Identification of EPHX2 and RMI2 as two novel key genes in cervical squamous cell carcinoma by an integrated bioinformatic analysis

Cervical cancer is the fourth most common malignancy in women worldwide and cervical squamous cell carcinoma (CESC) is the most common histological type of cervical cancer. The dysregulation of genes plays a significant role in cancer. In the present study, we screened out differentially expressed genes (DEGs) of CESC in the GSE63514 data set from the Gene Expression Omnibus database. An integrated bioinformatics analysis was used to select hub genes, as well as to investigate their related prognostic signature, functional annotation, methylation mechanism, and candidate molecular drugs. As a result, a total of 1907 DEGs were identified (944 were upregulated and 963 were downregulated). In the protein–protein interaction network, three hub modules and 30 hub genes were identified. And two hub modules and 116 hub genes were screened out from four CESC-related modules by the weighted gene coexpression network analysis. The gene ontology term enrichment analysis and Kyoto encyclopedia of genes and genomes pathway analysis were performed to better understand functions and pathways. Genes with a significant prognostic value were found by prognostic signature analysis. And there were five genes (EPHX2, CHAF1B, KIAA1524, CDC45, and RMI2) identified as significant CESC-associated genes after expression validation and survival analysis. Among them, EPHX2 and RMI2 were noted as two novel key genes for the CESC-associated methylation and expression. In addition, four candidate small molecule drugs for CESC (camptothecin, resveratrol, vorinostat, and trichostatin A) were defined. Further studies are required to explore these significant CESC-associated genes for their potentiality in diagnosis, prognosis, and targeted therapy.     

Proteinase-activated Receptor 2 Promotes Cancer Cell Migration through RNA Methylation-mediated Repression of miR-125b

Proteinase activated-receptor 2 (PAR₂) participates in cancer metastasis promoted by serine proteinases. The current study aimed to test the molecular mechanism by which PAR₂ promotes cancer cell migration. In different cancer cells, activation of PAR₂ by activating peptide (PAR₂-AP) dramatically increased cell migration, whereas knock down of PAR₂ inhibited cellular motility. The PAR₂ activation also repressed miR-125b expression while miR-125b mimic successfully blocked PAR₂-induced cell migration. Moreover, Grb associated-binding protein 2 (Gab2) was identified as a novel target gene of miR-125b and it mediated PAR₂-induced cell migration. The correlation of PAR₂ with miR-125b and Gab2 was further supported by the findings obtained from human colorectal carcinoma specimens. Remarkably, knock down of NOP2/Sun domain family, member 2 (NSun2), a RNA methyltransferase, blocked the reduction in miR-125b induced by PAR₂. Furthermore, PAR₂ activation increased the level of N⁶-methyladenosine (m⁶A)-containing pre-miR-125b in NSun2-dependent manner. Taken together, our results demonstrated that miR-125b mediates PAR₂-induced cancer cell migration by targeting Gab2 and that NSun2-dependent RNA methylation contributes to the down-regulation of miR-125b by PAR₂ signaling. These findings suggest a novel epigenetic mechanism by which microenvironment regulates cancer cell migration by altering miRNA expression.     

Delayed production of arachidonic acid contributes to the delay of proteinase-activated receptor-1 (PAR1)-triggered prostaglandin E2 release in rat gastric epithelial RGM1 cells

