Chenghu Liu

SOX2 promotes tumorigenesis and increases the anti-apoptotic property of human prostate cancer cell

SRY-related HMG-box gene 2 (SOX2) is one of the key regulatory genes that maintain the pluripotency and self-renewal properties in embryonic stem cells. Here we used immunohistochemistry to analyze the expression of SOX2 in human prostate tissues and found it contributed to tumorigenesis and correlated with histologic grade and Gleason score. We further investigated SOX2s function in cell growth and apoptosis process by using a human prostate cancer cell line DU145 with SOX2 overexpression or down-regulation. Cell cycle assay revealed that SOX2 promoted cell growth and increased the percentage of cells in S phase. In vitro and in vivo xenograft experiments in NOD/SCID mice further demonstrated that SOX2 increased the apoptosis-resistant properties of DU145 cells with decreased function of store-operated Ca(2+) entry and reduced expression of Orai1 at both mRNA and protein levels, suggesting a potential mechanism that contributes to the anti-apoptotic property of SOX2. To our knowledge, this study is the first to investigate SOX2s function in tumorigenesis and apoptosis of human prostate cancer and to elucidate its regulatory effect on the activity of store-operated Ca(2+) channels. Our results support the concept that SOX2 has the potential to be a significant marker to evaluate the progression of prostate cancer and serve as a potentially useful target for prostate cancer therapy.

ATF4 is directly recruited by TLR4 signaling and positively regulates TLR4-trigged cytokine production in human monocytes.

Toll-like receptors (TLRs) are sentinels of the host defense system, which recognize a large number of microbial pathogens. The host defense system may be inefficient or inflammatory diseases may develop if microbial recognition by TLRs and subsequent TLR-triggered cytokine production are deregulated. Activating transcription factor 4 (ATF4), a member of the ATF/CREB transcription factor family, is an important factor that participates in several pathophysiological processes. In this report, we found that ATF4 is also involved in the TLR-mediated innate immune response, which participates in TLR4 signal transduction and mediates the secretion of a variety of cytokines. We observed that ATF4 is activated and translocates to the nucleus following lipopolysaccharide (LPS) stimulation via the TLR4-MyD88-dependent pathway. Additionally, a cytokine array assay showed that some key inflammatory cytokines, such as IL-6, IL-8 and RANTES, are positively regulated by ATF4. We also demonstrate that c-Jun directly binds to ATF4, thereby promoting the secretion of inflammatory cytokines. Taken together, these results indicate that ATF4 acts as a positive regulator in TLR4-triggered cytokine production.

A loop-mediated isothermal amplification method targets the phoP gene for the detection of Salmonella in food samples

A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the specific detection of Salmonella in food. The assay used specifically designed primers to target within the phoP gene and correctly identified all 66 strains of Salmonella and 73 non-Salmonella strains tested. The phoP gene regulates the expression of genes involved in virulence and the survival of Salmonella from destruction by macrophage. The probability of detection was 100% when a Salmonella cell suspension containing 10(1) CFU/ml was used as a template in the LAMP assay. Prior to the LAMP assay, a sample preparation protocol was applied that included a pre-enrichment step in buffered peptone water, followed by extraction and purification of DNA. In this way, 85 various food samples were investigated for Salmonella including minced meat of pig and raw milk. The diagnostic accuracy was shown to be 100% when compared to the traditional culture method. This combination of sample enrichment, and LAMP assay can detect 35 CFU per 250 ml of prepared food samples. The overall analysis time for the LAMP assay method was approximately 24 h. This is in contrast to 5 to 7 days of analysis time required for the traditional culture method. Consequently, the LAMP described here has the potential to become a standardized method for the rapid detection of Salmonella in diagnostic laboratories once further validated by inter-laboratory studies.

Fra-1 Promotes Breast Cancer Chemosensitivity by Driving Cancer Stem Cells from Dormancy

Fra-1 is a member of the Fos transcription factor family that is highly expressed in multiple cancers, playing important roles in transformation, proliferation, and metastasis. In this study, we observed an inverse correlation between the expression of Fra-1 in human stage II breast cancer tissues and the corresponding level of clinical chemoresistance. Extending these findings in vitro, we found that knockdown of Fra-1 in breast tumor cells was sufficient to confer resistance to doxorubicin and cyclophosphamide, whereas enhanced Fra-1 expression could render these cells chemosensitive. The tumor cell side population, which is enriched for cancer stem cells, was found to be associated with chemoresistance. Increased side population fractions were detected among tumor cell lines subjected to Fra-1 knockdown. In contrast, enhanced expression of Fra-1 was correlated with a decreased side population fraction, and significantly, this finding was recapitulated in vivo, where tumors with enhanced expression of Fra-1 were found to have blunted growth. Tumor cells subjected to Fra-1 knockdown grew faster and were larger in size. Taken together, our findings suggest that Fra-1 may be an important prognostic marker for breast cancer therapy.

