurrent translocations into the immunoglobulin heavy chain locus. Novel methods, such as massively parallel sequencing, have begun to describe the pattern of tumor-acquired mutations, but their clinical relevance has yet to be established.
METHODS: We performed whole-exome sequencing for 463 patients who presented with myeloma and were enrolled onto the National Cancer Research Institute Myeloma XI trial, for whom complete molecular cytogenetic and clinical outcome data were available.
RESULTS: We identified 15 significantly mutated genes: IRF4, KRAS, NRAS, MAX, HIST1H1E, RB1, EGR1, TP53, TRAF3, FAM46C, DIS3, BRAF, LTB, CYLD, and FGFR3. The mutational spectrum is dominated by mutations in the RAS (43%) and nuclear factor-κB (17%) pathways, but although they are prognostically neutral, they could be targeted therapeutically. Mutations in CCND1 and DNA repair pathway alterations (TP53, ATM, ATR, and ZNFHX4 mutations) are associated with a negative impact on survival. In contrast, those in IRF4 and EGR1 are associated with a favorable overall survival. We combined these novel mutation risk factors with the recurrent molecular adverse features and international staging system to generate an international staging system mutation score that can identify a high-risk population of patients who experience relapse and die prematurely.
CONCLUSION: We have refined our understanding of genetic events in myeloma and identified clinically relevant mutations that may be used to better stratify patients at presentation.

BACKGROUND: To detect genetic expression profile alterations after papillary thyroid carcinoma (PTC) cells transfected with chemokine receptor CXCR7 gene by gene microarray, and gain insights into molecular mechanisms of how CXCR7 regulating PTC growth and metastasis.
METHODS: The Human OneArray microarray was used for a complete genome-wide transcript profiling of CXCR7 transfected PTCs (K1-CXCR7 cells), defined as experimental group. Non CXCR7 transfected PTCs (K1 cells) were used as control group. Differential analysis for per gene was performed with a random variance model and t test, p values were adjusted to control the false discovery rate. Gene ontology (GO) on differentially expressed genes to identify the biological processes in modulating the progression of papillary thyroid carcinoma. Pathway analysis was used to evaluate the signaling pathway that differentially expressed genes were involved in. In addition, quantitative real-time polymerase chain reaction (q-PCR) and Western blot were used to verify the top differentially expression genes.
RESULTS: Comparative analysis revealed that the expression level of 1149 genes was changed in response to CXCR7 transfection. After unsupervised hierarchical clustering analysis, 270 differentially expressed genes were filtered, of them 156 genes were up-regulated whereas 114 genes were down-regulated in K1-CXCR7 cells. GO enrichment analysis revealed the differentially expressed genes were mainly involved in biopolymer metabolic process, signal transduction and protein metabolism. Pathway enrichment analysis revealed differentially expressed genes were mainly involved in ECM-receptor interaction, Focal adhesion, MAPK signaling pathway and Cytokine-cytokine receptor interaction pathway. More importantly, the expression level of genes closely associated with tumor growth and metastasis was altered significantly in K1-CXCR7 cells, including up-regulated genes FN1, COL1A1, COL4A1, PDGFRB, LTB, CXCL12, MMP-11, MT1-MMP and down-regulated genes ITGA7, and Notch-1.
CONCLUSIONS: Gene expression profiling analysis of papillary thyroid carcinoma can further delineate the mechanistic insights on how CXCR7 regulating papillary thyroid carcinoma growth and metastasis. CXCR7 may regulate growth and metastasis of papillary thyroid carcinoma via the activation of PI3K/AKT pathway and its downstream NF-κB signaling, as well as the down-regulation of Notch signaling.

