YAF2 exerts anti-apoptotic effect in human tumor cells in a FANK1- and phosphorylation-dependent manner.

YY1-associated factor 2 (YAF2) was frequently reported to modulate target gene transcription through both epigenetic and non-epigenetic means. However, other mechanisms were also utilized by YAF2 to carry out its biological functions. Here, we demonstrated that YAF2 from human tumor and non-tumor cells were mainly expressed as Serine 167 phosphorylated form. Further studies showed that the phosphorylated YAF2 up-regulated while its knockdown by specific siRNAs reduced fibronectin type III and ankyrin repeat domains 1 (FANK1) protein level. Mechanistic exploration disclosed that phosphorylated YAF2 inhibit proteasomal degradation of polyubiquitinated FANK1, leading to its increased stability. We then validated their interaction, and displayed that the FN3 domain of FANK1 binds to amino-terminal of YAF2. Functional studies showed that phosphorylated YAF2 inhibits tumor cell apoptosis in a FANK1-dependent manner. Taken together, our current findings demonstrated that phosphorylated YAF2 exhibits anti-apoptotic activity through targeting FANK1 expression in human tumor cells.

[Clinical significance of RYBP expression in primary hepatocellular carcinoma].

OBJECTIVE: To explore the clinical significance of the altered expression of polycomb group (PcG)-associated protein RYBP in hepatocellular carcinoma (HCC) specimens.
 Methods: The expression levels of RYBP in tumor tissues and adjacent normal tissues in 77 HCC cases were detected by immunohistochemistry (IHC), and the relationships between RYBP expression levels and HCC clinicopathological characteristics, five-year survival rates or prognosis of HCC patients were analyzed.
 Results: RYBP expression level was significantly decreased in HCC tumor tissues than that in the adjacent normal tissues (P<0.05). The expression levels of RYBP in HCC specimens were highly correlated with HBsAg, ALT, GGT, Type III procollagen, tumor size, distant metastasis, and tumor differentiation (P<0.05). The RFS and OS for patients with RYBP-low expression were markedly lower than those with RYPB-high expression (P<0.05). Both age and RYBP expression level were protective factors for RFS, while GGT, lymph node metastasis, TNM stage, tumor differentiation and tumor size were risk factors for RFS (P<0.05). As to OS, RYBP expression level was a protective factor, while tumor number, ALT, GGT, AFP, pCEA, lymph node metastasis, TNM stage, tumor differentiation and tumor size were risk factors (P<0.05). The age, GGT, lymph node metastasis and TNM stage were independent prognostic factors for RFS (P<0.05), and both lymph node metastasis and TNM stage were independent risk factors for OS (P<0.05). Comparing to serum alpha fetoprotein (AFP) level, RYBP expression level in tumor tissues was applied to predict the prognosis of HCC patients more accurately.
 Conclusion: PcG associated protein RYBP displays a reduced expression in HCC tissues, which is related to poor prognosis of HCC patients. It might be a promising therapeutic target for HCC treatment.

ETS1 targets RYBP transcription to inhibit tumor cell proliferation.

ETS1 (E26 transformation specific-1) is the founding member of ETS transcriptional factor family. It transcriptionally modulates numerous gene expressions, and is involved in cellular differentiation, tissue remodeling, angiogenesis, drug resistance and tumorigenesis. ETS1 is usually regarded as an oncogene. However, its apoptosis-inducing activity was also frequently reported. Here, we identified RYBP (Ring1 and YY1 binding protein), a critical epigenetic regulator and apoptosis enhancer, as a novel transcriptional target of ETS1. Specifically, we found that overexpression of ETS1 up-regulates the promoter activity of RYBP in HEK293T and tumor cells from different tissue origins, indicating a universal transcriptional regulatory effect. Subsequently, both overexpression and RNA interfering experiments demonstrated that ETS1 positively modulates RYBP expression from both mRNA and protein levels. Bioinformatics analysis combined with site-directed mutagenesis suggested that there probably exist a multiple of ETS1 binding sites in RYBP promoter region, and chromatin immunoprecipitation assay validates the physical association between ETS1 protein and RYBP promoter region. Functional studies showed that ectopic expression of ETS1 significantly suppresses tumor cell proliferation. However, this inhibitory effect was partially compromised when RYBP was concomitantly knocked down by its specific short hairpin RNA. Meanwhile, we provide evidence to demonstrate that cyclin-dependent kinase inhibitor p21 is possibly involved in this regulatory loop. Taken together, our current study identified RYBP as a new transcriptional target which is utilized by ETS1 to carry out its tumor cell growth inhibitory effect.

