Anti-PD-L1 antibody reverses the immune tolerance induced by multiple MUC1-MBP vaccine immunizations by increasing the CD80/PD-L1 ratio, resulting in DC maturation, and decreasing Treg activity in B16-MUC1 melanoma-bearing mice.

In this study, we explored the possible mechanism of tumor tolerance induced by multiple repeated immunizations with a tumor vaccine (MUC1-MBP fusion protein plus CpG2006). We first analyzed the mechanism of tolerance by immunizing tumor-bearing mice 2, 5, or 8 times and found that compared with five immunizations with the M-M vaccine, eight immunizations increased tumor volume and weight and Treg levels, while the proportions of Th1 and Tc1 cells in the spleen and lymph nodes were decreased. In particular, the M-M vaccine induced PD-L1 expression in CD11c + DCs and decreased their CD80/PD-L1 ratio. Therefore, the mechanism of tolerance induction by multiple immunizations with the M-M vaccine was investigated by focusing on the CD80/PD-L1 ratio, and an anti-PD-L1 antibody (αPD-L1) and the M-M vaccine were used in combination to treat melanoma. The results showed that αPD-L1 increased the CD80/PD-L1 ratio and enhanced the maturation of cDC1s by blocking PD-L1 on DCs, which potentially increased the activity of Th1 and Tc1 cells. Furthermore, the combination of the M-M vaccine with αPD-L1 decreased the activity and proportion of Tregs, which reversed the immune tolerance induced by eight immunizations with the vaccine. This study reveals the mechanism of the combination of M-M and αPD-L1 and provides a new combination strategy for improving the therapeutic effect of the M-M vaccine, laying a theoretical basis for the clinical application of the vaccine.

Expression of MUC1 in different tumours and its clinical significance (Review).

Mucin 1 (MUC1) was the first discovered transmembrane protein of the mucin family; it normally covers epithelial cells of the mucous membrane, providing lubrication and protection. However, aberrant expression of MUC1 is involved in cancer development, invasion and metastasis. It has been reported that MUC1 upregulation is highly associated with the progression of different epithelial cancer types, such as lung, liver, pancreatic and breast cancer. Therefore, MUC1 can be used as a specific marker and a target for immunotherapy in clinical applications, and the detection of MUC1 expression levels can be used to diagnose the occurrence, metastasis, prognosis and recurrence of cancer. The present review summarizes the abnormal expression of MUC1 in different tumours and discusses its clinical significance, thereby highlighting the potential diagnostic and therapeutic significance of MUC1 in cancer.

MF59 Promoted the Combination of CpG ODN1826 and MUC1-MBP Vaccine-Induced Antitumor Activity Involved in the Enhancement of DC Maturation by Prolonging the Local Retention Time of Antigen and Down-Regulating of IL-6/STAT3.

Our previous study found that CpG oligodeoxynucleotides 1826 (CpG 1826), combined with mucin 1 (MUC1)-maltose-binding protein (MBP) (M-M), had certain antitumor activity. However, this combination is less than ideal for tumor suppression (tumors vary in size and vary widely among individuals), with a drawback being that CpG 1826 is unstable. To solve these problems, here, we evaluate MF59/CpG 1826 as a compound adjuvant with M-M vaccine on immune response, tumor suppression and survival. The results showed that MF59 could promote the CpG 1826/M-M vaccine-induced tumor growth inhibition and a Th1-prone cellular immune response, as well as reduce the individual differences of tumor growth and prolonged prophylactic and therapeutic mouse survival. Further research showed that MF59 promotes the maturation of DCs stimulated by CpG1826/M-M, resulting in Th1 polarization. The possible mechanism is speculated to be that MF59 could significantly prolong the retention time of CpG 1826, or the combination of CpG 1826 and M-M, as well as downregulate IL-6/STAT3 involved in MF59 combined CpG 1826-induced dendritic cell maturation. This study clarifies the utility of MF59/CpG 1826 as a vaccine compound adjuvant, laying the theoretical basis for the development of a novel M-M vaccine.