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin's lymphoma (NHL). In cancers, tumor suppressive microRNAs may be silenced by DNA hypermethylation. By microRNA profiling of representative EBV-negative MCL cell lines before and after demethylation treatment, miR-155-3p was found significantly restored. Methylation-specific PCR, verified by pyrosequencing, showed complete methylation of miR-155-3p in one MCL cell line (REC-1). 5-aza-2'-deoxycytidine treatment of REC-1 led to demethylation and re-expression of miR-155-3p. Over-expression of miR-155-3p led to increased sub-G1 apoptotic cells and reduced cellular viability, demonstrating its tumor suppressive properties. By luciferase assay, lymphotoxin-beta (LT-β) was validated as a miR-155-3p target. In 31 primary MCL, miR-155-3p was found hypermethylated in 6(19%) cases. To test if methylation of miR-155-3p was MCL-specific, miR-155-3p methylation was tested in an additional 191 B-cell, T-cell and NK-cell NHLs, yielding miR-155-3p methylation in 66(34.6%) including 36(27%) non-MCL B-cell, 24(53%) T-cell and 6(46%) of NK-cell lymphoma. Moreover, in 72 primary NHL samples with RNA, miR-155-3p methylation correlated with miR-155-3p downregulation (p=0.024), and LT-β upregulation (p=0.043). Collectively, miR-155-3p is a potential tumor suppressive microRNA hypermethylated in MCL and other NHL subtypes. As miR-155-3p targets LT-β, which is an upstream activator of the non-canonical NF-kB signaling, miR-155-3p methylation is potentially important in lymphomagenesis.

OBJECTIVE: The immune system is critical for controlling the progression of HPV cervical disease and the development of cancer. This study aimed to identify cervical cancer susceptibility alleles in candidate immune-modulating genes.
METHODS: Our family-based study involved a cohort of 641 probands (women with ICC/CIN III) and their biologic parents or siblings (641 trios). In the discovery phase (stage 1), involving 288 of the trios, 80 tag single nucleotide polymorphisms (SNPs) in 11 immune-modulating genes (IFNG, IFNGR1, IFNGR2, JAK1, JAK2, STAT1, STAT6, IL12A, TNF, LTA and LTB) were evaluated on the GoldenGate platform. We used the combined dataset for a total of 641 trios (stage 2) and the Taqman platform to validate the SNPs that had proved significant in the discovery dataset. The transmission disequilibrium test was used to detect significant shifts in allelic transmissions in the datasets.
RESULTS: Two SNPs in JAK2 and one SNP in STAT6 showed significant allelic association with cervical cancer in the stage 1 discovery dataset and were replicated in the larger joint analysis stage 2 dataset (JAK2 rs10815144, P=0.0029 and rs12349785, P=0.0058; and STAT6 rs3024971, P=0.0127). An additional SNP in exon 19 of JAK2 (rs2230724) was also examined in the combined dataset due to its strong linkage disequilibrium (LD) with rs10815144. It was also significant (P=0.0335).
CONCLUSIONS: Our results suggest an association of SNPs in JAK2 and STAT6 with cervical cancer. This association should be investigated in additional cervical cancer populations.

Uterine intravenous leiomyomatosis (IVL) is a distinct smooth muscle neoplasm with a potential of clinical aggressiveness due to its ability to extend into intrauterine and extrauterine vasculature. In this study, chromosomal alterations analyzed by oligonucleotide array comparative genomic hybridization were performed in 9 cases of IVL. The analysis was informative in all cases with multiple copy number losses and/or gains observed in each tumor. The most frequent recurrent loss of 22q12.3-q13.1 was observed in 6 tumors (66.7%), followed by losses of 22q11.23-q13.31, 1p36.13-p33, 2p25.3-p23.3, and 2q24.2-q32.2 and gains of 6p22.2, 2q37.3 and 10q22.2-q22.3, in decreasing order of frequency. Copy number variants were identified at 14q11.2, 15q11.1-q11.2, and 15q26.2. Genes mapping to the regions of loss include CHEK2, EWS, NF2, PDGFB, and MAP3K7IP1 on chromosome 22q, HEI10 on chromosome 14q, and succinate dehydrogenase subunit B, E2F2, ARID1A KPNA6, EIF3S2 , PTCH2, and PIK3R3 on chromosome 1p. Regional losses on chromosomes 22q and 1p and gains on chromosomes 12q showed overlaps with those previously observed in uterine leiomyosarcomas. In addition, presence of multiple chromosomal aberrations implies a higher level of genetic instability. Follow-up polymerase chain reaction (PCR) sequencing analysis of MED12 gene revealed absence of G> A transition at nucleotides c.130 or c.131 in all 9 cases, a frequent mutation found in uterine leiomyoma and its variants. In conclusion, this is the first report of high-resolution, genome-wide investigation of IVL by oligonucleotide array comparative genomic hybridization. The presence of high frequencies of recurrent regional loss involving several chromosomes is an important finding and likely related to the pathogenesis of the disease.

