Exon Junction Complex Mediates the Cap-Independent Translation of Circular RNA.

Evidence that circular RNAs (circRNA) serve as protein template is accumulating. However, how the cap-independent translation is controlled remains largely uncharacterized. Here, we show that the presence of intron and thus splicing promote cap-independent translation. By acquiring the exon junction complex (EJC) after splicing, the interaction between circRNA and ribosomes was promoted, thereby facilitating translation. Prevention of splicing by treatment with spliceosome inhibitor or mutating splicing signal hindered cap-independent translation of circRNA. Moreover, EJC-tethering using Cas13 technology reconstituted EJC-dependent circRNA translation. Finally, the level of a coding circRNA from succinate dehydrogenase assembly factor 2 (circSDHAF2) was found to be elevated in the tumorous tissues from patients with colorectal cancer, and shown to be critical in tumorigenesis of colorectal cancer in both cell and murine models. These findings reveal that EJC-dependent control of circSDHAF2 translation is involved in the regulation of oncogenic pathways. IMPLICATIONS: EJC-mediated cap-independent translation of circRNA is implicated in the tumorigenesis of colorectal cancer.

A Novel 3D Culture Scaffold to Shorten Development Time for Multicellular Tumor Spheroids.

Multicellular tumor spheroids and tumoroids are considered ideal in vitro models that reflect the features of the tumor microenvironment. Biomimetic components resembling the extracellular matrix form scaffolds to provide structure to 3-dimensional (3D) culture systems, supporting the growth of both spheroids and tumoroids. Although Matrigel has long been used to support 3D culture systems, batch variations, component complexity, and the use of components derived from tumors are complicating factors. To address these issues, we developed the ACD 3D culture system to provide better control and consistency. We evaluated spheroid and tumoroid formation using the ACD 3D culture system, including the assessment of cell viability and cancer marker expression. Under ACD 3D culture conditions, spheroids derived from cancer cell lines exhibited cancer stem cell characteristics, including a sphere-forming size and the expression of stem cell marker genes. The ACD 3D culture system was also able to support patient-derived primary cells and organoid cell cultures, displaying adequate cell growth, appropriate morphology, and resistance to oxaliplatin treatment. These spheroids could also be used for drug screening purposes. In conclusion, the ACD 3D culture system represents an efficient tool for basic cancer research and therapeutic development.

The Functional Roles and Regulation of Circular RNAs during Cellular Stresses.

Circular RNAs (circRNAs) are a novel class of regulatory RNA involved in many biological, physiological and pathological processes by functioning as a molecular sponge, transcriptional/epigenetic/splicing regulator, modulator of protein-protein interactions, and a template for encoding proteins. Cells are constantly dealing with stimuli from the microenvironment, and proper responses rely on both the precise control of gene expression networks and protein-protein interactions at the molecular level. The critical roles of circRNAs in the regulation of these processes have been heavily studied in the past decades. However, how the microenvironmental stimulation controls the circRNA biogenesis, cellular shuttling, translation efficiency and degradation globally and/or individually remains largely uncharacterized. In this review, how the impact of major microenvironmental stresses on the known transcription factors, splicing modulators and epitranscriptomic regulators, and thereby how they may contribute to the regulation of circRNAs, is discussed. These lines of evidence will provide new insight into how the biogenesis and functions of circRNA can be precisely controlled and targeted for treating human diseases.

CircVIS: a platform for circRNA visual presentation.

BACKGROUND: The collection of circRNAs mostly focused on their sequence composition such as protein/miRNA binding motif, and/or regulatory elements such as internal ribosome entry site. However, less attention was paid to subcellular localization. CircVIS aimed to provide a collection of circRNAs with information of subcellular compartments and also integrated the circRNA entries from previous circRNA databases. RESULTS: A collection of circRNAs from public circRNA databases and de novo identification were annotated according to subcellular localizations including nucleoplasm, chromatin-associated parts, cytoplasm and polyribosome. All circRNAs were aligned to a selected major transcript, and if presence, the circRNA-derived open reading frame with annotation of functional domain were compared to its parental protein. The results showed that distinct circRNAs may exert their molecular and cellular functions in different subcellular compartments. The web service is made freely available at http://lab-x-omics.nchu.edu.tw/circVIS . CONCLUSIONS: CircVIS allows users to visualize the alignment between a given circRNA and its most relevant reference transcript along with information of subcellular localization.

