Dual Inhibition of DKC1 and MEK1/2 Synergistically Restrains the Growth of Colorectal Cancer Cells.

Colorectal cancer, one of the most commonly diagnosed cancers worldwide, is often accompanied by uncontrolled proliferation of tumor cells. Dyskerin pseudouridine synthase 1 (DKC1), screened using the genome-wide RNAi strategy, is a previously unidentified key regulator that promotes colorectal cancer cell proliferation. Enforced expression of DKC1, but not its catalytically inactive mutant D125A, accelerates cell growth in vitro and in vivo. DKC1 knockdown or its inhibitor pyrazofurin attenuates cell proliferation. Proteomics, RNA immunoprecipitation (RIP)-seq, and RNA decay analyses reveal that DKC1 binds to and stabilizes the mRNA of several ribosomal proteins (RPs), including RPL10A, RPL22L1, RPL34, and RPS3. DKC1 depletion significantly accelerates mRNA decay of these RPs, which mediates the oncogenic function of DKC1. Interestingly, these DKC1-regulated RPs also interact with HRAS and suppress the RAS/RAF/MEK/ERK pathway. Pyrazofurin and trametinib combination synergistically restrains colorectal cancer cell growth in vitro and in vivo. Furthermore, DKC1 is markedly upregulated in colorectal cancer tissues compared to adjacent normal tissues. Colorectal cancer patients with higher DKC1 expression has consistently poorer overall survival and progression-free survival outcomes. Taken together, these data suggest that DKC1 is an essential gene and candidate therapeutic target for colorectal cancer.

Inhibition of DKC1 induces telomere-related senescence and apoptosis in lung adenocarcinoma.

BACKGROUND: Lung cancer is one of the most widely spread cancers in the world and half of the non-small cell lung cancers are lung adenocarcinoma (LUAD). Although there were several drugs been approved for LUAD therapy, a large portion of LUAD still cannot be effectively treated due to lack of available therapeutic targets. Here, we investigated the oncogenic roles of DKC1 in LUAD and its potential mechanism and explored the possibility of targeting DKC1 for LUAD therapy. METHODS: The Gene Expression Omnibus (GEO) and The Cancer Genome Atlas Program (TCGA) databases were used to examine the DKC1 transcript levels. Gene expression with clinical information from tissue microarray of LUAD were analyzed for associations between DKC1 expression and LUAD prognosis. In addition, loss- and gain-of-function assays were used for oncogenic function of DKC1 both in vitro and in vivo. RESULTS: DKC1 is overexpressed in LUAD compared with adjacent normal tissues. High expression of DKC1 predicts the poor overall survival. DKC1 knockdown in LUAD cell lines induced G1 phase arrest and inhibited cell proliferation. Ectopic expression of DKC1 could rescue the growth of LUAD cell lines. In addition, the abundance of DKC1 is positively correlated with telomerase RNA component (TERC) and telomerase reverse transcriptase (TERT) levels in LUAD. DKC1 downregulation resulted in decreased TERC expression, reduced telomerase activity and shorten telomere, and thus eventually led to cell senescence and apoptosis. CONCLUSIONS: Our results show that high DKC1 expression indicates poor prognosis of LUAD and DKC1 downregulation could induce telomere-related cell senescence and apoptosis. This study suggests that DKC1 could serve as a candidate diagnostic biomarker and therapeutic target for LUAD.

RNAi Screening Identifies that TEX10 Promotes the Proliferation of Colorectal Cancer Cells by Increasing NF-κB Activation.

Colorectal cancer (CRC) has become a predominant cancer worldwide. To understand the process of carcinogenesis, a short hairpin RNA library screening is employed to search for candidate genes that promote proliferation in the CRC cell line HT29. The candidate genes overlap with differentially expressed genes in 32 CRC tumor tissues in the GEO dataset GSE8671. The seventh-ranked testis expressed 10 (TEX10) is upregulated in CRC and its knockdown decreases cell proliferation. The TEX10 high-expression group exhibits worse overall survival (P = 0.003) and progression-free survival (P = 0.001) than the TEX10 low-expression group. TEX10 depletion decreases the growth of CRC cells in vitro and in vivo. Gene set enrichment analysis indicates that the nuclear factor-kappa B pathway is significantly enriched in the genes downregulated by TEX10 knockdown. Mechanistically, TEX10 interacts with RELA and increases its nuclear localization. TEX10 promotes RELA occupancy at gene promoters and regulates the expression of a subset of RELA-targeted genes, including TNFAIP8, SAT1, and IL6ST. Taken together, this study identifies that TEX10 promotes the proliferation of CRC cells in an RELA-dependent manner. In addition, high TEX10 expression is associated with poor prognosis in CRC patients.