Proteinase-activated receptor-1 (PAR1), upon activation, exerts prostanoid-dependent gastroprotection, and increases prostaglandin E(2) (PGE(2)) release through cyclooxygenase-2 (COX-2) upregulation in rat gastric mucosal epithelial RGM1 cells. However, there is a big time lag between the PAR1-triggered PGE(2) release and COX-2 upregulation in RGM1 cells; that is, the former event takes 18 h to occur, while the latter rapidly develops and reaches a plateau in 6 h. The present study thus aimed at clarifying mechanisms for the delay of PGE(2) release after PAR1 activation in RGM1 cells. Although a PAR1-activating peptide, TFLLR-NH(2), alone caused PGE(2) release at 18 h, but not 6 h, TFLLR-NH(2) in combination with arachidonic acid dramatically enhanced PGE(2) release even for 1-6 h. TFLLR-NH(2) plus linoleic acid caused a similar rapid response. CP-24879, a Δ(5)/Δ(6)-desaturase inhibitor, abolished the PGE(2) release induced by TFLLR-NH(2) plus linoleic acid, but not by TFLLR-NH(2) alone. The TFLLR-NH(2)-induced PGE(2) release was not affected by inhibitors of cytosolic phospholipase A(2) (cPLA(2)), Ca(2+)-independent PLA(2) (cPLA(2)) or secretory PLA(2) (sPLA(2)), but was abolished by their mixture or a pan-PLA(2) inhibitor. Among PLA(2) isozymes, mRNA of group IIA sPLA(2) (sPLA(2)-IIA) was upregulated following PAR1 stimulation for 6-18 h, whereas protein levels of PGE synthases were unchanged. These data suggest that the delay of PGE(2) release after COX-2 upregulation triggered by PAR1 is due to the poor supply of free arachidonic acid at the early stage in RGM1 cells, and that plural isozymes of PLA(2) including sPLA(2)-IIA may complementarily contribute to the liberation of free arachidonic acid.     

miR-125a/b inhibits tumor-associated macrophages mediated in cancer stem cells of hepatocellular carcinoma by targeting CD90

Cancer stem cells promote tumorigenesis and progression of hepatocellular carcinoma (HCC). Recently, emerging evidence indicates tumor-associated macrophages (TAMs) play an important role in tumor progression. However, TAMs often occurs with unknown mechanisms. As an important mediator in intercellular communications, exosomes secreted by host cells mediate the exchange of genetic materials and proteins, which involves tumor aggressiveness. The aim of the study was to investigate whether exosomes derived from TAMs mediate stem cell properties in HCC. TAMs were isolated from the tissues of HCC. microRNA (miRNA) expression profiles of TAMs were analyzed using miRNA microarray. In vitro cell coculture was further conducted to investigate the crosstalk between TAMs and tumor cells mediated by TAMs exosomes. In this study, we showed that TAMs exosomes promote HCC cell proliferation and stem cell properties. Using miRNA profiles assay, we identified significantly lower levels of miR-125a and miR-125b in exosomes and cell lysate isolated from TAMs. Functional studies revealed that the HCC cells were treated with TAM exosomes or transfected with miR-125a/b suppressed cell proliferation and stem cell properties by targeting CD90, a stem cell marker of HCC stem cells. The study indicated that miR-125a/b targeting CD90 played important roles in cancer stem cells of HCC.     

Neutrophil Elastase Activates Protease-activated Receptor-2 (PAR2) and Transient Receptor Potential Vanilloid 4 (TRPV4) to Cause Inflammation and Pain

Proteases that cleave protease-activated receptor-2 (PAR₂) at Arg³⁶↓Ser³⁷ reveal a tethered ligand that binds to the cleaved receptor. PAR₂ activates transient receptor potential (TRP) channels of nociceptive neurons to induce neurogenic inflammation and pain. Although proteases that cleave PAR₂ at non-canonical sites can trigger distinct signaling cascades, the functional importance of the PAR₂-biased agonism is uncertain. We investigated whether neutrophil elastase, a biased agonist of PAR₂, causes inflammation and pain by activating PAR₂ and TRP vanilloid 4 (TRPV4). Elastase cleaved human PAR₂ at Ala⁶⁶↓Ser⁶⁷ and Ser⁶⁷↓Val⁶⁸_. Elastase stimulated PAR₂-dependent cAMP accumulation and ERK1/2 activation, but not Ca²⁺ mobilization, in KNRK cells. Elastase induced PAR₂ coupling to Gα_s but not Gα_q in HEK293 cells. Although elastase did not promote recruitment of G protein-coupled receptor kinase-2 (GRK2) or β-arrestin to PAR₂, consistent with its inability to promote receptor endocytosis, elastase did stimulate GRK6 recruitment. Elastase caused PAR₂-dependent sensitization of TRPV4 currents in Xenopus laevis oocytes by adenylyl cyclase- and protein kinase A (PKA)-dependent mechanisms. Elastase stimulated PAR₂-dependent cAMP formation and ERK1/2 phosphorylation, and a PAR₂- and TRPV4-mediated influx of extracellular Ca²⁺ in mouse nociceptors. Adenylyl cyclase and PKA-mediated elastase-induced activation of TRPV4 and hyperexcitability of nociceptors. Intraplantar injection of elastase to mice caused edema and mechanical hyperalgesia by PAR₂- and TRPV4-mediated mechanisms. Thus, the elastase-biased agonism of PAR₂ causes Gα_s-dependent activation of adenylyl cyclase and PKA, which activates TRPV4 and sensitizes nociceptors to cause inflammation and pain. Our results identify a novel mechanism of elastase-induced activation of TRPV4 and expand the role of PAR₂ as a mediator of protease-driven inflammation and pain.     

