Honghui Han

CADPE Inhibits PMA-Stimulated Gastric Carcinoma Cell Invasion and Matrix Metalloproteinase-9 Expression by FAK/MEK/ERK–Mediated AP-1 Activation

Metastasis is one of the main causes of death for patients with malignant tumors. Aberrant expression of matrix metalloproteinase-9 (MMP-9) has been implicated in the invasion and metastasis of various cancer cells. Here, we found that caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE) could inhibit the migration and invasion of human gastric carcinoma cells in Transwell migration assays. To understand the underlying mechanism, we showed that CADPE significantly inhibited phorbol 12-myristate 13-acetate (PMA)–induced increases in MMP-9 expression and activity in a dose-dependent manner. The inhibitory effect of CADPE on MMP-9 expression correlated well with the suppression of MMP-9 promoter activity and the reduction of MMP-9 mRNA. Reporter gene assay and electrophoretic mobility shift assay showed that CADPE inhibited MMP-9 expression by suppressing the activation of the nuclear transcription factor activator protein-1 (AP-1) and c-Fos, but not NF-κB. Moreover, CADPE inhibited PMA-induced phosphorylation of protein kinases involved in AP-1 activation, such as focal adhesion kinase (FAK), mitogen-activated protein kinase/extracellular signal–regulated kinase (ERK) kinase (MEK), and ERK1/2, whereas CADPE had little effect on the phosphorylation of p38 and c-jun NH2-terminal kinase. Taken together, our findings indicate that CADPE could be a unique antitumor agent that specifically inhibits MMP-9 activity by targeting the activation of FAK/MEK/ERK protein kinases and AP-1 transcription factor. Mol Cancer Res; 8(11); 1477–88. ©2010 AACR.

Nitidine Chloride inhibits breast cancer cells migration and invasion by suppressing c-Src/FAK associated signaling pathway.

Nitidine is a benzophenanthridine alkaloid, which has been shown to have anti-tumor properties. Here, we demonstrated that Nitidine Chloride (NC) could inhibit breast cancer cells migration and invasion both in vitro and in vivo. Meanwhile, the protrusion formation and partial proteolytic activity of MMP-9 and MMP-2 were attenuated by NC in a dose-dependent manner in MDA-MB-231 cells. Furthermore, addition NC to cells significantly decreases PDGF induced phosphorylation of c-Src, FAK, MAPKs, activation of RhoA, Rac1 and AP-1 transcriptional activity. Taken together, our results indicate that NC could have potential as a novel anti-metastasis drug to breast cancer.

CRISPR/Cas9‐mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse

The X‐linked genetic bleeding disorder caused by deficiency of coagulator factor IX, hemophilia B, is a disease ideally suited for gene therapy with genome editing technology. Here, we identify a family with hemophilia B carrying a novel mutation, Y371D, in the human F9 gene. The CRISPR/Cas9 system was used to generate distinct genetically modified mouse models and confirmed that the novel Y371D mutation resulted in a more severe hemophilia B phenotype than the previously identified Y371S mutation. To develop therapeutic strategies targeting this mutation, we subsequently compared naked DNA constructs versus adenoviral vectors to deliver Cas9 components targeting the F9 Y371D mutation in adult mice. After treatment, hemophilia B mice receiving naked DNA constructs exhibited correction of over 0.56% of F9 alleles in hepatocytes, which was sufficient to restore hemostasis. In contrast, the adenoviral delivery system resulted in a higher corrective efficiency but no therapeutic effects due to severe hepatic toxicity. Our studies suggest that CRISPR/Cas‐mediated in situ genome editing could be a feasible therapeutic strategy for human hereditary diseases, although an efficient and clinically relevant delivery system is required for further clinical studies.