Multiple myeloma is an incurable plasma cell malignancy with a complex and incompletely understood molecular pathogenesis. Here we use whole-exome sequencing, copy-number profiling and cytogenetics to analyse 84 myeloma samples. Most cases have a complex subclonal structure and show clusters of subclonal variants, including subclonal driver mutations. Serial sampling reveals diverse patterns of clonal evolution, including linear evolution, differential clonal response and branching evolution. Diverse processes contribute to the mutational repertoire, including kataegis and somatic hypermutation, and their relative contribution changes over time. We find heterogeneity of mutational spectrum across samples, with few recurrent genes. We identify new candidate genes, including truncations of SP140, LTB, ROBO1 and clustered missense mutations in EGR1. The myeloma genome is heterogeneous across the cohort, and exhibits diversity in clonal admixture and in dynamics of evolution, which may impact prognostic stratification, therapeutic approaches and assessment of disease response to treatment.

Several cancer vaccine efforts have been directed to simultaneously cotarget multiple tumor antigens, with the intent to achieve broader immune responses and more effective control of cancer growth. Genetic cancer vaccines based on in vivo muscle electro-gene-transfer of plasmid DNA (DNA-EGT) and adenoviral vectors represent promising modalities to elicit powerful immune responses against tumor-associated antigens (TAAs) such as carcinoembryonic antigen (CEA) and human epidermal growth factor receptor-2 (HER2)/neu. Combinations of these modalities of immunization (heterologous prime-boost) can induce superior immune reactions as compared with single-modality vaccines. We have generated a dual component-dual target genetic cancer vaccine consisting of a DNA moiety containing equal amounts of two plasmids, one encoding the extracellular and transmembrane domains of HER2 (ECD.TM) and the other encoding CEA fused to the B subunit of Escherichia coli heat-labile toxin (LTB), and of an adenoviral subtype 6 dicistronic vector carrying the same two tumor antigens gene constructs. The CEA/HER2 vaccine was tested in two different CEA/HER2 double-transgenic mouse models and in NOD/scid-DR1 mice engrafted with the human immune system. The immune response was measured by enzyme-linked immunospot assay, flow cytometry, and ELISA. The CEA/HER2 vaccine was able to break immune tolerance against both antigens. Induction of a T cell and antibody immune response was detected in immune-tolerant mice. Most importantly, the vaccine was able to slow the growth of HER2/neu⁺ and CEA⁺ tumors. A significant T cell response was measured in NOD/scid-DR1 mice engrafted with human cord blood cells. In conclusion, the CEA/HER2 genetic vaccine was immunogenic and able to confer significant therapeutic effects. These data warrant the evaluation of this vaccination strategy in human clinical trials.

Exposure of the telomere overhang acts as a DNA damage signal, and exogenous administration of an 11-base oligonucleotide homologous to the 3'-telomere overhang sequence (T-oligo) mimics the effects of overhang exposure by inducing senescence and cell death in non-small cell lung cancer (NSCLC) cells, but not in normal bronchial epithelial cells. T-oligo-induced decrease in cellular proliferation in NSCLC is likely directed through both p53 and its homolog, p73, with subsequent induction of senescence and expression of senescence-associated proteins, p21, p33(ING), and p27(Kip1) both in vivo and in vitro. Additionally, T-oligo decreases tumor size and inhibits angiogenesis through decreased VEGF signaling and increased TSP-1 expression.