Tumor-suppressive microRNA-10a inhibits cell proliferation and metastasis by targeting Tiam1 in esophageal squamous cell carcinoma

Aberrant microRNAs (miRNAs) expressions could contribute to the progression of numerous cancers, including esophageal squamous cell carcinoma, while miR-10a participates in multiple biological processes on cancers. However, the molecular mechanism of miR-10a in esophageal squamous cell carcinoma (ESCC) has not been investigated. Herein, miR-10a was significantly reduced in ESCC clinical tissues and ESCC cell lines (EC109 and TE-3). In addition, immunohistochemistry indicated that the expressions of α-SMA, Ki-67, and PCNA in tumor tissues were higher than that of controls. In vitro, overexpression of miR-10a dramatically suppressed cell proliferation and enhanced cell apoptosis, while the decrease of miR-10a expressed the opposite outcome. Specially, overexpression of miR-10a caused a G0/G1 peak accumulation. Moreover, miR-10a also negatively regulated ESCC cell migration and invasion. Furthermore, targetscan bioinformatics predictions and the dual-luciferase assay confirmed that Tiam1 was a direct target gene of miR-10a. The statistical analysis showed Tiam1 was negatively in correlation with miR-10a in ESCC patient samples. And silencing Tiam1 could lead to a decline on cell growth, invasion, and migration in ESCC cell lines, while it could enhance cell apoptosis and cause a G0/G1 peak accumulation. In vivo, it revealed that miR-10a notably decreased the tumor growth and metastasis in xenograft model and pulmonary metastasis model. And it showed a lower expressions of Tiam1 in the miR-10a mimics group by immunohistochemistry. Taken together the results, they indicated that miR-10a might function as a novel tumor suppressor in vitro and in vivo via targeting Tiam1, suggesting miR-10a to be a candidate biomarker for the ESCC therapy.     

Redox regulation of human protease-activated receptor-2 by activated factor X

Activated factor X (FXa) exerts coagulation-independent actions such as proliferation of vascular smooth muscle cells (SMCs) through the protease-activated receptors PAR-1 and PAR-2. Both receptors are upregulated upon vascular injury but the underlying mechanisms have not been defined. We examined if FXa regulates PAR-1 and PAR-2 in human vascular SMCs. FXa increased PAR-2 mRNA, protein, and cell-surface expression and augmented PAR-2-mediated mitogenesis. PAR-1 was not influenced. The regulatory action of FXa on PAR-2 was concentration-dependent and mimicked by a PAR-2-selective activating peptide. PAR-2 regulation was not influenced by the thrombin inhibitor argatroban or PAR-1 siRNA. FXa increased dichlorofluorescein diacetate fluorescence and 8-isoprostane formation and induced expression of the NADPH oxidase subunit NOX-1. NOX-1 siRNA prevented FXa-stimulated PAR-2 regulation, as did ebselen and cell-permeative and impermeative forms of catalase. Exogenous H₂O₂ increased PAR-2 expression and mitogenic activity. FXa promoted nuclear translocation and PAR-2/DNA binding of nuclear factor κB (NF-κB); NF-κB inhibition prevented PAR-2 regulation by FXa. FXa also promoted PAR-2 mRNA stabilization through increased human antigen R (HuR)/PAR-2 mRNA binding and cytoplasmic shuttling. HuR siRNA abolished FXa-stimulated PAR-2 expression. Thus FXa induces functional expression of PAR-2 but not of PAR-1 in human SMCs, independent of thrombin formation, via a mechanism involving NOX-1-containing NADPH oxidase, H₂O₂, NF-κB, and HuR.     

