Zhuhong Zhang

MicroRNA-19a-3p inhibits breast cancer progression and metastasis by inducing macrophage polarization through downregulated expression of Fra-1 proto-oncogene

One of the hallmarks of malignancy is the polarization of tumor-associated macrophages (TAMs) from a pro-immune (M1-like) phenotype to an immune-suppressive (M2-like) phenotype. However, the molecular basis of the process is still unclear. MicroRNA (miRNA) comprises a group of small, non-coding RNAs that are broadly expressed by a variety of organisms and are involved in cell behaviors such as suppression or promotion of tumorigenesis. Here, we demonstrate that miR-19a-3p, broadly conserved among vertebrates, was downregulated in RAW264.7 macrophage cells of the M2 phenotype in conditoned medium of 4T1 mouse breast tumor cells. This downregulation correlated with an increased expression of the Fra-1 gene, which was reported to act as a pro-oncogene by supporting the invasion and progression of breast tumors. We found significant upregulation of miR-19a-3p in RAW264.7 macrophages after transfection with a miR-19a-3p mimic that resulted in a significant suppression of the expression of this gene. In addition, we could measure the activity of binding between miR-19a-3p and Fra-1 with a psiCHECK luciferase reporter system. Further, transfection of RAW264.7 macrophage cells with the miR-19a-3p mimic decreased the expression of the Fra-1 downstream genes VEGF, STAT3 and pSTAT3. Most importantly, the capacity of 4T1 breast tumor cells to migrate and invade was impaired in vivo by the intratumoral injection of miR-19a-3p. Taken together, these findings indicate that miR-19a-3p is capable of downregulating the M2 phenotype in M2 macrophages and that the low expression of this miRNA has an important role in the upregulation of Fra-1 expression and induction of M2 macrophage polarization.

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.

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.

miR-885-5p suppresses hepatocellular carcinoma metastasis and inhibits Wnt/β-catenin signaling pathway

MicroRNAs (miRNAs) inhibit or improve the malignant progression of hepatocellular carcinoma (HCC). We previously reported that compared to health controls, patients with liver cirrhosis present the highest levels of circulating miR-885-5p, followed by those with chronic hepatitis B and those with HCC. However, the molecular involvement of miR-885-5p in HCC metastasis is presently unclear. Here, we demonstrated that the expression of miR-885-5p negatively correlated with the invasive and metastatic capabilities of human HCC tissue samples and cell lines. We found that miR-885-5p expression levels correlated with the survival of patients with HCC. Overexpression of miR-885-5p decreased metastasis of HCC cells in vitro and in vivo. Inhibition of miR-885-5p improved proliferation of non-metastatic HCC cells. Furthermore, we disclosed that miR-885-5p targeted gene encoding β-catenin CTNNB1, leading to decreased activity of the Wnt/β-catenin signaling pathway. The present study indicates that miR-885-5p suppresses the metastasis of HCC and inhibits Wnt/β-catenin signaling pathway by its CTNNB1 target, which suggests that miR-885-5p to be a promising negative regulator of HCC progression and as a novel therapeutic agent to treat HCC.

Prognostic value of serum leptin in advanced lung adenocarcinoma patients with cisplatin/pemetrexed chemotherapy