RYBP modulates stability and function of Ring1B through targeting UBE3A.

Ring1 and yin yang 1-binding protein (RYBP) are central components of noncanonical polycomb-repressive complex 1 (nc-PRC1), which represses target gene expression and is required for normal organismal development. However, the molecular function of RYBP in this complex is obscure. In this study, we showed that RYBP inhibits the polyubiquitination-mediated proteasomal degradation of Ring1B independently of its ubiquitin (Ub)-protein isopeptide ligase (E3) ligase activity, leading to its stabilization and increased catalytic activity toward monoubiquitination of histone H2A at lysine 119. Mechanistic dissection further disclosed that RYBP directly binds to ubiquitin protein ligase E3A (UBE3A) to promote its ubiquitination and proteasomal degradation in an autoubiquitination-independent manner. The resultant reduction of UBE3A protein level alleviates its effect on ubiquitination-mediated degradation of Ring1B, therefore resulting in increased stability and enhanced transcriptional repressor activity on its target genes. Thus, our current findings lay a foundation for understanding how RYBP functions in nc-PRC1 complexes, which is involved in development, stem cell maintenance, and carcinogenesis.-Li, M., Zhang, S., Zhao, W., Hou, C., Ma, X., Li, X., Huang, B., Chen, H., Chen, D. RYBP modulates stability and function of Ring1B through targeting UBE3A.

[Expression, purification and characterization of N-glycosylation mutant human IFN-λ1 in Pichia pastoris].

IFN-λ1 is a member of a new family of interferons called type Ⅲ IFNs with similar functions to type ⅠIFNs. Previously we obtained recombinant soluble human rhIFN-λ1 from Pichia pastoris. However, the hyper-glycosylation from P. pastoris brings immunogenicity and low purification recovery rate. To overcome this disadvantage, in this study, we constructed an rhIFN-λ1 mutant (rhIFN-λ1-Nm) devoid of the potential N-glycosylation sites by site-directed mutagenesis. rhIFN-λ1-Nm was successfully expressed and secreted extracellularly in P. pastoris (GS115) using methanol inducible AOX1 promoter with α-mating factor signal sequence. rhIFN-λ1-Nm was purified and characterized. There was no significant difference between rhIFN-λ1-Nm and rhIFN-λ1 in structure and bioactivity. The molecular weight was low after N-glycosylation mutation whereas the glycosylation was much lower. The mutational rhIFN-λ1 (rhIFN-λ1-Nm) could legitimately be developed as substitutes for rhIFN-λ1, and thus it may be developed into a more promising therapeutic reagent in the future.

Ran binding protein 9 (RanBPM) binds IFN-λR1 in the IFN-λ signaling pathway.

Like the type I interferons (IFNs), the recently discovered cytokine IFN-λ displays antiviral, antiproliferative, and proapoptotic activities, mediated by a heterodimeric IFN-λ receptor complex composed of a unique IFN-λR1 chain and the IL-10R2 chain. However, the molecular mechanism of the IFN-λ-regulated pathway remains unclear. In this study, we newly identified RAN-binding protein M (RanBPM) as a binding partner of IFN-λR1. The interaction between RanBPM and IFN-λR1 was identified with a glutathione S-transferase pull-down assay and coimmunoprecipitation experiments. IFN-λ1 stimulates this interaction and affects the cellular distribution of RanBPM. However, the interaction between RanBPM and IFN-λR1 does not correlate with their conserved TRAF6-binding sites. Furthermore, we also found that RanBPM is a scaffolding protein with a modulatory function that regulates the activities of IFN-stimulated response elements. Therefore, RanBPM plays a novel role in the IFN-λ-regulated signaling pathway.