Targeted Drug Delivery to Hepatocarcinoma In vivo by Phage-Displayed Specific Binding Peptide

Hepatocellular carcinoma is one of the deadliest cancers in the world. In this study, a hepatocarcinoma-specific binding peptide, which could be used for drug delivery in targeting therapy, was obtained by in vivo phage display technology. After three rounds of panning, only the potential motif Pro-Ser was found in 80 sequenced phage clones. Phage A54 (sequence AGKGTPSLETTP) was shown to be the most effective and specific to the liver cancer cells by cell-based ELISA in all 130 tested clones. After phage A54 was injected i.v. into the xenograft-bearing mice for in vivo distribution, phage enrichment was found in tumor tissues compared with control phage C10 and normal liver tissues through phage titering and immunohistochemical staining. Next, the specific binding ability of synthesized peptide A54 was further confirmed by fluorescence microscopy, competition binding, and fluorescence-activated cell sorting assay. A54 and A54M (sequence AGKGTAALETTP) were synthesized and coupled to doxorubicin (DOX) to do the preliminary targeting therapy. After the treatment, the proliferation of liver cancer cells treated with A54-DOX was restrained significantly in vitro when compared with A54M-DOX–treated group. Reduction in tumor size and prolongation of long-term survival were also found in xenograft-bearing models compared with free DOX–treated group. In conclusion, the specific binding peptide A54, which was screened from phage display library, represents a promising approach for the development of novel target therapy strategies against hepatocellular carcinoma. Mol Cancer Res; 8(2); 135–44

Lgr4/Gpr48 Negatively Regulates TLR2/4-associated Pattern Recognition and Innate Immunity by Targeting CD14 Expression

Background: Lgr4/Gpr48 has been found to play an important role in development of different organs. Results: Overactivated innate immune response was found in Lgr4-deficient mice. Conclusion: Lgr4/Gpr48 could be a negative regulator in TLR2/4-associated immune responses. Significance: Our novel findings provide new insights into the physiological functions of Lgr4/Gpr48 in immune diseases. The recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs) is pivotal in both innate and adaptive immune responses. Here we demonstrate that deletion of Lgr4/Gpr48 (G-protein-coupled receptor 48), a seven-transmembrane glycoprotein hormone receptor, potentiates TLR2/4-associated cytokine production and attenuates mouse resistance to septic shock. The expression of CD14, a co-receptor for TLR2/4-associated pathogen-associated molecular patterns, is increased significantly in Lgr4-deficient macrophages, which is consistent with the increased immune response, whereas the binding activity of cAMP-response element-binding protein is decreased significantly in Lgr4-deficient macrophages, which up-regulate the expression of CD14 at the transcriptional level. Together, our data demonstrate that Lgr4/Gpr48 plays a critical role in modulating the TLR2/4 signaling pathway and represents a useful therapeutic approach of targeting Lgr4/Gpr48 in TLR2/4-associated septic shock and autoimmune diseases.

Norcantharidin facilitates LPS-mediated immune responses by up-regulation of AKT/NF-κB signaling in macrophages.

Norcantharidin (NCTD), a demethylated analog of cantharidin, is a common used clinical drug to inhibit proliferation and metastasis of cancer cells. But the role of NCTD in modulating immune responses remains unknown. Here, we investigated the function and mechanism of NCTD in regulation of TLR4 associated immune response in macrophages. We evaluated the influence of NCTD on host defense against invaded pathogens by acute peritonitis mouse model, ELISA, Q-PCR, nitrite quantification, phagocytosis assay and gelatin zymography assay. Our data showed that the survival and the serum concentrations of IL-6 and TNF-α were all enhanced by NCTD significantly in peritonitis mouse model. Accordingly, LPS-induced cytokine, nitric oxide and MMP-9 production as well as the phagocytosis of bacteria were all up-regulated by NCTD in a dose dependent manner in both RAW264.7 cells and bone marrow-derived macrophages (BMMs). Then we further analyzed TLR4 associated signaling pathway by Western blot, Immunofluorescence and EMSA in the presence or absence of LPS. The phosphorylation of AKT and p65 at serine 536 but not serine 468 was enhanced obviously by NCTD in a dose dependent manner, whereas the degradation of IκBα was little effected. Consequently, the nuclear translocation and DNA binding ability of NF-κB was also increased by NCTD obviously in RAW264.7 cells. Our results demonstrated that NCTD could facilitate LPS-mediated immune response through promoting the phosphorylation of AKT/p65 and transcriptional activity of NF-κB, thus reprofiling the traditional anti-tumor drug NCTD as a novel immune regulator in promoting host defense against bacterial infection.

