Integrin β1-dependent invasive migration of irradiation-tolerant human lung adenocarcinoma cells in 3D collagen matrix

Radiotherapy is one of the effective therapies used for treating various malignant tumors. However, the emergence of tolerant cells after irradiation remains problematic due to their high metastatic ability, sometimes indicative of poor prognosis. In this study, we showed that subcloned human lung adenocarcinoma cells (A549P-3) that are irradiation-tolerant indicate high invasive activity in vitro, and exhibit an integrin β1 activity-dependent migratory pattern. In collagen gel overlay assay, majority of the A549P-3 cells displayed round morphology and low migration activity, whereas a considerable number of A549P-3IR cells surviving irradiation displayed a spindle morphology and high migration rate. Blocking integrin β1 activity reduced the migration rate of A549P-3IR cells and altered the cell morphology allowing them to assume a round shape. These results suggest that the A549P-3 cells surviving irradiation acquire a highly invasive integrin β1-dependent phenotype, and integrin β1 might be a potentially effective therapeutic target in combination with radiotherapy.     

小鼠肺腺癌模型的建立及肿瘤病理分析

目的用乙基亚硝脲（ENU）在BABL/c小鼠建立肺腺癌模型并对ENU所诱发的肺腺癌进行病理观察。方法妊娠17d的SPF级母鼠腹腔接受ENU或缓冲液注射,在子代鼠的鼠龄满32周时收获其全肺标本,对肺组织进行常规石蜡半连续切片,HE染色,镜下观察肿瘤病理。结果 ENU经胎盘一次性诱发子代鼠多发性肺肿瘤形成,病理显示这些肿瘤为处于不同发展阶段的腺瘤和腺癌,腺癌的类型有细支气管肺泡癌样腺癌（雌性：5/6,雄性：4/6）和分化不等的腺癌（雌性：4/6,雄性：5/6）,诱癌频率在雌、雄性小鼠均为5/6,癌变频率在雌性16/43,雄性12/31。结论成功建立了小鼠肺腺癌模型,肿瘤病理的多样性提示癌变机制在分子水平的复杂性。     

S100A4 is frequently overexpressed in lung cancer cells and promotes cell growth and cell motility

S100A4, a small calcium-binding protein belonging to the S100 protein family, is commonly overexpressed in a variety of tumor types and is widely accepted to associate with metastasis by regulating the motility and invasiveness of cancer cells. However, its biological role in lung carcinogenesis is largely unknown. In this study, we found that S100A4 was frequently overexpressed in lung cancer cells, irrespective of histological subtype. Then we performed knockdown and forced expression of S100A4 in lung cancer cell lines and found that specific knockdown of S100A4 effectively suppressed cell proliferation only in lung cancer cells with S100A4-overexpression; forced expression of S100A4 accelerated cell motility only in S100A4 low-expressing lung cancer cells. PRDM2 and VASH1, identified as novel upregulated genes by microarray after specific knockdown of S100A4 in pancreatic cancer, were also analyzed, and we found that PRDM2 was significantly upregulated after S100A4-knockdown in one of two analyzed S100A4-overexpressing lung cancer cells. Our present results suggest that S100A4 plays an important role in lung carcinogenesis by means of cell proliferation and motility by a pathway similar to that in pancreatic cancer.     

Toll-like receptor 4 signaling plays a role in triggering periodontal infection

Toll-like receptors (TLRs) are a group of sensors on the surface of antigen-presenting cells, such as dendritic cells and macrophages, which recognize microbial pathogens and induce innate and adaptive immune responses. Periodontitis is an inflammatory disease characterized by the destruction of tooth-supporting structures. In order to address whether TLR4 signaling plays a role in periodontitis, we studied the gene expression change in human periodontal ligament cells (HPDLCs) in response to TLR4 ligand, lipopolysaccharide treatment by microarray analysis. Expression of TLR4 was detected in HPDLCs. Lipopolysaccharide treatment increased the expression of 12 genes (more than twofold), including TLR4, TLR5, TLR7, Pellino 1, colony stimulating factor 2 (CSF2) and IL-6. In addition, the expression of 15 genes (less than equal to twofold) was decreased, including Fos, LY64 and LY86. In addition, real-time PCR was used to confirm the change of gene expression of TLR4, IL-6 and Fos. We also showed that the upregulation of IL-6 by lipopolysaccharide treatment was TLR4-dependent. This pattern of gene expression indicates that pathogens may trigger TLR4 signaling and cause periodontitis. Manipulating TLR4 signaling may potentially become one of the recognized therapies for periodontitis.     