Tumor suppressor RYBP harbors three nuclear localization signals and its cytoplasm-located mutant exerts more potent anti-cancer activities than corresponding wild type.

Ectopically expressed Ring1 and YY1 binding protein (RYBP) induces tumor cell apoptosis through promoting the formation of the death-inducing signaling complex (DISC) in the cytoplasm. However, transiently overexpressed as well as endogenous RYBP in tumor tissues were observed to be mainly located in the nucleus while that in adjacent non-tumor tissues distributed majorly in the cytoplasm. Currently, we do not know the nuclear localization signals and biological function of different subcellular location of RYBP. In this study, we employed bioinformatic analysis, deletion, point mutation, enhanced green fluorescence protein (EGFP) fusion and others, to investigate the elements responsible for RYBP nuclear import and to explore the anti-tumor activities of cytoplasm- and nuclear-located RYBP. Herein, we identified three functional monopartite nuclear localization signals (NLSs), all of which located at the N-terminus of RYBP. Through four basic amino acid replacements within the NLSs, we obtained a cytoplasm-located RYBP mutant (RYBPmut). Compared with wild-type counterpart, RYBPmut exhibited more potent abilities to bind to caspase 8, to prevent MDM2-mediated polyubiquitination and degradation of p53, thereby leading to its stabilization. Further investigation revealed that, in contrast to its wild type, RYBPmut showed more potentials to inhibit tumor cell proliferation and to induce apoptosis, in both p53-dependent and -independent manner. Collectively, our current study revealed the molecular mechanism responsible for RYBP nuclear translocation, and provided evidences to support that RYBPmut could be a more promising candidate agent for cancer treatment.

RYBP Expression Is Regulated by KLF4 and Sp1 and Is Related to Hepatocellular Carcinoma Prognosis.

The expression of Ring1- and YY1-binding protein (RYBP) is reduced in several human cancers, but the molecular mechanism(s) have remained elusive. In this study, we used human hepatocellular carcinoma (HCC) cell lines and tissue specimens to study the mechanism and herein report several new findings. First, we cloned and characterized the basal promoter region of the human RYBP gene. We found that the decreased RYBP expression in HCC tissues was not due to promoter sequence variation/polymorphisms or CpG dinucleotide methylation. We identified two transcription factors, KLF4 and Sp1, which directly bind the promoter region of RYBP to induce and suppress RYBP transcription, respectively. We mapped the binding sites of KLF4 and Sp1 on the RYBP promoter. Studies in vitro showed that KLF4 suppresses whereas Sp1 promotes HCC cell growth through modulating RYBP expression. Deregulated KLF4 and Sp1 contributed to decreased expression of RYBP in HCC tumor tissues. Our studies of human HCC tissues indicated that a diminished RYBP level in the tumor (in association with altered KLF4 and Sp1 expression) was statistically associated with a larger tumor size, poorer differentiation, and an increased susceptibility to distant metastasis. These findings help to clarify why RYBP is decreased in HCC and indicate that deregulated KLF4, Sp1, and RYBP may lead to a poorer prognosis. Our findings support the idea that RYBP may represent a target for cancer therapy and suggest that it may be useful as a prognostic biomarker for HCC, either alone or in combination with KLF4 and Sp1.

Proapoptotic RYBP interacts with FANK1 and induces tumor cell apoptosis through the AP-1 signaling pathway.