The role of autophagy in pyocyanin (PCN)-induced toxicity in the central nervous system (CNS) remains unclear, with only evidence from our group identifying it as a mechanism underlying toxicity in 1321N1 astrocytoma cells. Therefore, the aim of this study was to further examine the role of autophagy in PCN-induced toxicity in the CNS. To achieve this, we exposed 1321N1 astrocytoma and SH-SY5Y neuroblastoma cells to PCN (0-100 μmol/L) and tested the contribution of autophagy by measuring the impact of the autophagy inhibitor 3-methyladenine (3-MA) using a series of biochemical and molecular markers. Pretreatment of 1321N1 astrocytoma cells with 3-MA (5 mmol/L) decreased the PCN-induced acidic vesicular organelle and autophagosome formation as measured using acridine orange and green fluorescent protein-LC3 -LC3 fluorescence, respectively. Furthermore, 3-MA (5 mmol/L) significantly protected 1321N1 astrocytoma cells against PCN-induced toxicity. In contrast pretreatment with 3-MA (5 mmol/L) increased PCN-induced toxicity in SH-SY5Y neuroblastoma cells. Given the influence of autophagy in inflammatory responses, we investigated whether the observed effects in this study involved inflammatory mediators. The PCN (100 μmol/L) significantly increased the production of interleukin-8 (IL-8), prostaglandin E2 (PGE₂), and leukotriene B4 (LTB₄) in both cell lines. Consistent with its paradoxical role in modulating PCN-induced toxicity, 3-MA (5 mmol/L) significantly reduced the PCN-induced production of IL-8, PGE₂, and LTB₄ in 1321N1 astrocytoma cells but augmented their production in SH-SY5Y neuroblastoma cells. In conclusion, we show here for the first time the paradoxical role of autophagy in mediating PCN-induced toxicity in 1321N1 astrocytoma and SH-SY5Y neuroblastoma cells and provide novel evidence that these actions may be mediated by effects on IL-8, PGE₂, and LTB₄ production.

Perinuclear reorganization via phosphorylation of specific serine residues in keratin is involved in the deformability of metastatic cancer cells. The level of leukotriene B₄ is high in pancreatic cancers. However, the roles of LTB₄ and its cognate receptors in keratin reorganization of pancreatic cancers are not known. LTB₄ dose-dependently induced phosphorylation and reorganization of Keratin 8 (K8) and these processes were reversed by LY255283 (BLT2 antagonist). BLT2 agonists such as Comp A and 15(S)-HETE also induced phosphorylation of serine 431 in K8. Moreover, Comp A-induced K8 phosphorylation and reorganization were blocked by LY255283. Gene silencing of BLT2 suppressed Comp A-induced K8 phosphorylation and reorganization in PANC-1 cells. Over-expression of BLT2 promoted K8 phosphorylation. Comp A promoted the migration of PANC-1 cells in a dose-dependent manner, and LY255283 blocked Comp A-induced migration, respectively. PD98059 (ERK inhibitor) suppressed Comp A-induced phosphorylation of serine 431 and reorganization of K8. Gene silencing of BLT2 suppressed the expression of pERK, and over-expression of BLT2 increased the expression of pERK even without Comp A. Comp A induced the expression of active ERK (pERK) and BLT2. These inductions were blocked by PD98059. Comp A decreased PP2A expression and hindered the binding of PP2A to the K8, leading to the activation of ERK. PD98059 suppressed the Comp A-induced migration of PANC-1 cells and BLT2 over-expression-induced migration of PANC-1 cells. Overall, these results suggest that BLT2 is involved in LTB(4)-induced phosphorylation and reorganization through ERK activation by PP2A downregulation, leading to increased migration of PANC-1 cells.