Evidence that PAR2-triggered prostaglandin E2 (PGE2) formation involves the ERK-cytosolic phospholipase A2-COX-1-microsomal PGE synthase-1 cascade in human lung epithelial cells

We investigated possible involvement of three isozymes of prostaglandin E synthase (PGES), microsomal PGES-1 (mPGES-1), mPGES-2 and cytosolic PGES (cPGES) in COX-2-dependent prostaglandin E(2) (PGE(2)) formation following proteinase-activated receptor-2 (PAR2) stimulation in human lung epithelial cells. PAR2 stimulation up-regulated mPGES-1 as well as COX-2, but not mPGES-2 or cPGES, leading to PGE(2) formation. The PAR2-triggered up-regulation of mPGES-1 was suppressed by inhibitors of COX-1, cytosolic phospholipase A(2) (cPLA(2)) and MEK, but not COX-2. Finally, a selective inhibitor of mPGES-1 strongly suppressed the PAR2-evoked PGE(2) formation. PAR2 thus appears to trigger specific up-regulation of mPGES-1 that is dependent on prostanoids formed via the MEK/ERK/cPLA(2)/COX-1 pathway, being critical for PGE(2) formation.     

Mechanisms for modulation of mouse gastrointestinal motility by proteinase-activated receptor (PAR)-1 and -2 in vitro

Proteinase-activated receptor (PAR)-1 or -2 modulates gastrointestinal transit in vivo. To clarify the underlying mechanisms, we characterized contraction/relaxation caused by TFLLR-NH2 and SLIGRL-NH2, PAR-1- and -2-activating peptides, respectively, in gastric and small intestinal (duodenal, jejunal and ileal) smooth muscle isolated from wild-type and PAR-2-knockout mice. Either SLIGRL-NH2 or TFLLR-NH2 caused both relaxation and contraction in the gastrointestinal preparations from wild-type animals. Apamin, a K+ channel inhibitor, tended to enhance the peptide-evoked contraction in some of the gastrointestinal preparations, whereas it inhibited relaxation responses to either peptide completely in the stomach, but only partially in the small intestine. Indomethacin reduced the contraction caused by SLIGRL-NH2 or TFLLR-NH2 in both gastric and ileal preparations, but unaffected apamin-insensitive relaxant effect of either peptide in ileal preparations. Repeated treatment with capsaicin suppressed the contractile effect of either peptide in the stomach, but not clearly in the ileum, whereas it enhanced the apamin-insensitive relaxant effect in ileal preparations. In any gastrointestinal preparations from PAR-2-knockout mice, SLIGRL-NH2 produced no responses. Thus, the inhibitory component in tension modulation by PAR-1 and -2 involves both apamin-sensitive and -insensitive mechanisms in the small intestine, but is predominantly attributable to the former mechanism in the stomach. The excitatory component in the PAR-1 and -2 modulation may be mediated, in part, by activation of capsaicin-sensitive sensory nerves and/or endogenous prostaglandin formation. Our study thus clarifies the multiple mechanisms for gastrointestinal motility modulation by PAR-1 and -2, and also provides ultimate evidence for involvement of PAR-2.     