Ring1 and YY1 Binding Protein (RYBP) induces tumor-specific cell apoptosis, but the underlying molecular mechanism has not been fully understood. Here we conducted a yeast two hybrid screen and identified FANK1 (Fibronectin type III and ankyrin repeat domains 1) as a novel RYBP-interacting protein. This interaction was confirmed by coimmunoprecipitation, GST pulldown and immunofluorescence assays. We mapped that the FNIII domain at the N-terminal of FANK1 binds to the Serine/Threonine-rich region at the C-terminal of RYBP. Further studies showed that overexpression of RYBP stabilized, whereas knockdown of RYBP by its specific shRNAs reduced, the expression of FANK1. Mechanistic studies revealed that RYBP inhibited the proteasome degradation of polyubiquitinated FANK1, thus prolonging the half-life of FANK1 protein. Functional studies indicated that RYBP activates FANK1-mediated activator protein 1 (AP-1) signaling pathway which contributes to tumor cell apoptosis. Taken together, our current study uncovered a new mechanism which RYBP utilizes to exert its pro-apoptotic activity in human tumor cells.

Suppression of hepatocellular carcinoma proliferation and hepatitis B surface antigen secretion with interferon-λ1 or PEG-interferon-λ1.

Hepatocellular carcinoma (HCC) is a common cancer associated with chronic hepatitis B virus (HBV) infection. Conventional interferon-α (IFN-α) and pegylated IFNs (PEG-IFNs) approved for chronic HBV infection treatment can reduce the risk of HCC but are not suitable for the majority of patients and cause significant side effects. IFN-λ1 is a type III IFN with antiviral, antiproliferative, and immunomodulatory functions similar to type I IFNs but with fewer side effects. However, the tolerability and antitumor activity of PEG-IFN-λ1 in HCC xenograft mice are unknown. In vitro IFN-λ1 treatment of Hep3B and Huh7 human hepatoma cell lines increased MHC class I expression, activated JAK-STAT signaling pathways, induced IFN-stimulated gene expression, and inhibited hepatitis B surface antigen (HBsAg) expression. IFN-λ1 treatment also caused 23.2 and 19.9% growth inhibition of Hep3B and Huh7 cells, respectively, and promoted cellular apoptosis. PEG-IFN-λ1, but not IFN-λ1 treatment, significantly suppressed tumor growth (P=0.002) and induced tumor cell apoptosis in a Hep3B cell xenograft mouse model without significant weight loss or toxicity. PEG-IFN-λ1 also significantly inhibited (P=0.000) serum HBsAg secretion from Hep3B xenograft tumors in vivo. Thus, PEG-IFN-λ1 can suppress Hep3B xenograft tumor growth and inhibit HBsAg production and may be a potential treatment for HBV-related HCC.

RanBPM interacts with TßRI, TRAF6 and curbs TGF induced nuclear accumulation of TßRI.

Transforming growth factor ß (TGF-ß), a cytokine, and its receptors play a vital role during normal embryogenesis, cell proliferation, differentiation, apoptosis and migration. Ran-binding protein in the microtubule-organizing center (RanBPM) serves as a scaffold protein that has been shown to interact with many other proteins, such as MET, Axl/Sky, TRAF6, IFNR, TrKA and TrkB in addition to p75NTR. In the current study, we have identified RanBPM as a novel binding partner of TßRI by yeast two-hybrid assay. The TßRI and RanBPM association was confirmed by co-immunoprecipitation and GST pull-down experiments. Additionally, expression of RanBPM abrogated the interaction between TßRI and TRAF6. Furthermore, RanBPM could depress TGF-ß induced TRAF6 ubiquitination, subsequent NF-κB signaling pathway, and block TGF-ß induced TßRI nuclear accumulation. Taken together, our results reveal that RanBPM may modulate TGF-ß-mediated downstream signaling and biological functions.

Interaction of IFNλR1 with TRAF6 regulates NF-κB activation and IFNλR1 stability.