Recombinant MUC1-MBP fusion protein combined with CpG2006 vaccine induces antigen-specific CTL responses through cDC1-mediated cross-priming mainly regulated by type I IFN signaling in mice.

In this study, we explored the initiation and regulation mechanism of antigen-specific CTL responses induced by a novel cancer vaccine containing recombinant human mucin1-maltose-binding protein fusion protein (MUC1-MBP) and CpG2006. First, DC subsets were analyzed by flow cytometry in vivo and in vitro. After vaccination, the proportion and maturation of cDC1s in mouse dLNs were upregulated, and the proportion of cDC2s and pDCs was also increased. In vitro studies on vaccine components showed similar changs, which may mainly depend on the activity of CpG2006. Subsequently, the regulatory effect of type Ⅰ IFN signaling on CTL triggering was confirmed through co-culture of sorted DC subsets and T cells and subsequent CTL activity experiments. CTL killing activity exhibited a 61.9% decrease once type I IFN signaling was blocked. Further analysis showed that blocking IFNAR1 on cDC1s but not on CTLs resulted in significant defects in CTL killing activity. Collectively, M-M combined with CpG2006 vaccine promotes MUC1-specific CTL responses by increasing the cDC1 activity in mice, and this is mainly regulated by type Ⅰ IFN signaling in cDC1s.

TRAF6-overexpressing dendritic cells loaded with MUC1 peptide enhance anti-tumor activity in B16-MUC1 melanoma-bearing mice.

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) signaling is a critical positive mechanism for the development, homeostasis and activation of immune cells. We investigated the effect of TRAF6 overexpression on dendritic cells (DCs) maturation. TRAF6-overexpressing DCs had increased expression of costimulatory molecules, major histocompatibility complex (MHC) molecules and IL-12 expression. This indicated that TRAF6 promoted the maturation of DCs and indirectly promoted Th1 activation. The antitumor activities between TRAF6-overexpressing DCs and control DCs were compared by administering DCs pulsed with mucin 1 (MUC1) Ag peptide in a therapeutic human MUC1-overexpressing mouse B16 melanoma cells (B16-MUC1) model. Administration of TRAF6-overexpressing DCs significantly inhibited the growth of B16-MUC1 tumors, accompanied by an increase in MUC1-specific Th1 responses and Tc1 responses, as well as a decrease in Tregs levels. TRAF6 signaling has been found to be involved in DCs maturation and Th1 activation in vitro, as well as therapeutic tumor models in vivo, indicating that TRAF6-overexpressing DCs may be a promising approach for cancer immunotherapy.

Anti-PD1 antibody enhances the anti-tumor efficacy of MUC1-MBP fusion protein vaccine via increasing Th1, Tc1 activity and decreasing the proportion of MDSC in the B16-MUC1 melanoma mouse model.

In previous studies, we have obtained a notable anti-tumor efficacy of the recombinant MUC1-MBP vaccine in the process of mouse B16-MUC1 melanoma treatment. However, the tumor cannot be eliminated completely. We found that the tumor inhibition rate decreased from 81.67% (five immunizations) to 43.67% (eight immunizations) after more than five immunizations, indicating persistent vaccine stimulation may activate immunosuppressive factors. In the present study, we revealed that programmed cell death 1 (PD1), an inhibitory molecule suppressing T cell function, expressed on splenic and tumor-infiltrating T cells were up-regulated by the vaccine. Therefore, to optimize the anti-tumor efficacy of the vaccine, we employed combination immunotherapy with MUC1-MBP vaccine and αPD1 (anti-PD1 antibody). Results showed that combination immunotherapy induced a more remarkable anti-tumor efficacy, the tumor clearance being increased to 80% from 20% which obtain by MUC1-MBP vaccine immunizations. To investigate the possible underlying mechanism, IFN-γ secretion and cytotoxic T lymphocyte (CTL) cytotoxicity were measured by enzyme-linked immunosorbent assay (ELISA) and xCELLigence real-time cell analyzer (RTCA) respectively. T cell subsets and immunosuppressive cells in the mouse spleen and tumor microenvironment were analyzed by FACS. Results showed that the proportion of splenic CD8+T cells and tumor infiltration was increased and the activity of CTL killing, T helper 1 (Th1), Type 1 CD8+T (Tc1) was enhanced, indicating that the anti-tumor efficacy enhanced by combination immunotherapy was mainly through boosting CD8+T cells mediated anti-tumor cellular immunity. Additionally, combination immunotherapy significantly decreased the splenic and tumor-infiltrating myeloid derived suppressor cells (MDSCs). These results demonstrated that combination immunotherapy with MUC1-MBP vaccine and αPD1 was capable to invoke a more potent anti-tumor immune response and provide a foundation for further research.