Extracellular vesicle-associated VEGF-C promotes lymphangiogenesis and immune cells infiltration in endometriosis.

Endometriosis is a highly prevalent gynecological disease with severe negative impacts on life quality and financial burden. Unfortunately, there is no cure for this disease, which highlights the need for further investigation about the pathophysiology of this disease to provide clues for developing novel therapeutic regimens. Herein, we identified that vascular endothelial growth factor (VEGF)-C, a potent lymphangiogenic factor, is up-regulated in endometriotic cells and contributes to increased lymphangiogenesis. Bioinformatic analysis and molecular biological characterization revealed that VEGF-C is negatively regulated by an orphan nuclear receptor, chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII). Further studies demonstrated that proinflammatory cytokines, via suppression of COUP-TFII level, induce VEGF-C overexpression. More importantly, we show that functional VEGF-C is transported by extracellular vesicles (EVs) to enhance the lymphangiogenic ability of lymphatic endothelial cells. Autotransplanted mouse model of endometriosis showed lenvatinib treatment abrogated the increased lymphatic vessels development in the endometriotic lesion, enlarged retroperitoneal lymph nodes, and immune cells infiltration, indicating that blocking VEGF-C signaling can reduce local chronic inflammation and concomitantly endometriosis development. Evaluation of EV-transmitted VEGF-C from patients' sera demonstrates it is a reliable noninvasive way for clinical diagnosis. Taken together, we identify the vicious cycle of inflammation, COUP-TFII, VEGF-C, and lymphangiogenesis in the endometriotic microenvironment, which opens up new horizons in understanding the pathophysiology of endometriosis. VEGF-C not only can serve as a diagnostic biomarker but also a molecular target for developing therapeutic regimens.

Oxaliplatin-Induced DHX9 Phosphorylation Promotes Oncogenic Circular RNA CCDC66 Expression and Development of Chemoresistance.

Circular RNA (circRNA), generated through backsplicing in which the downstream splice donor joins the upstream splice acceptor, is a novel class of RNA molecules. Our previous study found that a novel oncogenic circRNA-consisting exon 8-10 of CCDC66-is aberrantly expressed in colorectal cancer (CRC) tissues and cells. The failure of treatment for colorectal cancer is typically associated with recurrent and chemoresistant cancerous tissues. In this study, we aimed to investigate the role(s) of circCCDC66 during the development of chemoresistance. We discovered that the expression level of circCCDC66 is elevated in colorectal cancer cells with resistance to oxaliplatin. Knockdown of circCCDC66 caused the downregulation of a subset of genes which are regulated by circCCDC66-associated miRNAs and related to the modulation of apoptosis and the cell cycle, suppressing cell survival, promoting oxaliplatin-induced apoptosis and, thus, hindering the development of oxaliplatin-resistance (OxR). The induction of circCCDC66 was dependent on the time-course and dose of oxaliplatin treatment. Our analyses revealed that DHX9 harbors two phosphorylation sites of phosphatidylinositol 3-kinase-related kinases (PI3KKs) close to substrate-binding domains. Blockage of phosphorylation by either PI3KK inhibitors or nonphosphorable mutants of DHX9 decreased the oxaliplatin-induced circCCDC66 expression and the ability to develop chemoresistant cells. Taken together, we demonstrated and linked the functional role of DHX9 phosphorylation to oncogenic circCCDC66 expression during the development of resistance to oxaliplatin, providing a mechanistic insight for the development of therapeutic strategies to recurring/chemoresistant colorectal cancer.

Analysis of common targets for circular RNAs.

BACKGROUND: The studies of functions of circular RNAs (circRNAs) are heavily focused on the regulation of gene expression through interactions with multiple miRNAs. However, the number of predicted target genes is typically overwhelming due to the synergistic complexity caused by two factors ─ the binding of multiple miRNAs to a circRNA and the existence of multiple targets for each miRNA. Analysis of common targets (ACT) was designed to facilitate the identification of potential circRNA targets. RESULTS: We demonstrated the feasibility of the proposed feature/measurement to assess which genes are more likely to be regulated by circRNAs with given sequences by calculating the level of co-regulation by multiple miRNAs. The web service is made freely available at http://lab-x-omics.nchu.edu.tw/ACT_Server . CONCLUSIONS: ACT allows users to identify potential circRNA-regulated genes and their associated pathways for further investigation.

Hypoxia: The force of endometriosis.