IFNλR1 is a member of the class II cytokine receptor family, and it associates with IL-10R2 to form a functional receptor complex, IFNλR. This receptor complex transduces signals from IFNλs (IFNλ1, IFNλ2, and IFNλ3), promoting antiviral and antiproliferative activities similar to those of type I IFNs. In an effort to further understand signal transduction through IFNλR1, we used bioinformatics analysis and identified a tumor necrosis factor receptor-associated factor 6 (TRAF6)-binding motif in the intracellular domain of IFNλR1. In subsequent immunoprecipitation and GST pull-down assays, IFNλR1 was shown to immunoprecipitate with TRAF6 and was pulled down by GST-TRAF6. Endogenous IFNλR1 and TRAF-6 interaction implies that these proteins really interact in the cells. This interaction was abrogated upon mutation of the TRAF6-binding motif in IFNλR1. Furthermore, the interaction between IFNλR1 and TRAF6 inhibited TRAF6-induced NF-κB activation, likely due to a reduction in TRAF6 autoubiquitination. Moreover, co-expression of IFNλR1 with TRAF6 significantly increased the stability of IFNλR1, thereby prolonging its half-life and enhancing its steady-state level in cultured cells.

The Ran-binding protein RanBPM can depress the NF-κB pathway by interacting with TRAF6.

Ran-binding protein in microtubule-organizing center (RanBPM) has been reported to interact with the neurotrophin receptors p75NTR and TrkA, meanwhile p75NTR and TrkA can also interact with TRAF6. Whether RanBPM interacts directly with TRAF6 has not yet been established. In this study, using a yeast two-hybrid system and glutathione-S: -transferase pull-down assays, we determined that RanBPM binds to the TRAF6 C-terminus through its SPRY motif. Complex formation between overexpressed RanBPM and TRAF6 was also confirmed with a co-immunoprecipitation assay, laser scanning confocal and fluorescence resonance energy transfer. Additional co-immunoprecipitation experiments verified that endogenous RanBPM and TRAF6 interact in several mammalian cell lines. A series of experiments revealed that RanBPM influences TRAF6 ubiquitination and the TRAF6-triggered NF-κB signaling pathway through RanBPM's interaction with TRAF6. These data suggest that RanBPM participates in gene transcription by binding to TRAF6.

Insulin-like growth factor binding protein-6 interacts with the thyroid hormone receptor α1 and modulates the thyroid hormone-response in osteoblastic differentiation.

Insulin-like growth factor binding protein-6 (IGFBP-6) is a member of the insulin-like growth factor binding protein family, which has both Insulin-like growth factor-dependent and independent effects on cell growth. In previous studies, we have shown that recombinant IGFBP-6 could be translocated into the cell nucleus. But the effect in the nucleus of IGFBP-6 is not clear. In the present study, we use multiple methodologies including Glutathione S-transferase pull-down assay, co-immunoprecipitation, fluorescence resonance energy transfer to demonstrate that IGFBP-6 can directly interact with thyroid hormone receptor alpha 1 (TRα1) in vitro and in vivo. We also demonstrate that the DNA-binding domains and Ligand-binding domains of TRα1 and N-terminal domains and C-terminal domains of IGFBP-6 are involved in the interaction. This interaction also can block the formation of TR: retinoid X receptor heterodimers. Furthermore, immunofluorescence co-localization studies show IGFBP-6 and TRα1 could co-localize in the nucleus of the cells. Reporter gene experiment shows that IGFBP-6 negatively regulates the growth hormone promoter activity induced by ligand activated TRα1. Moreover, real-time RT-PCR demonstrates that IGFBP-6 could inhibit the osteocalcin mRNA transcription induced by Triiodothyronine (3,3',5-Triiodo-L-thyronine, T3) in osteoblastic cells. Finally, alkaline phosphatase activity was significantly decreased in osteoblastic cells when the cells were transfected with IGFBP-6 in the presence of T3. In conclusion, these studies provide evidence that overexpression of IGFBP-6 suppresses osteoblastic differentiation regulated by TR in the present of T3.

Antitumor activities of recombinant human interferon (IFN)-λ1 in vitro and in xenograft models in vivo for colon cancer.