Phenylalanine 93 of the human UGT1A10 plays a major role in the interactions of the enzyme with estrogens

Little is currently known about the substrate binding site of the human UDP-glucuronosyltransferases (UGTs) and the structural elements that affect their complex substrate selectivity. In order to further understand and extend our earlier findings with phenylalanines 90 and 93 of UGT1A10, we have replaced each of them with Gly, Ala, Val, Leu, Ile or Tyr, and tested the activity of the resulting 12 mutants toward eight different substrates. Apart from scopoletin glucuronidation, the F90 mutants other than F90L were nearly inactive, while the F93 mutants’ activity was strongly substrate dependent. Hence, F93L displayed high entacapone and 1-naphthol glucuronidation rates, whereas F93G, which was nearly inactive in entacapone glucuronidation, was highly active toward estradiol, estriol and even ethinylestradiol, a synthetic estrogen that is a poor substrate for the wild-type UGT1A10. Kinetic analyses of 4-nitrophenol, estradiol and ethinylestradiol glucuronidation by the mutants that catalyzed the respective reactions at considerable rates, revealed increased K_m values for 4-nitrophenol and estradiol in all the mutants, whilst the K_m values of F93G and F93A for ethinylestradiol were lower than in control UGT1A10. Based on the activity results and a new molecular model of UGT1A10, it is suggested that both F90 and F93 are located in a surface helix at the far end of the substrate binding site. Nevertheless, only F93 directly affects the selectivity of UGT1A10 toward large and rigid estrogens, particularly those with substitutions at the D ring. The effects of F93 mutations on the glucuronidation of smaller or less rigid substrates are indirect, however.     

Plectin regulates the signaling and trafficking of the HIV-1 co-receptor CXCR4 and plays a role in HIV-1 infection

The CXC chemokine CXCL12 and its cognate receptor CXCR4 play an important role in inflammation, human immunodeficiency virus (HIV) infection and cancer metastasis. The signal transduction and intracellular trafficking of CXCR4 are involved in these functions, but the underlying mechanisms remain incompletely understood. In the present study, we demonstrated that the CXCR4 formed a complex with the cytolinker protein plectin in a ligand-dependent manner in HEK293 cells stably expressing CXCR4. The glutathione-S-transferase (GST)-CXCR4 C-terminal fusion proteins co-precipitated with the full-length and the N-terminal fragments of plectin isoform 1 but not with the N-terminal deletion mutants of plectin isoform 1, thereby suggesting an interaction between the N-terminus of plectin and the C-terminus of CXCR4. This interaction was confirmed by confocal microscopic reconstructions showing co-distribution of these two proteins in the internal vesicles after ligand-induced internalization of CXCR4 in HEK293 cells stably expressing CXCR4. Knockdown of plectin with RNA interference (RNAi) significantly inhibited ligand-dependent CXCR4 internalization and attenuated CXCR4-mediated intracellular calcium mobilization and activation of extracellular signal regulated kinase 1/2 (ERK1/2). CXCL12-induced chemotaxis of HEK293 cells stably expressing CXCR4 and of Jurkat T cells was inhibited by the plectin RNAi. Moreover, CXCR4 tropic HIV-1 infection in MAGI (HeLa-CD4-LTR-Gal) cells was inhibited by the RNAi of plectin. Thus, plectin appears to interact with CXCR4 and plays an important role in CXCR4 signaling and trafficking and HIV-1 infection.     

Construction of a mitochondrial-associated protein DRP1 and a lung cancer-associated protein Erbb4 combined regulatory network

ObjectiveThis research was to establish a mitochondrial-related Drp1 gene and a lung cancer-related Erbb4 gene to participate in the regulatory network of lung cancer cell apoptosis, and to provide theoretical support for mitochondria to participate in tumor regulation.MethodThe GO and KEGG methods were used to construct the regulatory networks of lung cancer related Drp1 and Erbb4 proteins that involved in the apoptosis of tumor cells, and to combine with the Bayesian network theory to screen out the largest possible action path acting on this network; The information about Drp1 in Oncomine database was collected, and the data in current database were analyzed twice. The role of Drp1 in lung cancer was meta-analyzed.ResultA regulatory network of Drp1 and Erbb4 involved in the apoptosis of tumor cells was successfully constructed; the optimal pathway was optimized using Bayesian theory; a total of 446 different types of research results were collected in the Oncomine database, of which there were 18 studies with statistical differences in Drp1 expression, 13 studies with increased Drp1’s expression, and 5 studies with decreased expression. Compared with the control group, Drp1 was expressed in lung cancer tissues highly (P < 0.05).ConclusionEstablishment and optimization of mitochondrial-related Drp1 and tumor-related Erbb4 genes involved in the regulation of apoptosis of cancer cells. It was proposed that Drp1 was expressed in lung cancer tissues highly through in-depth excavation of tumor-associated gene information in the Oncomine gene chip database.     