CP-31398 inhibits the progression of cervical cancer through reversing the epithelial mesenchymal transition via the downregulation of PAX2s

CP-31398, a styrylquinazoline, emerges from a screen for therapeutic agents that restore the wild-type DNA-binding conformation of mutant p53 to suppress tumors in vivo, but its effects on cervical cancer (CC) remain unknown. Hence, this study aimed to explore the effects CP-31398 has on the CC cells and to investigate whether it is associated with paired box 2 (PAX2) expression. CC cells were treated with different concentrations of CP-31398 (1, 2, 4, 6, 8, and 10 μg/ml) to determine the optimum concentration using fluorometric microculture cytotoxicity assay. After constructing the sh-PAX2 vector, CC cells were transfected with sh-PAX2 or treated with CP-31398. The effects of CP-31398 or PAX2 silencing on CC cell proliferation, apoptosis, invasion, and migration were evaluated. Epithelial mesenchymal transition (EMT)-related genes such as E-cadherin, vimentin, N-cadherin, snail, and twist in CC cells were detected. Tumor formation experiment in nude mice was performed to observe tumor growth. The optimum concentration of CP-31398 was 2 μg/ml. PAX2 was overexpressed in CC cells. CC cells treated with CP-31398 or treated with sh-PAX2 inhibited proliferation, invasion, and migration but promoted apoptosis with decreased PAX2 expression. The EMT process in CC cells was also reversed after treatment with CP-31398 or sh-PAX2. Moreover, the tumor formation experiment in nude mice revealed the inhibitory activity of CP-31398 in CC tumor in nude mice by suppressing PAX2. Our results provide evidence that CP-31398 could inhibit EMT and promote apoptosis of CC cells to curb CC tumor growth by downregulating PAX2.     

SOX9/miR-130a/CTR1 axis modulates DDP-resistance of cervical cancer cell

Cisplatin (DDP) -based chemotherapy is a standard strategy for cervical cancer, while chemoresistance remains a huge challenge. Copper transporter protein 1 (CTR1), a copper influx transporter required for high affinity copper (probably reduced Cu I) transport into the cell, reportedly promotes a significant fraction of DDP internalization in tumor cells. In the present study, we evaluated the function of CTR1 in the cell proliferation of cervical cancer upon DDP treatment. MicroRNAs (miRNAs) have been regarded as essential regulators of cell proliferation, apoptosis, migration, as well as chemoresistance. By using online tools, we screened for candidate miRNAs potentially regulate CTR1, among which miR-130a has been proved to promote cervical cancer cell proliferation through targeting PTEN in our previous study. In the present study, we investigated the role of miR-130a in cervical cancer chemoresistance to DDP, and confirmed the binding of miR-130a to CTR1. SOX9 also reportedly act on cancer chemoresistance. In the present study, we revealed that SOX9 inversely regulated miR-130a through direct targeting the promoter of miR-130a. Consistent with previous studies, SOX9 could affect cervical cancer chemoresistance to DDP. Taken together, we demonstrated a SOX9/miR-130a/CTR1 axis which modulated the chemoresistance of cervical cancer cell to DDP, and provided promising targets for dealing with the chemoresistance of cervical cancer.     

Specificity protein 1 is a novel target of 2, 4-bis (p-hydroxyphenyl)-2-butenal for the suppression of human oral squamous cell carcinoma cell growth

Background

The Maillard reaction is a chemical reaction occurring between a reducing sugar and an amino acid, generally requiring thermal processing. Maillard reaction products (MRPs) have antioxidant, antimutagenic, and antibacterial effects though 2,4-bis (p-hydroxyphenyl)-2-butenal (HPB242), a fructose-tyrosine MRP, appears to inhibit proliferation of cancer cells, its mechanism of action has not been studied in detail. The purpose of this study was to investigate the anti-proliferative effects of 2,4-bis (p-hydroxyphenyl)-2-butenal (HPB242) on two oral squamous cell carcinoma (OSCC) cell lines, HN22 and HSC4, through regulation of specificity protein 1 (Sp1).