AIM: Summarize recent findings of how hypoxia regulates numerous important processes to facilitate the implantation, proliferation and progression of ectopic endometriotic lesions. METHODS: Most up-to-date evidences about how hypoxia contributes to the disease pathogenesis of endometriosis and potential therapeutic approaches were collected by conducting a comprehensive search of medical literature electronic databases. Quality of data was analyzed by experienced experts including gynecologist and basic scientists. RESULTS: Uterus is a highly vascularized organ, which makes endometrial cells constantly expose to high concentration of oxygen. When endometrial tissues shed off from the eutopic uterus and retrograde to the peritoneal cavity, they face severe hypoxic stress. Even with successful implantation to ovaries or peritoneum, the hypoxic stress remains as a critical issue because endometrial cells are used to live in the well-oxygenated environment. Under the hypoxia condition, cells undergo epigenetic modulation and evolve several survival processes including steroidogenesis, angiogenesis, inflammation and metabolic switch. The complex gene regulatory network driven by hypoxia ensures endometriotic cells can survive under the hostile peritoneal microenvironment. CONCLUSION: Hypoxia plays critical roles in promoting pathological processes to facilitate the development of endometriosis. Targeting hypoxia-mediated gene network represents an alternative approach for the treatment of endometriosis.

Suppression of COUP-TFII upregulates angiogenin and promotes angiogenesis in endometriosis.

STUDY QUESTION: How does hypoxia-mediated downregulation of chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) promote angiogenesis in endometriosis? SUMMARY ANSWER: Suppression of COUP-TFII by hypoxia stimulates angiogenesis through induction of angiogenin (ANG). WHAT IS KNOWN ALREADY: The level of COUP-TFII is downregulated in endometriotic tissues, and downregulation of COUP-TFII contributes to the development of endometriosis. STUDY DESIGN, SIZE, DURATION: Twenty-seven patients of reproductive age with endometriosis were recruited in this study. Eutopic endometrial and ectopic endometriotic stromal cells were isolated, cultured and subjected to various treatments. PARTICIPANTS/MATERIALS, SETTING, METHODS: Microarray hybridization, quantitative RT-PCR, and Western blot were used to detect gene expression in normal and endometriotic samples. A luciferase reporter assay and chromatin immunoprecipitation in normoxia- or hypoxia-treated primary cultures of human endometrial stromal cells were performed. Tube formation analysis was performed using primary human umbilical vein endothelial cells (HUVECs). MAIN RESULTS AND THE ROLE OF CHANCE: Protein level of COUP-TFII was downregulated by hypoxia (P < 0.05, normoxia versus hypoxia). Loss of COUP-TFII increased the angiogenic capacity of endometrial stromal cells (P < 0.05, COUP-TFII knockdown versus knockdown control). A novel COUP-TFII target gene, ANG, was identified through microarray analysis. Chromatin immunoprecipitation and promoter activity assays demonstrated that the ANG promoter was bound and suppressed by COUP-TFII (P < 0.05, COUP-TFII overexpression versus empty vector). The levels of ANG mRNA and protein were elevated in ectopic endometriotic stromal cells and negatively correlated with COUP-TFII (P < 0.05, endometrial versus endometriotic tissues/stromal cells). Both knockdown and forced-expression of COUP-TFII further demonstrated that ANG expression and ANG-mediated angiogenic activity were negatively regulated by COUP-TFII (P < 0.05, COUP-TFII knockdown versus knockdown control, and COUP-TFII overexpression versus empty vector). LIMITATIONS, REASONS FOR CAUTION: This study was conducted in primary human endometrial stromal cell cultures and HUVECs, therefore, may not fully reflect the situation in vivo. LARGE SCALE DATA: The raw data were submitted to Gene Expression Omnibus (GSE107469). WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to highlight that the aberrant expression of ANG in endometriotic lesions is mediated by hypoxia-suppressed COUP-TFII expression, which reveals an as yet unidentified molecular pathogenesis of endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by research grants (MOST 105-2314-B-006-059-MY3 to M.H.W. and MOST 104-2320-B-006-036-MY3 to S.J.T.) from the Ministry of Science and Technology, Taiwan. The authors declare that there is no conflict of interest.

Epigenetic regulation of the pathological process in endometriosis.