ARHGEF19 regulates MAPK/ERK signaling and promotes the progression of small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor survival. To identify genes that differentially expressed in SCLC with tumor promotion activity as candidate therapeutic targets, we analyzed the expression of 50 RhoGEFs family genes in published microarray data of SCLC and normal tissues (Gene Expression Omnibus (GEO) dataset GSE43346). We identified ARHGEF19, a member of RhoGEFs family, as an overexpressed oncogene in SCLC. ARHGEF19 is up-regulated in SCLC tissues and ranks first in RhoGEFs family genes. Enforced ARHGEF19 expression promotes SCLC cell proliferation in vitro and its knockdown decreases cell proliferation in vitro and in vivo. ARHGEF19-DH and -PD domain interacts with HRAS and activates the MAPK/ERK pathway in SCLC cells and SCLC xenografts. Our study presents evidences that ARHGEF19 overexpression promotes SCLC cell growth and activates the MAPK/ERK pathway. These findings would shed light on the development of new therapeutics for SCLC management.

CALML6 Controls TAK1 Ubiquitination and Confers Protection against Acute Inflammation.

Proper regulation of innate immune response is important for individual health. The NF-κB signaling pathway plays crucial roles in innate immunity and inflammation, and its aberrant activation is implicated in diverse diseases and disorders. In this study, we report that calmodulin-like 6 (CALML6), a member of the EF-hand protein family, is a negative regulator of the NF-κB signaling pathway. CALML6 attenuated TNF-stimulated phosphorylation of proteins downstream of TGF-ß-activated kinase 1 (TAK1) and inhibited TAK1-induced NF-κB activation. Further studies showed that CALML6 interacted with TAK1 and recruited the deubiquitylating enzyme cylindromatosis to repress the K63-linked polyubiquitination of TAK1. CALML6 transgenic mice had higher tolerances to lethal LPS treatment in vivo. These findings suggest that CALML6 is a negative regulator of the NF-κB signaling pathway, which is important for maintaining the balance of the innate immune response.

A MicroRNA Signature in Gestational Diabetes Mellitus Associated with Risk of Macrosomia.

BACKGROUND/AIMS: MicroRNA (miRNA) is a small non-coding RNA molecule that functions in regulation of gene expression by targeting mRNA to affect its stability and/or translation. The aim of this study was to evaluate the miRNAs involvement in gestational diabetes mellitus (GDM), a well known risk factor for fetal overgrowth. METHODS: Differential microRNA expression in placental tissues of normal controls and women with GDM were identified by miRNA micorarray analysis and further confirmed by quantitative real-time PCR (qRT-PCR) on an independent set of normal and GDM placental tissues. Target genes of microRNAs were bioinformatically predicted and verified in vitro by Western blotting. RESULTS: Our results uncovered 9 miRNAs that were significantly deregulated in GDM samples: miR-508-3p was up-regulated and miR-27a, miR-9, miR-137, miR-92a, miR-33a, miR-30d, miR-362-5p and miR-502-5p were down-regulated. Bioinformatic approaches revealed that the microRNAs signature identifies gene targets involved in EGFR (epidermal growth factor receptor)-PI3K (phosphoinositide 3-Kinase)-Akt (also known as protein kinase B) pathway, a signal cascade which plays important roles in placental development and fetal growth. We found that the protein levels of EGFR, PI3K and phospho-Akt were up-regulated and PIKfyve (a FYVE finger-containing phosphoinositide kinase), a negative regulator of EGFR signaling, was down-regulated significantly in GDM tissues. We also confirmed PIKfyve was a direct target of miR-508-3p. CONCLUSION: Our data identified a miRNA signature involvement in GDM which may contribute to macrosomia through enhancing EGFR signaling.

SOCS3 Drives Proteasomal Degradation of TBK1 and Negatively Regulates Antiviral Innate Immunity.

TANK-binding kinase 1 (TBK1)-mediated induction of type I interferon (IFN) plays a critical role in host antiviral responses and immune homeostasis. The negative regulation of TBK1 activity is largely unknown. We report that suppressor of cytokine signaling 3 (SOCS3) inhibits the IFN-ß signaling pathway by promoting proteasomal degradation of TBK1. Overexpression and knockdown experiments indicated that SOCS3 is a negative regulator of IFN regulatory factor 3 (IRF3) phosphorylation and IFN-ß transcription. Moreover, SOCS3 directly associates with TBK1, and they colocalize in the cytoplasm. SOCS3 catalyzes K48-linked polyubiquitination of TBK1 at Lys341 and Lys344 and promotes subsequent TBK1 degradation. On the contrary, SOCS3 knockdown markedly increases the abundance of TBK1. Interestingly, both the BOX domain of SOCS3 and Ser172 phosphorylation of TBK1 are indispensable for the processes of ubiquitination and degradation. Ectopic expression of SOCS3 significantly inhibits vesicular stomatitis virus (VSV) and influenza A virus strain A/WSN/33 (WSN)-induced IRF3 phosphorylation and facilitates the replication of WSN virus by detecting the transcription of its viral RNA (vRNA). Knockdown of SOCS3 represses WSN replication. Collectively, these results demonstrate that SOCS3 acts as a negative regulator of IFN-ß signal by ubiquitinating and degrading TBK1, shed light on the understanding of antiviral innate immunity, and provide a potential target for developing antiviral agents.