Dual roles of myeloid-derived suppressor cells induced by Toll-like receptor signaling in cancer.

Myeloid-derived suppressor cells (MDSCs) are one of the major components of the tumor microenvironment (TME), and are the main mediators of tumor-induced immunosuppression. Recent studies have reported that the survival, differentiation and immunosuppressive activity of MDSCs are affected by the Toll-like receptor (TLR) signaling pathway. However, the regulatory effect of TLR signaling on MDSCs remains controversial. TLR-induced MDSC can acquire different immunosuppressive activities to influence the immune response that can be either beneficial or detrimental to cancer immunotherapy. The present review summarizes the effects of TLR signals on the number, phenotype and inhibitory activity of MDSCs, and their role in cancer immunotherapy, which cannot be ignored if effective cancer immunotherapies are to be developed for the immunosuppression of the TME.

Mechanism by which TRAF6 Participates in the Immune Regulation of Autoimmune Diseases and Cancer.

Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase, is a signal transduction molecule shared by the interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) family and the TNFR superfamily. TRAF6 has a unique TRAF domain and RING finger domain that mediate intracellular signaling events. In the immune system, TRAF6-mediated signaling has been shown to be critical for the development, homeostasis, and activation of a variety of immune cells, including B cells, T cells, dendritic cells, and macrophages. Although the pathogenesis and etiology of autoimmune diseases and cancer are not fully understood, it is worth noting that existing studies have shown that TRAF6 is involved in the pathogenesis and development of a variety of these diseases. Herein, we reviewed the role of TRAF6 in certain immune cells, as well as the function and potential effect of TRAF6 in autoimmune diseases and cancer. Our review indicates that TRAF6 may be a novel target for autoimmune diseases and cancer.

The Effect of Different Immunization Cycles of a Recombinant Mucin1-Maltose-Binding Protein Vaccine on T Cell Responses to B16-MUC1 Melanoma in Mice.

We explored the effect of a recombinant mucin1-maltose-binding protein vaccine, including immunization cycles of recombinant mucin1-maltose-binding protein (MUC1-MBP) and CpG 2006 on T cell responses to human MUC1-overexpressing mouse melanoma B16 cells (B16-MUC1) melanoma in mice. We found that the vaccine had a significant antitumor effect, with the most obvious tumor-suppressive effect being observed in mice immunized five times. After more than five immunizations, the tumor inhibition rate decreased from 81.67% (five immunizations) to 43.67% (eight immunizations). To study the possible mechanism, Mucin-1(MUC1)-specific antibodies, IFN-γ secretion by lymphocytes, and cytotoxic T lymphocyte (CTL) cytotoxicity were measured by enzyme-linked immunosorbent assay (ELISA) and a real-time cell analyzer (RTCA). T cell subsets and immunosuppressive cells in the mouse spleen and tumor microenvironment were analyzed by FACS. These results showed that five immunizations activated MUC1-specific Th1 and CTL and reduced the ratio of myeloid-derived suppressor cells (MDSCs) and Th17 in mice more significantly than eight immunizations, indicating that excessive frequency of the immune cycle leads to the increased numbers of immunosuppressive cells and decreased numbers of immunostimulatory cells, thereby inhibiting antitumor immune activity. This data provide an experimental foundation for the clinical application of a recombinant MUC1-MBP vaccine.

An 8­gene signature predicts the prognosis of cervical cancer following radiotherapy.