MicroRNA-133 regulates the expression of GLUT4 by targeting KLF15 and is involved in metabolic control in cardiac myocytes

GLUT4 shows decreased levels in failing human adult hearts. We speculated that GLUT4 expression in cardiac muscle may be fine-tuned by microRNAs. Forced expression of miR-133 decreased GLUT4 expression and reduced insulin-mediated glucose uptake in cardiomyocytes. A computational miRNA target prediction algorithm showed that KLF15 is one of the targets of miR-133. It was confirmed that over-expression of miR-133 reduced the protein level of KLF15, which reduced the level of the downstream target GLUT4. Cardiac myocytes infected with lenti-decoy, in which the 3′UTR with tandem sequences complementary to miR-133 was linked to the luciferase reporter gene, had decreased miR-133 levels and increased levels of GLUT4. The expression levels of KLF15 and GLUT4 were decreased at the left ventricular hypertrophy and congestive heart failure stage in a rat model. The present results indicated that miR-133 regulates the expression of GLUT4 by targeting KLF15 and is involved in metabolic control in cardiomyocytes.     

SNP rs10248565 in HDAC9 as a novel genomic aberration biomarker of lung adenocarcinoma in non-smoking women

Background

Numerous efforts have been made to elucidate the etiology and improve the treatment of lung cancer, but the overall five-year survival rate is still only 15%. Although cigarette smoking is the primary risk factor for lung cancer, only 7% of female lung cancer patients in Taiwan have a history of smoking. Since cancer results from progressive accumulation of genetic aberrations, genomic rearrangements may be early events in carcinogenesis.

Results

In order to identify biomarkers of early-stage adenocarcinoma, the genome-wide DNA aberrations of 60 pairs of lung adenocarcinoma and adjacent normal lung tissue in non-smoking women were examined using Affymetrix Genome-Wide Human SNP 6.0 arrays. Common copy number variation (CNV) regions were identified by ≥30% of patients with copy number beyond 2 ± 0.5 of copy numbers for each single nucleotide polymorphism (SNP) and at least 100 continuous SNP variant loci. SNPs associated with lung adenocarcinoma were identified by McNemar’s test. Loss of heterozygosity (LOH) SNPs were identified in ≥18% of patients with LOH in the locus. Aberration of SNP rs10248565 at HDAC9 in chromosome 7p21.1 was identified from concurrent analyses of CNVs, SNPs, and LOH.

Conclusion

The results elucidate the genetic etiology of lung adenocarcinoma by demonstrating that SNP rs10248565 may be a potential biomarker of cancer susceptibility.     

NTF4 plays a dual role in breast cancer in mammary tumorigenesis and metastatic progression

Breast cancer metastasis can happen even when the primary tumor is relatively small. But the mechanism for such early metastasis is poorly understood. Herein, we report that neurotrophin 4 (NTF4) plays a dual role in breast cancer proliferation and metastasis. Clinical data showed high levels of NTF4, especially in the early stage, to be associated with poor clinical outcomes, supporting the notion that metastasis, rather than primary cancer, was the major determinant of breast cancer mortality for patients. NTF4 promoted epithelial-mesenchymal transition (EMT), cell motility, and invasiveness of breast cancer cells in vitro and in vivo. Interestingly, NTF4 inhibited cell proliferation while promoting cellular apoptosis in vitro and inhibited xenograft tumorigenicity in vivo. Mechanistically, NTF4 elicited its pro-metastatic effects by activating PRKDC/AKT and ANXA1/NF-κB pathways to stabilize SNAIL protein, therefore decreasing the level of E-cadherin. Conversely, NTF4 increased ANXA1 phosphorylation and sumoylation and the interaction with importin β, leading to nuclear import and retention of ANXA1, which in turn activates the caspase-3 apoptosis cascade. Our findings identified an unexpected dual role for NTF4 in breast cancer which contributes to early metastasis of the disease. Therefore, NTF4 may serve as a prognostic marker and a potential therapeutic target for breast cancer.     