BACKGROUND: Inhibitor of growth 1b (ING1b) is considered to be a class II tumor suppressor gene. Although reduced expression of p33(ING1b) has been reported in many human malignancies, including gastric cancers, the effect of p33(ING1b) on gastric cancer cells has yet to be investigated.
METHODS: Expression of p33(ING1b) in gastric adenocarcinoma tissues and their adjacent non-malignant gastric mucosa, as well as in gastric adenocarcinoma cell lines and normal gastric epithelial cells, was detected by using Western blotting. Recombinant adenoviruses were prepared to mediate the ectopic expression of p33(ING1b) (Ad-ING1b) and green fluorescent protein (GFP)(Ad-GFP) in the gastric adenocarcinoma cell lines, SGC-7901, MKN28, and MKN45 and the normal gastric epithelial cell line GES-1. Alterations in the proliferation and apoptosis of the cells after adenoviral infection were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively, and cell cycle distribution was analyzed in a fluorescence-activated cell sorter.
RESULTS: Western blotting confirmed the reduced expression of p33(ING1b) in gastric adenocarcinoma tissues and gastric adenocarcinoma cell lines. The ectopic expression of p33(ING1b) mediated by Ad-ING1b resulted in decreased growth, increased apoptosis, and cell cycle arrest at the G1 phase in both benign and malignant gastric epithelial cells regardless of their p53 status. Addition of a p53 inhibitor, pifithrin-α, did not abolish the pro-apoptotic and cell cycle-arresting effects of p33(ING1b) in p53 wild-type cells.
CONCLUSIONS: Down-regulation of p33(ING1b) might play an important role in the development of gastric adenocarcinoma. Targeted local expression of p33(ING1b) may offer a promising alternative therapeutic measure for gastric cancer.

15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is a key prostaglandin catabolic enzyme catalyzing the oxidation and inactivation of prostaglandin E(2) (PGE(2)) synthesized from the cyclooxygenase (COX) pathway. Accumulating evidence indicates that 15-PGDH may function as a tumor suppressor antagonizing the action of COX-2 oncogene. 15-PGDH has been found to be down-regulated contributing to elevated levels of PGE(2) in most tumors. The expression of 15-PGDH and COX-2 appears to be regulated reciprocally in cancer cells. Down-regulation of 15-PGDH in tumors is due, in part, to transcriptional repression and epigenetic silencing. Numerous agents have been found to up-regulate 15-PGDH by down-regulation of transcriptional repressors and by attenuation of the turnover of the enzyme. Up-regulation of 15-PGDH may provide a viable approach to cancer chemoprevention. Further catabolism of 15-keto-prostaglandin E(2) is catalyzed by 15-keto-prostaglandin-∆(13)-reductase (13-PGR), which also exhibits LTB(4)-12-hydroxydehydrogenase (LTB(4)-12-DH) activity. 13-PGR/LTB(4)-12-DH behaves as a tumor suppressor as well. This review summarizes current knowledge of the expression and function of 15-PGDH and 13-PGR/LTB(4)-12-DH in lung and other tissues during tumor progression. Future directions of research on these prostaglandin catabolic enzymes as tumor suppressors are also discussed.

The purpose of this study was to investigate the expressions of tumor inhibitor of growth (ING1) gene p33ING1, p53, and autophagy-related gene Beclin1 in human non-small cell lung cancer (NSCLC), and the correlation between their expressions with clinical pathological features and clinical significance. The research can provide new ideas and experimental evidence for early diagnosis and biotherapy for NSCLC in the future. The human NSCLC tissues and surrounding non-cancerous tissues were collected from surgical operation. The expressions of mRNA or protein of p33ING1, p53, and Beclin1 were detected by using of reverse transcription polymerase chain reaction or Western blot in these tissues. The results were used to analyze the relationships between these gene expressions with the developing of NSCLC and clinical pathological features. The expressions of mRNA or protein of p33ING1 and Beclin1 in NSCLC tissues were significantly lower than that in surrounding noncancerous tissues (p < 0.05). The expressions of mRNA or protein of p33ING1 and Beclin1 in well- and middle-differentiated NSCLC tissues were lower than those in poor-differentiated NSCLC tissues (p < 0.05). The expressions of mRNA or protein of p33ING1 and Beclin1 in presence of lymph nodes metastasis were lower than those in absence of lymph nodes metastasis (p < 0.05). The expressions of mRNA or protein of p33ING1 and Beclin1 in patients of pathological stage (stages I-II) were higher than those in pathological stage (stages III-IV) (p < 0.05). But the expression of protein of mutant-type p53 in NSCLC tissues was significantly higher than that in surrounding non-cancerous tissues (p < 0.05). The expressions of protein of mutant-type p53 in well- and middle-differentiated NSCLC tissues were higher than those in poor-differentiated NSCLC tissues (p < 0.05). The expressions of protein of mutant-type p53 in presence of lymph nodes metastasis were higher than those in absence of lymph nodes metastasis (p < 0.05). The expressions of protein of mutant-type p53 in patients of pathological stage (stages I-II) were lower than those in pathological stage (stages III-IV) (p < 0.05). These expression changes of p33ING1, p53, and autophagy-related Beclin1 genes were associated with tumor cell differentiation, lymph nodes metastasis, and pathological stage of NSCLC. But these expression changes of these three genes were not associated with gender, age, size of primary carcinoma, histological type of NSCLC (p > 0.05). The expression of mRNA of p53 and Beclin1 were correlated with p33ING1 mRNA expression in NSCLC tissues (p < 0.05). The activity changes of tumor inhibitor of growth, autophagy, and apoptosis may be related to the emergence and the development of NSCLC. The combined detection of p33ING1, p53, and Beclin1 genes and proteins will be helpful for early diagnosis and prognosis judgment for NSCLC, and can provide experimental evidence for biotherapy of NSCLC.