Gene expression and DNA methylation levels affect the outcomes of patients with cancer. The present study aimed to establish a multigene risk model for predicting the outcomes of patients with cervical cancer (CerC) treated with or without radiotherapy. RNA sequencing training data with matched DNA methylation profiles were downloaded from The Cancer Genome Atlas database. Patients were divided into radiotherapy and non­radiotherapy groups according to the treatment strategy. Differently expressed and methylated genes between the two groups were identified, and 8 prognostic genes were identified using Cox regression analysis. The optimized risk model based on the 8­gene signature was defined using the Cox's proportional hazards model. Kaplan­Meier survival analysis indicated that patients with higher risk scores exhibited poorer survival compared with patients with lower risk scores (log­rank test, P=3.22x10­7). Validation using the GSE44001 gene set demonstrated that patients in the high­risk group exhibited a shorter survival time comprared with the low­risk group (log­rank test, P=3.01x10­3). The area under the receiver operating characteristic curve values for the training and validation sets were 0.951 and 0.929, respectively. Cox regression analyses indicated that recurrence and risk status were risk factors for poor outcomes in patients with CerC treated with or without radiotherapy. The present study defined that the 8­gene signature was an independent risk factor for the prognosis of patients with CerC. The 8­gene prognostic model had predictive power for CerC prognosis.

CpG ODN1826 as a Promising Mucin1-Maltose-Binding Protein Vaccine Adjuvant Induced DC Maturation and Enhanced Antitumor Immunity.

Mucin 1 (MUC1), being an oncogene, is an attractive target in tumor immunotherapy. Maltose binding protein (MBP) is a potent built-in adjuvant to enhance protein immunogenicity. Thus, a recombinant MUC1 and MBP antitumor vaccine (M-M) was constructed in our laboratory. To enhance the antitumor immune activity of M-M, CpG oligodeoxynucleotides 1826 (CpG 1826), a toll-like receptor-9 agonist, was examined in this study as an adjuvant. The combination of M-M and CpG 1826 significantly inhibited MUC1-expressing B16 cell growth and prolonged the survival of tumor-bearing mice. It induced MUC1-specific antibodies and Th1 immune responses, as well as the Cytotoxic T Lymphocytes (CTL) cytotoxicity in vivo. Further studies showed that it promoted the maturation and activation of the dendritic cell (DC) and skewed towards Th1 phenotype in vitro. Thus, our study revealed that CpG 1826 is an efficient adjuvant, laying a foundation for further M-M clinical research.

Targeting MUC1 and JNK by RNA interference and inhibitor inhibit the development of hepatocellular carcinoma.

Mucin 1 (MUC1), as an oncogene, is overexpressed in hepatocellular carcinoma (HCC) cells and promotes the progression and tumorigenesis of HCC through JNK/TGF-ß signaling pathway. In the present study, RNA interference (RNAi) and JNK inhibitor SP600125, which target MUC1 and/or JNK, were used to treat HCC cells in vitro, and the results showed that both silencing the expression of MUC1 and blocking the activity of JNK inhibited the proliferation of HCC cells. In addition, MUC1-stable-knockdown and SP600125 significantly inhibited the growth of tumors in the subcutaneous transplant tumor models that established in BALB/c nude mice rather than MUC1 or JNK siRNAs transiently transfection. Furthermore, the results from immunohistochemical staining assays showed that the inhibitory effects of MUC1 gene silencing and SP600125 on the proliferation of HCC cells in vivo were through the JNK/TGF-ß signaling pathway. These results indicate that MUC1 and JNK are attractive targets for HCC therapy and may provide new therapeutic strategies for the treatment of HCC.

TLR9 played a more important role than TLR2 in the combination of maltose-binding protein and BCG-induced Th1 activation.