Yes-associated protein (YAP) increases chemosensitivity of hepatocellular carcinoma cells by modulation of p53

The yes-associated protein (YAP) transcription co-activator has been reported either as an oncogene candidate or a tumor suppressor. Liver tissue chips revealed that about 51.4% human hepatocellular carcinoma (HCC) samples express YAP and 32.9% HCC samples express phosphorylated YAP. In this study, we found that chemotherapy increased YAP protein expression and nuclear translocation in HepG2 cells, as well as p53 protein expression and nuclear translocation. However, little is known about YAP functions during chemotherapy. Our results show that overexpression of YAP increases chemosensitivity of HepG2 cells during chemotherapy. Dominant negative transfection of Flag-S94A (TEAD binding domain mutant) or Flag-W1W2 (WW domain mutant) to HepG2 cells decreases p53 expression/ nuclear translocation and chemosensitivity when compared with control HepG2 cells. Furthermore, rescue transfection of Flag-5SA-S94A or Flag-5SA-W1W2, respectively to HepG2 cells regains p53 expression/nuclear translocation and chemosensitivity. These results indicate that YAP promotes chemosensitivity by modulating p53 during chemotherapy and both TEAD and WW binding domains are required for YAP-mediated p53 function. ChIP assay results also indicated that YAP binds directly to the p53 promoter to improve its expression. In addition, p53 could positively feedback YAP expression through binding to the YAP promoter. Taken together, our current data indicate that YAP functions as a tumor suppressor that enhances apoptosis by modulating p53 during chemotherapy.

TLR2 signaling subpathways regulate TLR9 signaling for the effective induction of IL-12 upon stimulation by heat-killed Brucella abortus

Brucella abortus is a Gram-negative intracellular bacterium that induces MyD88-dependent IL-12 production in dentritic cells (DCs) and a subsequent protective Th1 immune response. Previous studies have shown that the Toll-like receptor 2 (TLR2) is required for tumor-necrosis factor (TNF) production, whereas TLR9 is responsible for IL-12 induction in DCs after exposure to heat-killed Brucella abortus (HKBA). TLR2 is located on the cell surface and is required for optimal microorganism-induced phagocytosis by innate immune cells; thus, phagocytosis is an indispensable preliminary step for bacterial genomic DNA recognition by TLR9 in late-endosomal compartments. Here, we hypothesized that TLR2-triggered signals after HKBA stimulation might cross-regulate TLR9 signaling through the indirect modulation of the phagocytic function of DCs or the direct modulation of cytokine gene expression. Our results indicate that HKBA phagocytosis was TLR2-dependent and an essential step for IL-12p40 induction. In addition, HKBA exposure triggered the TLR2-mediated activation of both p38 and extracellular signal-regulated kinase 1/2 (ERK1/2). Interestingly, although p38 was required for HKBA phagocytosis and phagosome maturation, ERK1/2 did not affect these processes but negatively regulated IL-12 production. Although p38 inhibitors tempered both TNF and IL-12 responses to HKBA, pre-treatment with an ERK1/2 inhibitor significantly increased IL-12p40 and abrogated TNF production in HKBA-stimulated DCs. Further experiments showed that the signaling events that mediated ERK1/2 activation after TLR2 triggering also required HKBA-induced Ras activation. Furthermore, Ras-guanine nucleotide-releasing protein 1 (RasGRP1) mediated the TLR2-induced ERK1/2 activation and inhibition of IL-12p40 production. Taken together, our results demonstrated that HKBA-mediated TLR2-triggering activates both the p38 and ERK1/2 signaling subpathways, which divergently regulate TLR9 activation at several levels to induce an appropriate protective IL-12 response.

Activating transcription factor 4 increases chemotherapeutics resistance of human hepatocellular carcinoma

It has been reported that activating transcription factor 4 (ATF4) increases the processes of tumor growth, metastasis and drug resistance. However, the role played by ATF4 in chemoresistance of hepatocellular carcinoma (HCC) remains unknown. Clarification of this role of ATF4 in HCC could greatly benefit the efficacy of clinical treatment of HCC. In this study, we found that ATF4 was overexpressed in about 50.7% of HCC tissues. In fact knockdown of ATF4 significantly increased the cytotoxicity of cisplatin in both in vitro and in vivo assays, while overexpression of this molecule dramatically decreased the sensitivity of HCC cell lines to cisplatin. Additionally, we found that synthesis of glutathione was significantly reduced in HCC cell lines subjected to ATF4 knockdown. Taken together, these results demonstrate that ATF4 can increase resistance to cisplatin in HCC by increased biosynthesis of glutathione, and that this may be a potent novel target for the future development of anti-HCC drugs.