Background: Endometriosis is one of the most common gynecological diseases that greatly compromises the quality of life in affected individuals. A growing body of evidence shows that the remodeling of retrograde endometrial tissues to the ectopic endometriotic lesions involves multiple epigenetic alterations, such as DNA methylation, histone modification, and microRNA expression. Methods: This article retrospectively reviewed the studies that were related to the epigenetic regulatory factors that contribute to the development and maintenance of endometriosis. A literature search was performed in order to collect scientific articles that were written in English by using the key words of "endometriosis," "epigenetics," "DNA methylation," "histone modification," and "microRNA." Results: Epigenetic modifications, including DNA methylation, histone modification, and microRNA expression, are involved in the pathogenesis of endometriosis. These epigenetic players are regulated or tuned by microenvironmental cues, such as locally produced estradiol, proinflammatory cytokines, and hypoxic stress, and reciprocally regulate the process or response to those stimuli. Conclusion: Understanding the molecular mechanisms that underlie these epigenetic regulatory processes would shed light on the etiology and/or progression of endometriosis and facilitate the development of novel therapeutic strategies.

Circular RNA - New member of noncoding RNA with novel functions.

A growing body of evidence indicates that circular RNAs are not simply a side product of splicing but a new class of noncoding RNAs in higher eukaryotes. The progression for the studies of circular RNAs is accelerated by combination of several advanced technologies such as next generation sequencing, gene silencing (small interfering RNAs) and editing (CRISPR/Cas9). More and more studies showed that dysregulated expression of circular RNAs plays critical roles during the development of several human diseases. Herein, we review the current advance of circular RNAs for their biosynthesis, molecular functions, and implications in human diseases. Impact statement The accumulating evidence indicate that circular RNA (circRNA) is a novel class of noncoding RNA with diverse molecular functions. Our review summarizes the current hypotheses for the models of circRNA biosynthesis including the direct interaction between upstream and downstream introns and lariat-driven circularization. In addition, molecular functions such as a decoy of microRNA (miRNA) termed miRNA sponge, transcriptional regulator, and protein-like modulator are also discussed. Finally, we reviewed the potential roles of circRNAs in neural system, cardiovascular system as well as cancers. These should provide insightful information for studying the regulation and functions of circRNA in other model of human diseases.

Hypoxia-inhibited DUSP2 expression promotes IL-6/STAT3 signaling in endometriosis.

PROBLEM: How does hypoxia-mediated downregulation of dual-specificity phosphatase-2 (DUSP2) promote the development of endometriotic lesions? METHOD OF STUDY: The levels of IL-6 and DUSP2 were assessed in eutopic stromal cells with DUSP2 knockdown or hypoxia treatment. Bromodeoxyuridine (BrdU) incorporation was applied for evaluating cell proliferation. The protein levels of DUSP2, cleaved caspase-3, phosphorylated STAT3, and STAT3 were analyzed using immunoblot. RESULTS: The genomewide analysis of cells with DUSP2 overexpression indicated IL-6 regulates multiple pathways related to inflammation, proliferation, and apoptosis. DUSP2 overexpression significantly suppressed IL-6 expression, while DUSP2 knockdown promoted IL-6 expression. The hypoxia-treated eutopic stromal cells expressed higher levels of IL-6, recapitulating the elevated levels of IL-6 in ectopic stromal cells. The treatment with IL-6 elicited the phosphorylation of STAT3, mimicking the elevated levels of phosphorylated STAT3 in the ectopic stromal cells. The IL-6-treated eutopic stromal cells showed more BrdU incorporation and less cleaved caspase-3, which can be reversed by STAT3 inhibitor. CONCLUSION: Hypoxia-induced IL-6 production in endometriotic lesions is mediated via downregulation of DUSP2, which causes aberrant activation of STAT3 signaling pathway and helps the endometriotic cells survive under the ectopic environment.

Noncoding Effects of Circular RNA CCDC66 Promote Colon Cancer Growth and Metastasis.

Circular RNA (circRNA) is a class of noncoding RNA whose functions remain mostly unknown. Recent studies indicate circRNA may be involved in disease pathogenesis, but direct evidence is scarce. Here, we characterize the functional role of a novel circRNA, circCCDC66, in colorectal cancer. RNA-Seq data from matched normal and tumor colon tissue samples identified numerous circRNAs specifically elevated in cancer cells, several of which were verified by quantitative RT-PCR. CircCCDC66 expression was elevated in polyps and colon cancer and was associated with poor prognosis. Gain-of-function and loss-of-function studies in colorectal cancer cell lines demonstrated that circCCDC66 controlled multiple pathological processes, including cell proliferation, migration, invasion, and anchorage-independent growth. In-depth characterization revealed that circCCDC66 exerts its function via regulation of a subset of oncogenes, and knockdown of circCCDC66 inhibited tumor growth and cancer invasion in xenograft and orthotopic mouse models, respectively. Taken together, these findings highlight a novel oncogenic function of circRNA in cancer progression and metastasis. Cancer Res; 77(9); 2339-50. ©2017 AACR.