Our previous study demonstrated that maltose-binding protein (MBP) combined with BCG induced synergistic mouse Th1 activation in vivo. Here, to explore the mechanism of MBP combined with BCG on Th1 activation, mouse purified CD4+ T cells were stimulated with MBP and BCG in vitro. The results showed that MBP combined with BCG synergistically increased IFN-γ production, accompanied with the upregulation of TLR2/9 expressions, suggesting that TLR2/9 were involved in the combination-induced Th1 activation. Next, TLR2 antibodies and TLR9 inhibitor were used to further analyze the effects of TLRs in Th1 activation. Results showed TLR2 antibody partly decreased MBP combined with BCG-induced IFN-γ production, MyD88 expression and IκB phosphorylation, indicating that TLR2-mediated MyD88-dependent pathway was involved in the MBP combined with BCG-induced Th1 activation. Moreover, MBP combined with BCG-induced Th1 activation was completely abrogated by TLR9 inhibitor, suggesting that TLR9-mediated MyD88-dependent pathway played a more important role than TLR2 in the combination-induced Th1 activation. Further study showed that TLR9 inhibitor downregulated TLR2 expression, suggesting that TLR9 signaling regulated TLR2 activation to favor Th1 resonse induced by MBP combined with BCG. Collectively, we demonstrated for the first time that the cross-talk of TLR2 and TLR9 triggered Th1 activation collaboratively and our findings provided valuable information about designing more effective adjuvant for cancer therapy.

Development and application of a double- antibody sandwich ELISA kit for the detection of serum MUC1 in lung cancer patients.

BACKGROUND: Mucin 1 (MUC1), as an oncogene, plays an important role in the diagnosis of lung cancer. OBJECTIVE: To establish a double-antibody sandwich enzyme-linked immune sorbent assay (ELISA) kit for the detection of serum MUC1 in lung cancer patients. METHODS: Commercial mouse anti-human MUC1 monoclonal antibody and rabbit anti-human MUC1 polyclonal antibody were used to construct a double-antibody sandwich ELISA kit. The serum MUC1 levels in peripheral blood of lung disease patients and healthy individuals were detected by the double-antibody sandwich ELISA kit and CA15-3 kit, respectively. RESULTS: A double-antibody sandwich ELISA kit was successfully constructed, and the sensitivity was up to 0.5 µ g/l. The cut-off value for the serum MUC1 levels in the peripheral blood was 1.98 µ g/l, the sensitivity was 62.5%, the specificity was 100% and the Youden index was 0.6250 when detected by the double-antibody sandwich ELISA kit, while the sensitivity was 18.75%, the specificity was 100% and the Youden index was 0.1875 when detected by CA15-3 kit. CONCLUSION: The double-antibody sandwich ELISA kit is superior to the CA15-3 kit in the detection of serum MUC1 in lung cancer patients, suggesting an attractive applying of the double-antibody sandwich ELISA kit in the early diagnosis of lung cancer.

Role of C-Jun N-terminal Kinase in Hepatocellular Carcinoma Development.

Hepatocellular carcinoma (HCC) is among the most frequently occurring cancers and the leading causes of cancer mortality worldwide. Identification of the signaling pathways regulating liver carcinogenesis is critical for developing novel chemoprevention and targeted therapies. C-Jun N-terminal kinase (JNK) is a member of a larger group of serine/threonine (Ser/Thr) protein kinases known as the mitogen-activated protein kinase (MAPK) family. JNK is an important signaling component that converts external stimuli into a wide range of cellular responses, including cell proliferation, differentiation, survival, migration, invasion, and apoptosis, as well as the development of inflammation, fibrosis, cancer growth, and metabolic diseases. Because of the essential roles of JNK in these cellular functions, deregulated JNK is often found to contribute to the development of HCC. Recently, the functions and molecular mechanisms of JNK in HCC development have been addressed using mouse models and human HCC cell lines. Furthermore, recent studies demonstrate that the activation of JNK by oncogenes can promote the development of cancers by regulating the transforming growth factor (TGF)-ß/Smad pathway, which makes the oncogenes/JNK/Smad signaling pathway an attractive target for cancer therapy. Additionally, JNK-targeted therapy has a broad potential for clinical applications. In summary, we are convinced that promising new avenues for the treatment of HCC by targeting JNK are on the horizon, which will undoubtedly lead to better, more effective, and faster therapies in the years to come.

Pre-clinical toxicity and immunogenicity evaluation of a MUC1-MBP/BCG anti-tumor vaccine.