BCAR1 plays critical roles in the formation and immunoevasion of invasive circulating tumor cells in lung adenocarcinoma

Background: We investigated the roles of breast cancer anti-estrogen resistance 1 (BCAR1/p130Cas) in the formation and immunoevasion of invasive circulating tumor cells (CTCs) in lung adenocarcinoma (LUAD).Methods: Biomarkers of CTCs including BCAR1 and CD274, were evaluated by the CanPatrol method. Proteomics analysis of LUAD cells and exosomes after BCAR1 overexpression (BCAR1-OE) was performed by mass spectrometry. Cell functions and relevant signaling pathways were investigated after BCAR1 knockdown (BCAR1-KO) or BCAR1-OE in LUAD cells. Lastly, in vitro and in vivo experiments were performed to confirm the roles of BCAR1 in the formation and immunoevasion of CTCs.Results:High expression of BCAR1 by CTCs correlated with CD274 expression and epithelial-to-mesenchymal transition (EMT). RAC1, together with BCAR1, was found to play an important role in the carcinogenesis of LUAD. RAC1 functioned with BCAR1 to induce EMT and to enhance cell proliferation, colony formation, cell invasion and migration, and anoikis resistance in LUAD cells. BCAR1 up-regulated CD274 expression probably by shuttling the short isoform of BRD4 (BRD4-S) into the nucleus. CTCs, as well as tumor formation, were prohibited in nude mice xenografted with BCAR1-KO cells. The co-expression of BCAR1/RAC1 and BCAR1/CD274 was confirmed in LUAD. BCAR1 expression in LUAD is an indicator of poor prognosis, and it associates with immunoevasion.Conclusion:BCAR1, as a new target for the treatment of LUAD, plays roles in the formation and immunoevasion of invasive CTCs. The mechanism includes triggering EMT via RAC1 signaling and up-regulating CD274 expression by shuttling BRD4-S into the nucleus.     

Protein disulfide isomerase activity is essential for viability and extracellular matrix formation in the nematode Caenorhabditis elegans

Protein disulfide isomerase (PDI) is a multifunctional protein required for many aspects of protein folding and transit through the endoplasmic reticulum. A conserved family of three PDIs has been functionally analysed using genetic mutants of the model organism Caenorhabditis elegans. PDI-1 and PDI-3 are individually non-essential, whereas PDI-2 is required for normal post-embryonic development. In combination, all three genes are synergistically essential for embryonic development in this nematode. Mutations in pdi-2 result in severe body morphology defects, uncoordinated movement, adult sterility, abnormal molting and aberrant collagen deposition. Many of these phenotypes are consistent with a role in collagen biogenesis and extracellular matrix formation. PDI-2 is required for the normal function of prolyl 4-hydroxylase, a key collagen-modifying enzyme. Site-directed mutagenesis indicates that the independent catalytic activity of PDI-2 may also perform an essential developmental function. PDI-2 therefore performs two critical roles during morphogenesis. The role of PDI-2 in collagen biogenesis can be restored following complementation of the mutant with human PDI.     

P120-catenin isoforms 1A and 3A differently affect invasion and proliferation of lung cancer cells

Different isoforms of p120-catenin (p120ctn), a member of the Armadillo gene family, are variably expressed in different tissues as a result of alternative splicing and the use of multiple translation initiation codons. When expressed in cancer cells, these isoforms may confer different properties with respect to cell adhesion and invasion. We have previously reported that the p120ctn isoforms 1 and 3 were the most highly expressed isoforms in normal lung tissues, and their expression level was reduced in lung tumor cells. To precisely define their biological roles, we transfected p120ctn isoforms 1A and 3A into the lung cancer cell lines A549 and NCI-H460. Enhanced expression of p120ctn isoform 1A not only upregulated E-cadherin and β-catenin, but also downregulated the Rac1 activity, and as a result, inhibited the ability of cells to invade. In contrast, overexpression of p120ctn isoform 3A led to the inactivation of Cdc42 and the activation of RhoA, and had a smaller influence on invasion. However, we found that isoform 3A had a greater ability than isoform 1A in both inhibiting the cell cycle and reducing tumor cell proliferation. The present study revealed that p120ctn isoforms 1A and 3A differently regulated the adhesive, proliferative, and invasive properties of lung cancer cells through distinct mechanisms.     