Cisplatin/pemetrexed chemotherapy has been established as a standard treatment in lung adenocarcinoma. However, the response to the cisplatin/pemetrexed combination varies considerably among patients due to individual variations. Thus, novel biomarkers are required to aid the prediction of the response to the cisplatin/pemetrexed combination. We hypothesized that leptin expression may be a determinant for prognosis in lung adenocarcinoma patients with cisplatin/pemetrexed chemotherapy. Serum from consenting patients with lung adenocarcinoma were obtained for the measurement of leptin and associated tumor biomarkers. Leptin expression was measured by radioimmunoassay. Carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), CA15-3, CA125, CA72-4, cytokeratin 19 fragment (CYFRA21-1) and neuron-specific enolase (NSE) expression were determined by electrochemiluminescence immunoassays. Serum squamous cell carcinoma antigen levels were measured using a microparticle enzyme immunoassay. The associations between serum leptin and tumor biomarker expression were evaluated by Spearman’s correlation analysis. Serum CEA, CA19-9, CA15-3, CA125, CA72-4, CYFRA21-1 and NSE levels showed no obvious difference among patients. However, a trend towards an improved prognosis was observed in patients with lower serum leptin at diagnosis and an increase during cisplatin/pemetrexed chemotherapy. The results indicated that the serum leptin level has prognostic indications in patients with advanced lung adenocarcinoma during cisplatin/pemetrexed chemotherapy, which indicates that it may be a useful marker for the prognosis of cancer patients undergoing chemotherapy treatment.

A Novel Differential Predict Model Based on Matrix-Assisted Laser Ionization Time-of-Flight Mass Spectrometry and Serum Ferritin for Acute Graft-versus-Host Disease

Clinical diagnosis of acute graft-versus-host disease (aGVHD) mainly depends on clinical manifestation and tissue biopsies, leading to a delayed diagnosis and treatment for aGVHD patients when the early symptom is insignificant. Our objective was to investigate the possibility of prewarning the risk of aGVHD before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT) by serum protein profiling combined with serum ferritin. The difference in polypeptide expression before and after transplantation had been compared by using CLINPROT technology, and serum ferritin levels have been analyzed simultaneously. Through combining serum ferritin and MS spectral data, the diagnosis sensitivity and specificity of our model for prewarning severe aGVHD (III~IV°aGVHD) before transplant all increased to 90.0%, while after transplant, the sensitivity and specificity are 78.3% and 86.4%. Our joint prewarning model could predict the risk of aGVHD, especially severe aGVHD before and after transplant, which also provides a reliable method to the continuous monitoring of the condition of patients.

Abstract 2828: miR-19a-3p inhibits breast carcinoma metastasis via reversing M2 phenotype of TAMs.

Tumor-associated macrophages (TAMs), most of which exhibit M2 phenotype, function as immunosuppressive cells in tumor microenvironment (TME). Polarization of TAMs from a pro-immune (M1 like) phenotype to an immune-suppressive (M2-like) phenotype is one of the hallmarks of malignancy, but their molecular basis is still remains unknown. It has been reported that microRNAs are involved in monocyte-macrophage differentiation. In this study, we found that miR-19a-3p, broadly conserved in vertebrate, could reverse the M2 phenotype of RAW macrophage cells. When mouse breast tumor cells such as 4T1, 4TO7 and EMT6 were co-cultured with RAW macrophage cells which over express miR-19a-3p, the invasion capacity was suppressed. Meanwhile, when the conditional medium of RAW cells which were transfected with miR-19a-3p mimic was added into culturing medium of tumor cells, the migration capacity of 4T1 and EMT6 breast cancer cells was inhibited. Moreover, when miR-19a-3p was injected intratumor, consistent with the in vitro experiments, we found that the M2 phenotype of TAMs was suppressed significantly. Although 4T1 xengraft breast tumor growth was not affected by miR-19a-3p, lung metastasis of tumor cells was significantly suppressed. Taken together, our findings indicate that miR-19a-3p is down-regulated in M2 phenotype RAW macrophage and TAMs in TME. The low expression of miR-19a-3p plays an important role in inducing M2 macrophage polarization and promoting migration and invasion capacity and metastasis. Citation Format: Jian Yang, Na Li, Zhuhong Zhang, Qin Si, Chong Chen, Yan Liu, Ralph A. Reisfeld, Peiqing Sun, Dwayne Stupack, Rong Xiang, Yunping Luo. miR-19a-3p inhibits breast carcinoma metastasis via reversing M2 phenotype of TAMs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2828. doi:10.1158/1538-7445.AM2013-2828 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

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