TLR4 Promotes Breast Cancer Metastasis via Akt/GSK3β/β-Catenin Pathway upon LPS Stimulation

Bacteria/virus‐induced chronic inflammation is involved in both tumor initiation and tumor progression. Toll‐like receptor 4 (TLR4) has been implicated in the development of several types of cancer. In this study, we explored the impact of TLR4 activation by lipopolysaccharide (LPS) on breast cancer metastasis and associated signaling molecules. We first examined TLR4 expression levels in breast tissue using a human breast tissue microarray and breast cell lines. We then studied the role of TLR4 activation by LPS stimulation in breast cancer metastasis using both in vitro and in vivo models. Finally, we investigated signaling molecules involved in the process using Western blotting and fluorescent immunohistochemistry staining. The results showed that TLR4 expression levels increased in breast cancer tissue compared to normal breast tissue. In addition, our results also showed that TLR4 pathway activation by LPS stimulation in MCF7 and MDA‐MB‐231 breast cancer cells caused the following actions: (1) promotes migration of breast cancer cells, (2) triggers the β‐catenin signaling pathway via PI3K/Akt/GSK3β, and (3) promotes transcription of downstream β‐catenin target genes leading to breast cancer metastasis. This study substantiates and further extends the relationship between TLR4 activation by LPS and breast cancer using both in vitro and in vivo models. The results suggest that the Akt/GSK3β/β‐catenin signal transduction pathway may serve as a viable clinical treatment target in breast cancer. Anat Rec, 300:1219–1229, 2017.

Ifit1 Protects Against Lipopolysaccharide and D-galactosamine–Induced Fatal Hepatitis by Inhibiting Activation of the JNK Pathway

Treatment of mice with lipopolysaccharide (LPS) and the liver-specific transcriptional inhibitor D-(+)-galactosamine (GalN) induces fatal hepatitis, which is mediated by tumor necrosis factor α (TNF-α) and characterized by massive hepatic apoptosis. Previous studies suggest that GalN increases the sensitivity to LPS/TNF-α, probably by blocking the transcription of protective factors, but the identity of most of these factors is still unclear. Here, we report that Ifit1 protects against LPS/GalN-induced fatal hepatitis. Forced expression of Ifit1 in hepatocytes significantly diminished TNF-α-mediated apoptosis. Moreover, targeted expression of Ifit1 in the liver by recombinant adeno-associated virus serotype 8 protected mice from LPS/GalN-induced lethal hepatitis, which was associated with the inhibition of TNF-α-mediated activation of the c-Jun N-terminal kinase (JNK)-Bim cascade. Furthermore, Ifit1 bound to a scaffolding protein Axin and inhibited its function to mediate JNK activation. Together, our data demonstrate that Ifit1 is a novel protective factor that inhibits LPS/GalN-induced (TNF-α-mediated) fatal hepatitis, suggesting that Ifit1 is a potential therapeutic target for treatment of inflammatory liver diseases.

Abstract 826: Yes-associated protein increases hepatocellular carcinoma chemosensitivity by modulating p53

The function of the yes-associated protein (YAP) transcription co-activator remains an enigma since reports indicate that YAP either acts as an oncogene or enhances apoptosis. In this study, we found that YAP expression occurs in 51% of human hepatocellular carcinoma (HCC) biopsies while phosphorylated YAP (pYAP) expression occurs in 33% of Grade II and III HCCs. In addition, we found that YAP and p53 expression/ nuclear translocation increase in chemo-treated HepG2 cells. In chemo-treated HepG2 cells transfected with WT-YAP or blank (control), the chemosensitivity of the HepG2 + WT-YAP cells increases compared to HepG2+blank cells. In chemo-treated HepG2 cells transfected with Flag-S94A (TEAD binding domain mutant), Flag-W1W2 (WW domain mutant), or blank (control), the chemosensitivity and p53 expression/ nuclear translocation of the HepG2+Flag-S94A cells and HepG2+Flag-W1W2 cells decrease compared to HepG2+blank cells. In chemo-treated HepG2 cells transfected with Flag-5SA-S94A or Flag-5SA-W1W2, the chemosensitivity and p53 expression/ nuclear translocation of the HepG2+Flag-5SA-S94A cells and HepG2+Flag-5SA-W1W2 cells increase again compared to HepG2+Flag-S94A cells or HepG2+Flag-W1W2 cells respectively. These results indicate that YAP promotes chemosensitivity by modulating p53 during chemo-treatment, and YAP-modulated p53 activity requires the TEAD binding domain and the WW domain. In summary, our data indicate that YAP functions as a tumor-suppressor that enhances apoptosis by modulating p53 activity during chemo-treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 826. doi:1538-7445.AM2012-826