Interferon (IFN)-λ1, as a newly identified IFN, interacts with the structurally unique IFN-λ1 receptor complex and exhibits antiviral and antiproliferation effects. The major focus of our work was to study the antitumor activities of recombinant human IFN-λ1 (rhIFN-λ1) in vivo and in vitro. The MTT analysis showed that rhIFN-λ1 inhibited the proliferation of HCT116 cells in a dose-dependent manner, but with a less efficacy in HT29 cells than HCT116 cells. IFN-λ1 also activated the STATs and induced apoptosis in both types of cells. In the in vivo study, we found that rhIFN-λ1 suppressed tumor growth in a dose-dependent fashion, with an inhibition rate of 52% of HCT116 (P<0.01) and 56% of HT29 (P<0.01). These results indicate that rhIFN-λ1 could be a new potential IFN reagent for cancer therapy.

[The recent progress in the studies of vitamin D receptor].

Vitamin D receptor (VDR) is a nuclear receptor which can regulate the expression of many kinds of genes by combining with its ligand. This article is about the recent progress in the studies of VDR, including its co-activator, co-repressor, and the function in development, differentiation, immuno-regulation and cancer repressor. In the end, we summarized the recent progress in research of drug development about the analog of VDR ligand.

A novel interaction between insulin-like growth factor binding protein-6 and the vitamin D receptor inhibits the role of vitamin D3 in osteoblast differentiation.

Insulin-like growth factor binding protein-6 (IGFBP-6) is a secreted glycoprotein that reduces the bioavailability of IGFs. It has both IGF-dependent and -independent effects on cell growth, however the mechanisms responsible for its IGF-independent actions of IGFBP-6 are not fully understood. In previous studies, we have shown that recombinant IGFBP-6 can be internalized and translocated to the nucleus. The present study shows that IGFBP-6 interacts with the vitamin D receptor (VDR). Physical interactions between IGFBP-6 and the VDR were confirmed by GST pulldown and co-immunoprecipitation assays. We also determined that the interaction binding sites were on the C-terminal region of the VDR. This interaction can influence retinoid X receptor (RXR):VDR heterodimerization. Furthermore, immunofluorescence colocalization studies showed that IGFBP-6 colocalized with the VDR predominantly in the cell's nucleus. Inductions of osteocalcin and growth hormone promoter activities by 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) were significantly decreased when cells were co-transfected with IGFBP-6 and the VDR compared with cells transfected with the VDR only. Moreover, we found that alkaline phosphatase activity (ALP, a general marker of osteoblast differentiation) was significantly decreased in osteoblast-like cells when they were transfected with IGFBP-6 in the presence of 1,25(OH)(2)D(3). No obvious difference in ALP activity was observed when cells were transfected with IGFBP-6 and endogenous VDR was knocked down by siRNA. These results demonstrate that IGFBP-6 inhibits osteoblastic differentiation mediated by 1,25(OH)(2)D(3) and the VDR through interacting with the VDR and inhibiting its function. This is a novel mechanism for IGFBP-6.

Insulin-like growth factor binding protein-3 interacts with the thyroid hormone receptor α1 and modulates transcription of thyroid hormone responsive gene / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the interaction between insulin-like growth factor binding protein-3 (IGFBP-3) and thyroid hormone receptor α1 (TRα1) and the modulatory effect of IGFBP-3 on transcription of the thyroid hormone responsive gene.</p><p><b>METHODS</b>The interaction between IGFBP-3 and TRα1 was detected with glutathione-S-transferase pull-down method, co-immunoprecipitation, fluorescence resonance energy transfer test. The cellular distribution of these two proteins was observed by confocal laser scanning microscopy. The effect of IGFBP-3 on the growth hormone promoter activity stimulated by triiodothyronine (T3) was determined by dual-luciferase reporter assay.</p><p><b>RESULTS</b>IGFBP-3 interacted with TRα1 both in vivo and in vitro. IGFBP-3 and TRα1 were shown to co-localize in the nucleus of HEK-293 cells. The overexpressed IGFBP-3 inhibited the growth hormone promoter activity stimulated by T3 (P<0.01).</p><p><b>CONCLUSIONS</b>IGFBP-3 interacts with TRα1 and inhibits T3 responsive gene transcription. This finding further confirms the insulin-like growth factor-independent role of IGFBP-3 in the nucleus.</p>