Virus-Triggered ATP Release Limits Viral Replication through Facilitating IFN-β Production in a P2X7-Dependent Manner

Accumulating evidence shows that innate immune responses are associated with extracellular nucleotides, particularly ATP. In this article, we demonstrate extensive protection of ATP/P2X7 signaling in a host against viral infection. Interestingly, we observed a significant increase in ATP as a danger signal in vesicular stomatitis virus (VSV)-infected cell supernatant and VSV-infected mice in an exocytosis- and pannexin channel–dependent manner. Furthermore, extracellular ATP reduces the replication of VSV, Newcastle disease virus, murine leukemia virus, and HSV in vivo and in vitro through the P2X7 receptor. Meanwhile, ATP significantly increases IFN-β expression in a concentration- and time-dependent manner. Mechanistically, ATP facilitates IFN-β secretion through P38/JNK/ATF-2 signaling pathways, which are crucial in promoting antiviral immunity. Taken together, these results demonstrate the protective role of extracellular ATP and P2X7 in viral infection and suggest a potential therapeutic role for ATP/P2X7 in viral diseases.

Leucine-rich repeat-containing G protein-coupled receptor 4 facilitates vesicular stomatitis virus infection by binding vesicular stomatitis virus glycoprotein

Vesicular stomatitis virus (VSV) and rabies and Chandipura viruses belong to the Rhabdovirus family. VSV is a common laboratory virus to study viral evolution and host immune responses to viral infection, and recombinant VSV-based vectors have been widely used for viral oncolysis, vaccination, and gene therapy. Although the tropism of VSV is broad, and its envelope glycoprotein G is often used for pseudotyping other viruses, the host cellular components involved in VSV infection remain unclear. Here, we demonstrate that the host protein leucine-rich repeat-containing G protein–coupled receptor 4 (Lgr4) is essential for VSV and VSV-G pseudotyped lentivirus (VSVG-LV) to infect susceptible cells. Accordingly, Lgr4-deficient mice had dramatically decreased VSV levels in the olfactory bulb. Furthermore, Lgr4 knockdown in RAW 264.7 cells also significantly suppressed VSV infection, and Lgr4 overexpression in RAW 264.7 cells enhanced VSV infection. Interestingly, only VSV infection relied on Lgr4, whereas infections with Newcastle disease virus, influenza A virus (A/WSN/33), and herpes simplex virus were unaffected by Lgr4 status. Of note, assays of virus entry, cell ELISA, immunoprecipitation, and surface plasmon resonance indicated that VSV bound susceptible cells via the Lgr4 extracellular domain. Pretreating cells with an Lgr4 antibody, soluble LGR4 extracellular domain, or R-spondin 1 blocked VSV infection by competitively inhibiting VSV binding to Lgr4. Taken together, the identification of Lgr4 as a VSV-specific host factor provides important insights into understanding VSV entry and its pathogenesis and lays the foundation for VSV-based gene therapy and viral oncolytic therapeutics.

Increasing targeting scope of adenosine base editors in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants

The clustered regularly interspaced short palindromic repeat (CRISPR) system has been widely adapted to genome editing to either introduce or correct genetic mutations (Wang et al., 2016). However, due to competition with the dominant non-homologous end-joining (NHEJ) pathway, precise genome modifications through Cas9-stimulated homologous recombination (HR) is inefficient. Through fusion of cytidine deaminases, APOBEC1 (apolipoprotein B editing complex 1) or AID (activation-induced deaminase), with Cas9 variants, several groups have developed the cytidine base editor (BE) systems (Komor et al., 2016; Li et al., 2018; Nishida et al., 2016). The BE system achieves programmable conversion of C•G base pairs to T•A without double-stranded DNA cleavage (Zhou et al., 2017). More recently, adenine base editors (ABEs), which efficiently convert A•T base pairs to G•C in genomic DNA, have been developed via fusion of an engineered tRNA adenosine deaminase (ecTadA from Escherichia coli) with nCas9 (Gaudelli et al., 2017). The ABE system has quickly been adapted to generate disease models and correction of genetic disease in mice (Ryu et al., 2017; Liu et al., 2018). However, whether the editing efficiency and the targeting scope of ABE could be improved is largely unexplored. In this study, we describe the efficient generation of base-edited mice and rats modeling human diseases through ABEs with highest efficiency up to 100%. We also demonstrate an increase of ABE activity through injection of chemically modified tracrRNA and crRNA in mouse zygotes, and the expansion of editing scope by fusion of an ecTadA mutant to SaCas9n-KKH and Cas9n-VQR variants in both cells and embryos. Our study suggests that the ABE system is a powerful and convenient tool to introduce precise base conversions in rodents. To test the ABE efficiency in embryos, we injected ABE mRNA (Fig. 1A) together with sgRNA targeting the TATA box of the Hbb-bs gene, into C57BL6 strain mouse zygotes (Fig. S1A and Table S1). Overlapping A/G peaks in the target sites were identified in 14/27 of F0 mice as determined by the chromatograms of Sanger sequencing (Figs. 2F and S1B). Further analysis by deep sequencing revealed allelic frequencies from 6%–71% among the founders (Fig. S1C). In individual allele, the editing window was extended from position A2–A9 in mouse embryos, which is broader than the window spanning position A4–A7 observed in mammalian cell lines (Gaudelli et al., 2017) (Fig. S1B and S1C). These data demonstrate that ABE is efficient to generate point mutant mice and its mutation window expands in embryos. Next, we tested the capability of ABE to precisely mutate A:T pairs for disrupting the stop codon of the gene encoding the fumarylacetoacetate hydrolase (Fah) (Fig. 1B), whose mutations cause hereditary tyrosinemia type I (HTI) in humans. We observed high A>G conversion efficiency (39/47) among F0 mice with allelic frequencies varying from 7%–99% as determined by deep sequencing (Figs. 1B, 1C, 2F and S2A). Increasing the sgRNA concentration from 50 ng/μL to 100 ng/μL results in 100% (13/13) point mutation rate in F0 mice (Figs. 2F and S2B). Since disruption of the stop codon usually affects mRNA stability and protein expression (Frischmeyer et al., 2002), the Fah mRNA and protein levels were dramatically impaired (Fig. S2C and S2D). Through immunohistochemistry analysis of the liver tissue from founder F0–F32, Fah protein expression was almost undetectable (Fig. 1D) suggesting this founder was a homozygote (Fig. 1B and 1C). To investigate germline transmission efficiency, founder mice were crossed with wild type or with other founders. We observed high germline transmission efficiency (Fig. S3A and S3B). In homozygous F1 mice, the expression of Fah mRNA and protein was lost (Fig. S3C–E). After withdrawal of the 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) treatment, the phenotypes of Fah mutant homozygotes were similar to previous HTI model, including loss of body weight and perturbation of serum biomarkers (Shao et al., 2018) (Fig. S3F and S3G). As mutations that generate premature stop codons are common drivers in various genetic diseases (Keeling et al., 2014), ABE has a promising potential for readthrough of premature stop codons in certain genetic diseases as demonstrated in the mouse DMD model (Ryu et al., 2017). Previous study demonstrated that 2’-O-methyl-3’-phosphorothioate (MS) modification on each ends of RNA can increase its stability, thus enhancing the Cas9 genome

Kisspeptin/GPR54 signaling restricts antiviral innate immune response through regulating calcineurin phosphatase activity

Blocking signaling by the neuropeptide hormone kisspeptin enhances antiviral immune responses. G protein–coupled receptor 54 (GPR54), the key receptor for the neuropeptide hormone kisspeptin, plays essential roles in regulating puberty development and cancer metastasis. However, its role in the antiviral innate immune response is unknown. We report that virus-induced type I interferon (IFN-I) production was significantly enhanced in Gpr54-deficient cells and mice and resulted in restricted viral replication. We found a marked increase of kisspeptin in mouse serum during viral infection, which, in turn, impaired IFN-I production and antiviral immunity through the GPR54/calcineurin axis. Mechanistically, kisspeptin/GPR54 signaling recruited calcineurin and increased its phosphatase activity to dephosphorylate and deactivate TANK [tumor necrosis factor receptor-associated factor (TRAF) family member-associated NF-κB activator]–binding kinase 1 (TBK1) in a Ca2+-dependent manner. Thus, our data reveal a kisspeptin/GPR54/calcineurin-mediated immune evasion pathway exploited by virus through the negative feedback loop of TBK1 signaling. These findings also provide insights into the function and cross-talk of kisspeptin, a known neuropeptide hormone, in antiviral innate immune response.