Mucin 1 (MUC1), as an oncogene, plays a key role in the progression and tumorigenesis of many human adenocarcinomas and is an attractive target in tumor immunotherapy. Our previous study showed that the MUC1-MBP/BCG anti-tumor vaccine induced a MUC1-specific Th1-dominant immune response, simulated MUC1-specific cytotoxic T lymphocyte killing activity, and could significantly inhibit MUC1-expression B16 cells' growth in mice. To help move the vaccine into a Phase I clinical trial, in the current study, a pre-clinical toxicity and immunogenicity evaluation of the vaccine was conducted. The evaluation was comprised of a single-dose acute toxicity study in mice, repeat-dose chronic toxicity and immunogenicity studies in rats, and pilot toxicity and immunogenicity studies in cynomolgus monkeys. The results showed that treatment with the MUC1-MBP/BCG anti-tumor vaccine did not cause any organ toxicity, except for arthritis or local nodules induced by BCG in several rats. Furthermore, the vaccine significantly increased the levels of IFN-γ in rats, indicating that Th1 cells were activated. In addition, the results showed that the MUC1-MBP/BCG anti-tumor vaccine induced a MUC1-specific IgG antibody response both in rats and cynomolgus monkeys. Collectively, these data are beneficial to move the MUC1-MBP/BCG anti-tumor vaccine into a Phase I clinical trial.

Cytotoxicity of various types of gold-mesoporous silica nanoparticles in human breast cancer cells.

Recently, gold nanoparticles (AuNPs) have shown promising biological applications due to their unique electronic and optical properties. However, the potential toxicity of AuNPs remains a major hurdle that impedes their use in clinical settings. Mesoporous silica is very suitable for the use as a coating material for AuNPs and might not only reduce the cytotoxicity of cetyltrimethylammonium bromide-coated AuNPs but might also facilitate the loading and delivery of drugs. Herein, three types of rod-like gold-mesoporous silica nanoparticles (termed bare AuNPs, core-shell Au@mSiO2NPs, and Janus Au@mSiO2NPs) were specially designed, and the effects of these AuNPs on cellular uptake, toxic behavior, and mechanism were then systematically studied. Our results indicate that bare AuNPs exerted higher toxicity than the Au@mSiO2NPs and that Janus Au@mSiO2NPs exhibited the lowest toxicity in human breast cancer MCF-7 cells, consistent with the endocytosis capacity of the nanoparticles, which followed the order, bare AuNPs > core-shell Au@mSiO2NPs > Janus Au@mSiO2NPs. More importantly, the AuNPs-induced apoptosis of MCF-7 cells exhibited features that were characteristic of intracellular reactive oxygen species (ROS) generation, activation of c-Jun-N-terminal kinase (JNK) phosphorylation, an enhanced Bax-to-Bcl-2 ratio, and loss of the mitochondrial membrane potential. Simultaneously, cytochrome c was released from mitochondria, and the caspase-3/9 cascade was activated. Moreover, both ROS scavenger (N-acetylcysteine) and JNK inhibitor (SP600125) partly blocked the induction of apoptosis in all AuNPs-treated cells. Taken together, these findings suggest that all AuNPs induce apoptosis through the ROS-/JNK-mediated mitochondrial pathway. Thus, Janus Au@mSiO2NPs exhibit the potential for applications in biomedicine, thus aiding the clinical translation of AuNPs.

Mucin 1 gene silencing inhibits the growth of SMMC-7721 human hepatoma cells through Bax-mediated mitochondrial and caspase-8-mediated death receptor apoptotic pathways.