BACKGROUND: It has recently been found that 5-lipoxygenase (5-LO) and cytochrome P450-2J2 (CYP2J2), molecules capable of arachidonic acid (AA) metabolism, might promote cancer cell viability through several mechanisms similar to those of cyclooxygenase-2 (COX-2). We found that not only COX-2 expression, but also the expression of 5-LO and CYP2J2 is up-regulated in head and neck squamous cell carcinoma (HNSCC) cell lines. From these observations, we hypothesized that AA metabolism by 5-LO and/or CYP2J2 may lower the efficacy of anti-cancer effect by COX-2 inhibition.
METHODS AND RESULTS: Although COX-2 was highly expressed in all cell lines tested, COX-2-specific inhibition showed little growth-inhibitory effect in some cell lines. Inhibition of COX-2 resulted in increased production of LTB(4) and 14-15-DHET/EET, metabolites of 5-LO and CYP2J2, respectively. Combined knock-down of COX-2 and 5-LO or CYP2J2 by siRNA results in a decrease in cell proliferation and VEGF production. Furthermore, these results are dependent on 5-LO and CYP2J2 expression in cells.
CONCLUSION: Therefore, combined inhibition of COX-2 and 5-LO or CYP2J2 may be one way to overcome low efficacy of single inhibition of COX-2 in cancer cells. In addition, combined therapies should be chosen based on the expression of members of other AA metabolism pathways.

The incidence rates of urinary bladder cancer continue to rise yearly, and thus new therapeutic approaches and early diagnostic markers for bladder cancer are urgently needed. Thus, identifying the key mediators and molecular mechanisms responsible for the survival of bladder cancer has valuable implications for the development of therapy. In this study, the role of BLT2, a receptor for leukotriene B((4)) (LTB((4))) and 12(S)-hydroxyeicosatetraenoic acid (HETE), in the survival of bladder cancer 253J-BV cells was investigated. We found that the expression of BLT2 is highly elevated in bladder cancer cells. Also, we observed that blockade of BLT2 with an antagonist or BLT2 siRNA resulted in cell cycle arrest and apoptotic cell death, suggesting a role of BLT2 in the survival of human bladder cancer 253J-BV cells. Further experiments aimed at elucidating the mechanism by which BLT2 mediates survival revealed that enhanced level of reactive oxygen species (ROS) are generated via a BLT2-dependent up-regulation of NADPH oxidase members NOX1 and NOX4. Additionally, we observed that inhibition of ROS generation by either NOX1/4 siRNAs or treatment with an ROS-scavenging agent results in apoptotic cell death in 253J-BV bladder cancer cells. These results demonstrated that a 'BLT2-NOX1/4-ROS' cascade plays a role in the survival of this aggressive bladder cancer cells, thus pointing to BLT2 as a potential target for anti-bladder cancer therapy.