Results

HPB242 treatment dramatically reduced the cell growth rate and apoptotic cell morphologies. Sp1 was significantly inhibited by HPB242 in a dose-dependent manner. Furthermore, cell cycle regulating proteins and anti-apoptotic proteins, which are known as Sp1 target genes, were altered at the molecular levels. The key important regulators in the cell cycle such as p27 were increased, whereas cell proliferation- and survival-related proteins such as cyclin D1, myeloid leukemia sequence 1 (Mcl-1) and survivin were significantly decreased by HPB242 or suppressed Sp1 levels, however pro-apoptotic proteins caspase3 and PARP were cleaved in HN22 and HSC4.

Conclusions

HPB242 may be useful as a chemotherapeutic agent for OSCC for the purpose of treatment and prevention of oral cancer and for the improvement of clinical outcomes.     

Epigenetic inactivation of SPINT2 is associated with tumor suppressive function in esophageal squamous cell carcinoma

Hepatocyte growth factor activator inhibitor type 2 (SPINT2), a Kunitz-type serine proteinase inhibitor, has been identified as a putative tumor suppressor gene silenced by promoter methylation. We aimed to investigate whether SPINT2 might act as an esophageal squamous cell carcinoma (ESCC) tumor suppressor gene. Four ESCC cell lines, Fifty-two ESCC tissues and twenty-nine neighboring non-cancerous tissues were included in this study. The expression of SPINT2 was monitored by real time PCR. Bisulfite genomic sequencing and methylation-specific PCR were used to analyze methylation status. The effect of SPINT2 on cell proliferation and apoptosis in EC109 and EC9706 cells was observed by CCK-8 assay and flow cytometric analysis. We found that silencing of SPINT2 was associated with promoter methylation in ESCC cell lines. The densely methylated SPINT2 promoter region was confirmed by bisulfite genomic sequencing. Ectopic expression of SPINT2 inhibited cell proliferation through inducing cell apoptosis in vitro. Furthermore, methylation-specific PCR analysis revealed that SPINT2 promoter methylation was prominent in carcinoma tissues (52.08%) compared with neighboring non-cancerous tissues (22.58%). Kaplan–Meier analysis showed that patients with SPINT2 hypermethylation had shorter survival time. The tumor suppressor gene of SPINT2 is commonly silenced by promoter hypermethylation in human ESCC and SPINT2 hypermethylation is correlated with poor overall survival, implicating SPINT2 is an underlying prognostic marker for human ESCC.     

Investigation of quinazolines as inhibitors of breast cancer resistance protein (ABCG2)

Chemotherapy is one of the major forms of cancer treatment. Unfortunately, tumors are prone to multidrug resistance leading to failure of treatment. Breast cancer resistance protein (BCRP), the second member of ABC transporter subfamily G, has been found to play a major role in drug efflux and hence multidrug resistance. Until now, very few potent and selective BCRP inhibitors like Ko143 have been identified. In the search for more potent and selective BCRP inhibitors, we synthesized and investigated a series of differently substituted quinazoline compounds. Several variations at positions 2, 4, 6 and 7 of the quinazoline scaffold were carried out to develop a structure–activity-relationship analysis for these compounds. It was found that compounds bearing a phenyl substituent at position 2 of the 4-anilinoquinazoline scaffold were most potent. On the aniline ring at position 4 of the quinazoline moiety substituents like NO₂, CN, CF₃ led to very high BCRP inhibition potencies. The most potent compounds were further investigated for their intrinsic cytotoxicity and their ability to reverse the multidrug resistance. Compound 20, an anilinoquinazoline bearing a phenyl ring at position 2 and meta-nitro substitution on the 4-anilino ring, was found to have the highest therapeutic ratio. The most active compounds from each variation were also investigated for their effect on BCRP expression. It was found that compound 20 has no significant effect on BCRP expression, while compound 31 decreased the surface BCRP expression. The only difference in the two compounds was the presence of a 3,4-dimethoxyphenyl ring in compound 31 instead of phenyl substitution at position 2 of the quinazoline moiety. From the study of all target compounds, compound 20 was the most prominent compound having inhibitory potency even higher than Ko143, the most potent BCRP inhibitor known. Compound 20 was also found to be selective towards BCRP with a very high therapeutic ratio.