The iron-induced cysteine proteinase TvCP4 plays a key role in Trichomonas vaginalis haemolysis

Trichomonas vaginalis has multiple proteinases, mainly of the cysteine type (CPs), including a 34 kDa precursor cathepsin L-like CP dubbed TvCP4. TvCP4 is an iron-up-regulated CP. The goal of this work was to identify the role of TvCP4 in the virulence of T. vaginalis. We cloned, expressed, and purified the recombinant mature enzyme region of TvCP4 (TvCP4r) to produce a rabbit polyclonal antibody (α-TvCP4r). This antibody reacted with a ∼24 kDa protein band in total protein extracts that could correspond to the mature enzyme. By two-dimensional western blot assays TvCP4 corresponded to three protein spots of ∼24 kDa with pI values of ∼6.7, 6.9, and 7.0 and two spots of ∼22 and ∼21 kDa with a pI of 6.9, as confirmed by mass spectrometry. As expected, a higher amount of TvCP4 was detected in cytoplasmic vesicles, lysosomes, and on the surface of iron-rich parasites when compared with normal and iron-depleted parasites. The α-TvCP4r antibody protected human erythrocytes from trichomonal lysis. Additionally, TvCP4 is expressed during infection and is part of the released products detected in vaginal fluids of patients with trichomonosis. Thus, data show that TvCP4 is an iron-induced CP that participates in T. vaginalis haemolysis.     

Identification of KIF4A and its effect on the progression of lung adenocarcinoma based on the bioinformatics analysis

Background: Lung adenocarcinoma (LUAD) is the most frequent histological type of lung cancer, and its incidence has displayed an upward trend in recent years. Nevertheless, little is known regarding effective biomarkers for LUAD.Methods: The robust rank aggregation method was used to mine differentially expressed genes (DEGs) from the gene expression omnibus (GEO) datasets. The Search Tool for the Retrieval of Interacting Genes (STRING) database was used to extract hub genes from the protein–protein interaction (PPI) network. The expression of the hub genes was validated using expression profiles from TCGA and Oncomine databases and was verified by real-time quantitative PCR (qRT-PCR). The module and survival analyses of the hub genes were determined using Cytoscape and Kaplan–Meier curves. The function of KIF4A as a hub gene was investigated in LUAD cell lines.Results: The PPI analysis identified seven DEGs including BIRC5, DLGAP5, CENPF, KIF4A, TOP2A, AURKA, and CCNA2, which were significantly upregulated in Oncomine and TCGA LUAD datasets, and were verified by qRT-PCR in our clinical samples. We determined the overall and disease-free survival analysis of the seven hub genes using GEPIA. We further found that CENPF, DLGAP5, and KIF4A expressions were positively correlated with clinical stage. In LUAD cell lines, proliferation and migration were inhibited and apoptosis was promoted by knocking down KIF4A expression.Conclusion: We have identified new DEGs and functional pathways involved in LUAD. KIF4A, as a hub gene, promoted the progression of LUAD and might represent a potential therapeutic target for molecular cancer therapy.     

Esophageal cancer related gene 4 (ECRG4) is a marker of articular chondrocyte differentiation and cartilage destruction

With the aim of identifying novel genes regulating cartilage development and degeneration, we screened a cartilage-specific expressed sequence tag database. Esophageal cancer related gene 4 (ECRG4) was selected, based on the criteria of ‘chondrocyte-specific’ and ‘unknown function.’ ECRG4 expression was particularly abundant in chondrocytes and cartilage, compared to various other mouse tissues. ECRG4 is a secreted protein that undergoes cleavage after secretion. The protein is specifically expressed in chondrocytes in a manner dependent on differentiation status. The expression is very low in mesenchymal cells, and dramatically increased during chondrogenic differentiation. The ECRG4 level in differentiated chondrocytes is decreased during hypertrophic maturation, both in vitro and in vivo, and additionally in dedifferentiating chondrocytes induced by interleukin-1β or serial subculture, chondrocytes of human osteoarthritic cartilage and experimental mouse osteoarthritic cartilage. However, ectopic expression or exogenous ECRG4 treatment in a primary culture cell system does not affect chondrogenesis of mesenchymal cells, hypertrophic maturation of chondrocytes or dedifferentiation of differentiated chondrocytes. Additionally, cartilage development and organization of extracellular matrix are not affected in transgenic mice overexpressing ECRG4 in cartilage tissue. However, ectopic expression of ECRG4 reduced proliferation of primary culture chondrocytes. While the underlying mechanisms of ECRG4 expression and specific roles remain to be elucidated in more detail, our results support its function as a marker of differentiated articular chondrocytes and cartilage destruction.