Karyotypic analysis and genomic copy number analysis with single nucleotide polymorphism (SNP)-based microarrays were compared with regard to the detection of recurrent genomic imbalances in 20 clear cell renal cell carcinomas (ccRCCs). Genomic imbalances were identified in 19 of 20 tumors by DNA copy number analysis and in 15 tumors by classical cytogenetics. A statistically significant correlation was observed between the number of genomic imbalances and tumor stage. The most common genomic imbalances were loss of 3p and gain of 5q. Other recurrent genomic imbalances seen in at least 15% of tumors included losses of 1p32.3-p33, 6q23.1-qter and 14q and gain of chromosome 7. The SNP-based arrays revealed losses of 3p in 16 of 20 tumors, with the highest frequency being at 3p21.31-p22.1 and 3p24.3-p25.3, the latter encompassing the VHL locus. One other tumor showed uniparental disomy of chromosome 3. Thus, altogether loss of 3p was identified in 17 of 20 (85%) cases. Fourteen tumors showed both overlapping losses of 3p and overlapping gains of 5q, and the karyotypic assessment performed in parallel revealed that these imbalances arose via unbalanced 3;5 translocations. Among the latter, there were common regions of loss at 3p21.3-pter and gain at 5q34-qter. These data suggest that DNA copy number analysis will supplant karyotypic analysis of tumor types such as ccRCC that are characterized by recurrent genomic imbalances, rather than balanced rearrangements. These findings also suggest that the 5q duplication/3p deficiency resulting from unbalanced 3;5 translocations conveys a proliferative advantage of particular importance in ccRCC tumorigenesis.

We studied a series of 68 subjects diagnosed with childhood acute myeloid leukemia (AML) using conventional cytogenetics and fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR) to analyze mutations in FLT3 and NPM1 genes, and/or array comparative genomic hybridization (CGH). Cytogenetic/FISH abnormalities were observed in 71% of subjects, FLT3-ITD mutations in 15%, and NPM1 mutations in 13%. The array CGH alterations (average 3.6 per case) were observed in 96% of the tested subjects. The most frequent alterations were gains of 8q24.3 and 11p15.5-p15.4 in 16% of the samples. Six genes (AKT1, RUNX1, LTB, SDC1, RUNX1T1, and JAK2) from the imbalanced regions have been reported to be involved in AML, whereas other 30 cancer genes, not previously reported in an AML context, were identified as imbalanced. They probably correspond to non passenger alterations that cooperate with the recurrent translocations. The clinical data and genetic changes were tested to find out the possible association with prognosis. Genomic instability (four or more genomic imbalances) was correlated with poor patient outcome (p = 0.029).

Hepatitis B and C viruses (HBV and HCV) cause chronic hepatitis and hepatocellular carcinoma (HCC) by poorly understood mechanisms. We show that cytokines lymphotoxin (LT) alpha and beta and their receptor (LTbetaR) are upregulated in HBV- or HCV-induced hepatitis and HCC. Liver-specific LTalphabeta expression in mice induces liver inflammation and HCC, causally linking hepatic LT overexpression to hepatitis and HCC. Development of HCC, composed in part of A6(+) oval cells, depends on lymphocytes and IKappa B kinase beta expressed by hepatocytes but is independent of TNFR1. In vivo LTbetaR stimulation implicates hepatocytes as the major LT-responsive liver cells, and LTbetaR inhibition in LTalphabeta-transgenic mice with hepatitis suppresses HCC formation. Thus, sustained LT signaling represents a pathway involved in hepatitis-induced HCC.

Leukotriene B(4)-12-hydroxydehydrogenase/15-oxo-prostaglandin 13-reductase (LTBDH/PGR) is a bifunctional enzyme capable of inactivating leukotriene B(4) (LTB(4)) and 15-oxo-prostaglandins (15-PGs). Its role in growth suppressive functions in lung cancer was studied in in vitro and in vivo systems. The LTBDH/PGR gene was expressed in lung cancer cell lines through recombinant adenovirus infection, and through a tetracycline-inducible expression system. After restoration of LTBDH/PGR expression in LTBDH/PGR-negative (H1299) or -low (A549) lung cancer cell lines, the restored enzyme induced apoptosis and growth inhibition in vitro. Ectopic expression of LTBDH/PGR caused also suppression of tumorigenicity of A549 cells in nude mice. In contrast, LTBDH/PGR over-expression in LTBDH/PGR-positive (H157) lung cancer cell line induced little apoptosis and growth inhibition. This study indicates that restoration of LTBDH/PGR expression is effective in preventing lung cancer growth in vitro and in vivo.