Mucin 1 (MUC1) is an oncogene that has a crucial role in the pathogenesis and progression of the majority of epithelial malignant tumors. Our previous study demonstrated that MUC1 gene silencing inhibited the growth of SMMC­7721 cells in vitro and in vivo, however, whether this growth inhibition is associated with apoptotic cell death remains to be elucidated. In the present study, it was found that MUC1 gene silencing not only resulted in the inhibition of SMMC­7721 cell growth, determined using a clone formation assay in vitro and a tumor xenograft mouse model with an in vivo imaging system, but also induced apoptotic alterations in SMMC­7721 cells, determined using Hoechst 33342 staining, flow cytometry with an Annexin V-PE staining and a DNA ladder assay. Further investigation using western blotting revealed that cytochrome c was released from the mitochondria into the cytoplasm, and caspase­8 and caspase­9 were activated in MUC1 gene­silenced SMMC­7721 cells. The pro­apoptotic protein Bcl­2­associated X protein (Bax) and the tumor suppressor p53 were increased, while the anti­apoptotic protein B­cell lymphoma 2 was decreased in MUC1 gene­silenced cells. In addition, results from the co­immunoprecipitation experiments demonstrated that the MUC1 cytoplasmic tail can bind directly to Bax or caspase­8 and these interactions were reduced upon MUC1 gene silencing in SMMC­7721 cells. The above results indicate that MUC1 gene silencing induces growth inhibition in SMMC­7721 cells through Bax­mediated mitochondrial and caspase-8-mediated death receptor apoptotic pathways.

Mucin1 promotes the migration and invasion of hepatocellular carcinoma cells via JNK-mediated phosphorylation of Smad2 at the C-terminal and linker regions.

Mucin1 (MUC1), as an oncogene, plays a key role in the progression and tumorigenesis of many human adenocarcinomas. In this study, wound-healing, transwell migration and matrigel invasion assays showed that MUC1 promotes human hepatocellular carcinoma (HCC) cell migration and invasion by MUC1 gene silencing and overexpressing. Treatment with exogenous transforming growth factor beta (TGF-ß)1, TGF-ß type I receptor (TßRI) inhibitor, TGF-ß1 siRNAs, or activator protein 1 (AP-1) inhibitor to MUC1-overexpressing HCC cells revealed that MUC1-induced autocrine TGF-ß via JNK/AP-1 pathway promotes the cell migration and invasion. In addition, the migration and invasion of HCC cells were more significantly inhibited by JNK inhibitor compared with that by TßRI inhibitor or TGF-ß1 siRNAs. Further studies demonstrated that MUC1-mediated JNK activation not only enhances the phosphorylation of Smad2 C-terminal at Ser-465/467 site (Smad2C) through TGF-ß/TßRI, but also directly enhances the phosphorylation of Smad2 linker region at Ser-245/250/255 site (Smad2L), and then both of them collaborate to upregulate matrix metalloproteinase (MMP)-9-mediated cell migration and invasion of HCC. These results indicate that MUC1 is an attractive target in liver cancer therapy.

Escherichia coli Maltose-Binding Protein Induces M1 Polarity of RAW264.7 Macrophage Cells via a TLR2- and TLR4-Dependent Manner.

Maltose-binding protein (MBP) is a critical player of the maltose/maltodextrin transport system in Escherichia coli. Our previous studies have revealed that MBP nonspecifically induces T helper type 1 (Th1) cell activation and activates peritoneal macrophages obtained from mouse. In the present study, we reported a direct stimulatory effect of MBP on RAW264.7 cells, a murine macrophage cell line. When stimulated with MBP, the production of nitric oxide (NO), IL-1ß, IL-6 and IL-12p70, and the expressions of CD80, MHC class II and inducible nitric oxide synthase (iNOS) were all increased in RAW264.7 cells, indicating the activation and polarization of RAW264.7 cells into M1 macrophages induced by MBP. Further study showed that MBP stimulation upregulated the expression of TLR2 and TLR4 on RAW264.7 cells, which was accompanied by subsequent phosphorylation of IκB-α and p38 MAPK. Pretreatment with anti-TLR2 or anti-TLR4 antibodies largely inhibited the phosphorylation of IκB-α and p38 MAPK, and greatly reduced MBP-induced NO and IL-12p70 production, suggesting that the MBP-induced macrophage activation and polarization were mediated by TLR2 and TLR4 signaling pathways. The observed results were independent of lipopolysaccharide contamination. Our study provides a new insight into a mechanism by which MBP enhances immune responses and warrants the potential application of MBP as an immune adjuvant in immune therapies.