Wedelolactone disrupts the interaction of EZH2-EED complex and inhibits PRC2-dependent cancer.

Polycomb repressive complex 2 (PRC2), which is responsible for the trimethylation of H3K27 (H3K27me3), plays a part in tumorigenesis, development and/or maintenance of adult tissue specificity. The pivotal role of PRC2 in cancer makes it a therapeutic target for epigenetic cancer therapy. However, natural compounds targeting the enhancer of zeste homolog 2 (EZH2) - embryonic ectoderm development (EED) interaction to disable PRC2 complex are scarcely reported. Here, we reported the screening and identification of natural compounds which could disrupt the EZH2-EED interaction. One of these compounds, wedelolactone, binds to EED with a high affinity (KD = 2.82 µM), blocks the EZH2-EED interaction in vitro, induces the degradation of PRC2 core components and modulates the expression of detected PRC2 downstream targets and cancer-related genes. Furthermore, some PRC2-dependent cancer cells undergone growth arrest upon treatment with wedelolactone. Thus, wedelolactone and its derivatives which target the EZH2-EED interaction could be candidates for the treatment of PRC2-dependent cancer.

[Expression, purification of recombinant human cryptochrome I and its application in preparation of protective agent for radiotherapy].

Radiotherapy is a treatment for cancer with undesired by-effects. In order to develop a new radiation protective agent that could reduce the by-effects, we tried to express and purify human cryptochrome 1 (hCRY1). The coding sequence of hCRY1 was inserted into prokaryotic expression plasmid pET28a(+), and this protein was purified from Escherichia coli BL21(DE3) after IPTG induction, ultrasonication, inclusion body dissolution, gradient dialysis, nickel column purification and ultrafiltration. The yield of hCRY1 in 1 L E. coli culture (LB medium) was about 10-15 mg. The radiation protective efficiency of hCRY1 was monitored by detecting X-ray-induced H2A.X foci in HaCaT cells. The results of immunofluorescence show that hCRY1 significantly reduces X-ray stimulated DNA damage response. The apoptosis of HaCaT cell was also detected, and the repression of H2A.X foci formation was not due to hCRY1's cytotoxity. All these data suggest a potential application of recombinant hCRY1 as a protective agent for radiotherapy.

Interaction of NS2 with AIMP2 facilitates the switch from ubiquitination to SUMOylation of M1 in influenza A virus-infected cells.

UNLABELLED: Influenza A viruses (IAVs) rely on host factors to support their life cycle, as viral proteins hijack or interact with cellular proteins to execute their functions. Identification and understanding of these factors would increase our knowledge of the molecular mechanisms manipulated by the viruses. In this study, we searched for novel binding partners of the influenza A virus NS2 protein, the nuclear export protein responsible for overcoming host range restriction, by a yeast two-hybrid screening assay and glutathione S-transferase-pulldown and coimmunoprecipitation assays and identified AIMP2, a potent tumor suppressor that usually functions to regulate protein stability, as one of the major NS2-binding candidates. We found that the presence of NS2 protected AIMP2 from ubiquitin-mediated degradation in NS2-transfected cells and AIMP2 functioned as a positive regulator of IAV replication. Interestingly, AIMP2 had no significant effect on NS2 but enhanced the stability of the matrix protein M1. Further, we provide evidence that AIMP2 recruitment switches the modification of M1 from ubiquitination to SUMOylation, which occurs on the same attachment site (K242) on M1 and thereby promotes M1-mediated viral ribonucleoprotein complex nuclear export to increase viral replication. Collectively, our results reveal a new mechanism of AIMP2 mediation of influenza virus replication. IMPORTANCE: Although the ubiquitination of M1 during IAV infection has been observed, the precise modification site and the molecular consequences of this modification remain obscure. Here, we demonstrate for the first time that ubiquitin and SUMO compete for the same lysine (K242) on M1 and the interaction of NS2 with AIMP2 facilitates the switch of the M1 modification from ubiquitination to SUMOylation, thus increasing viral replication.