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Abstract Background MicroRNAs (miRNAs) are important regulators involved in diverse physiological and pathological processes including cancer. SUMO (small ubiquitin-like modifier) is a reversible protein modifier. We recently found that SUMOylation of TARBP2 and DGCR8 is involved in the regulation of the miRNA pathway. KHSRP is a single stranded nucleic acid binding protein with roles in transcription and mRNA decay, and it is also a component of the Drosha-DGCR8 complex promoting the miRNA biogenesis. Methods The in vivo SUMOylation assay using the Ni2+-NTA affinity pulldown or immunoprecipitation (IP) and the in vitro E.coli-based SUMOylation assay were used to analyze SUMOylation of KHSRP. Nuclear/Cytosol fractionation assay and immunofluorescent staining were used to observe the localization of KHSRP. High-throughput miRNA sequencing, quantantive RT-PCR and RNA immunoprecipitation assay (RIP) were employed to determine the effects of KHSRP SUMO1 modification on the miRNA biogenesis. The soft-agar colony formation, migration, vasculogenic mimicry (VM) and three-dimensional (3D) cell culture assays were performed to detect the phenotypes of tumor cells in vitro, and the xenograft tumor model in mice was conducted to verify that SUMO1 modification of KHSRP regulated tumorigenesis in vivo. Results KHSRP is modified by SUMO1 at the major site K87, and this modification can be increased upon the microenvironmental hypoxia while reduced by the treatment with growth factors. SUMO1 modification of KHSRP inhibits its interaction with the pri-miRNA/Drosha-DGCR8 complex and probably increases its translocation from the nucleus to the cytoplasm. Consequently, SUMO1 modification of KHSRP impairs the processing step of pre-miRNAs from pri-miRNAs which especially harbor short G-rich stretches in their terminal loops (TL), resulting in the downregulation of a subset of TL-G-Rich miRNAs such as let-7 family and consequential tumorigenesis. Conclusions Our data demonstrate how the miRNA biogenesis pathway is connected to tumorigenesis and cancer progression through the reversible SUMO1 modification of KHSRP.

Background: It is known that the MDM2 protein is stabilized when it forms a heterodimer with its partner MDM4, but MDM2 protein stability in its homodimer form is not known. The MDM2 protein contains a C-terminal RING domain that not only functions as an E3 ligase to regulate ubiquitination of p53 and MDM2 itself, but also is characterized to be able to bind several specific cellular mRNAs to regulate gene expression. In this study, we evaluate whether the MDM2 protein stability is regulated by the binding of a specific small RNA (XIAP IRES mRNA). Methods: We performed chemical cross-linking and bimolecular fluorescence complementation (BiFC) assay to measure the human MDM2 protein stability in its homodimer form and the effect of XIAP IRES on MDM2 homodimerization and protein stabilization. Ubiquitination and pulse-chase assays were used to detect MDM2 self-ubiquitination and protein turn-over. Fluorescent titration and ITC were used to examine the binding between MDM2 RING protein and XIAP IRES. Western blot assay was used for determining protein expression. Clonogenic assay, WST and flow cytometry were used to test the effects of XIAP IRES, siXIAP and IR on cancer cell growth and apoptosis. Results: We found that self-association (homodimerization) of MDM2 occurs through the C-terminal RING domain of MDM2 and that the MDM2 protein becomes unstable when it is homodimerized. MDM2 homodimerization resulted in an increased function of the RING domain for MDM2 self-ubiquitination. Binding of XIAP IRES to the RING domain inhibited MDM2 homodimerization and self-ubiquitination, which resulted in stabilization of MDM2, as well as increased XIAP expression. Upregulation of XIAP and MDM2 that led to inhibition of p53 by the XIAP IRES resulted in cell growth and survival in both p53-normal and -deficient cancer cells. Conclusions: Our study identified a new IRES RNA that interacts with MDM2 protein and regulates its stabilization, which suggested that targeting of MDM2 through disruption of MDM2 protein-RNA interaction might be a useful strategy for developing novel anti-cancer therapeutics. [ABSTRACT FROM AUTHOR]

Background: A safe and effective adjuvant plays an important role in the development of a vaccine. However, adjuvants licensed for administration in humans remain limited. Here, for the first time, we developed a novel combination adjuvant alum-polysaccharide-HH2 (APH) with potent immunomodulating activities, consisting of alum, polysaccharide of Escherichia coli and the synthetic cationic innate defense regulator peptide HH2. Methods: The adjuvant effects of APH were examined using NY-ESO-1 protein-based vaccines in prophylactic and therapeutic models. We further determined the immunogenicity and anti-tumor effect of NY-ESO-1-APH (NAPH) vaccine using adoptive cellular/serum therapy in C57/B6 and nude mice. Cell-mediated and antibody-mediated immune responses were evaluated. Results: The APH complex significantly promoted antigen uptake, maturation and cross-presentation of dendritic cells and enhanced the secretion of TNF-α, MCP-1 and IFN-γ by human peripheral blood mononuclear cells compared with individual components. Vaccination of NAPH resulted in significant tumor regression or delayed tumor progression in prophylactic and therapeutic models. In addition, passive serum/cellular therapy potently inhibited tumor growth of NY-ESO-1-B16. Mice treated with NAPH vaccine produced higher antibody titers and greater antibody-dependent/ independent cellular cytotoxicity. Therefore, NAPH vaccination effectively stimulated innate immunity, and boosted both arms of the adaptive humoral and cellular immune responses to suppress tumorigenesis and growth of melanoma. Conclusions: Our study revealed the potential application of APH complex as a novel immunomodulatory agent for vaccines against tumor refractory and growth. [ABSTRACT FROM AUTHOR]

Background Rectal cancer is one of the most prevalent tumor types. Understanding the metabolic profile of rectal cancer is important for developing therapeutic approaches and molecular diagnosis. Methods Here, we report a metabonomics profiling of tissue samples on a large cohort of human rectal cancer subjects (n = 127) and normal controls (n = 43) using 1H nuclear magnetic resonance (1H NMR) based metabonomics assay, which is a highly sensitive and non-destructive method for the biomarker identification in biological systems. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal projection to latent structure with discriminant analysis (OPLS-DA) were applied to analyze the 1H-NMR profiling data to identify the distinguishing metabolites of rectal cancer. Results Excellent separation was obtained and distinguishing metabolites were observed among the different stages of rectal cancer tissues (stage I = 35; stage II = 37; stage III = 37 and stage IV = 18) and normal controls. A total of 38 differential metabolites were identified, 16 of which were closely correlated with the stage of rectal cancer. The up-regulation of 10 metabolites, including lactate, threonine, acetate, glutathione, uracil, succinate, serine, formate, lysine and tyrosine, were detected in the cancer tissues. On the other hand, 6 metabolites, including myo-inositol, taurine, phosphocreatine, creatine, betaine and dimethylglycine were decreased in cancer tissues. These modified metabolites revealed disturbance of energy, amino acids, ketone body and choline metabolism, which may be correlated with the progression of human rectal cancer. Conclusion Our findings firstly identify the distinguishing metabolites in different stages of rectal cancer tissues, indicating possibility of the attribution of metabolites disturbance to the progression of rectal cancer. The altered metabolites may be as potential biomarkers, which would provide a promising molecular diagnostic approach for clinical diagnosis of human rectal cancer. The role and underlying mechanism of metabolites in rectal cancer progression are worth being further investigated. [ABSTRACT FROM AUTHOR]