Loss of dual-specificity phosphatase-2 promotes angiogenesis and metastasis via up-regulation of interleukin-8 in colon cancer.

Dual-specificity phosphatase 2 (DUSP2) is a negative regulator of mitogen-activated protein kinases. Our previous study showed that DUSP2 expression is down-regulated in many human cancers and loss of DUSP2 promotes cancer progression; however, the underlying mechanism remains largely uncharacterized. Herein, we found that loss of DUSP2 induces angiogenesis, while forced expression of DUSP2 inhibits microvessel formation in xenografted mouse tumours. Genome-wide screening of expression profiles, and meta-analysis of clinical data, identified that the level of interleukin-8 (IL-8) correlated negatively with that of DUSP2, suggesting that it may be a downstream target of DUSP2. Molecular characterization revealed that DUSP2 inversely regulates IL-8 expression, mediated by ERK1/2 and C/EBPα-dependent transcriptional regulation. Further study showed that hypoxia-induced IL-8 expression in cancer cells is also mediated via down-regulation of DUSP2. Treatment with the IL-8 receptor inhibitor reparixin or knockdown of IL-8 in cancer cells abolished angiogenesis induced by loss of DUSP2. Functionally, knockdown of DUSP2 enhanced tumour growth and metastasis, which were abolished by treatment with reparixin or knockdown of IL-8 in an orthotopic mouse model. Taken together, our results demonstrate that hypoxia inhibits DUSP2 expression in colon cancer, leading to up-regulation of IL-8, which facilitates angiogenesis and tumour metastasis. Our findings suggest that blocking hypoxia-DUSP2-IL-8 signalling may be a plausible approach for therapeutic intervention in cancer. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Coordination of AUF1 and miR-148a destabilizes DNA methyltransferase 1 mRNA under hypoxia in endometriosis.

STUDY HYPOTHESIS: DNA methylation is regulated by hypoxia in endometriosis. STUDY FINDING: Hypoxia causes global hypomethylation through AU-rich element binding factor 1 (AUF1)/microRNA-148a (miR-148a)-mediated destabilization of DNA methyltransferase 1 (DNMT1) mRNA. WHAT IS KNOWN ALREADY: Eutopic endometrial and ectopic endometriotic stromal cells have the same genetic background, but differ in several cellular and molecular responses. Both hypoxia and DNA methylation regulate several genes involved in the development of endometriosis. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: This laboratory study included 15 patients of reproductive age with endometriosis or normal menstrual cycles. Paired endometrial and endometriotic tissues were collected for assaying the levels of DNMT1, 3a and 3b using quantitative RT-PCR, western blot and immunohistochemical (IHC) staining. Primary cultured endometrial stromal cells maintained in normoxia/hypoxia (1% O2) or treated with hypoxia-mimetic compounds were also assayed. The levels of DNA 5-methylcytosine were assayed by using IHC in clinical specimens and murine tissues, and by ELISA in cultured stromal cells. The 3'-untranslated region reporter assay was used to evaluate the effect of hypoxia, microRNAs (miRNAs) and human antigen R (HuR)/AUF1 on DNMT1 mRNA stability. RNA immunoprecipitation was used to assess the interaction of HuR/AUF1 and miR-148a/DNMT1 mRNA under hypoxia. Finally, a transplant-induced mouse model of endometriosis using 20 mice was used to elucidate the alteration of Dnmt1 levels and DNA methylation in the endometriotic tissues. MAIN RESULTS AND THE ROLE OF CHANCE: Levels of DNMT1 mRNA and protein and 5-methylcytosine were lower in the ectopic stromal cells (P < 0.05) than in the eutopic cells. Treatment with hypoxia and its mimetic compounds recapitulated the reduced levels of DNMT1 and 5-methylcytosine levels (P < 0.05 versus control). Hypoxia treatment destabilized DNMT1 mRNA through recruitment of miR-148a and AUF1. Mutations introduced to the miR-148a targeting site or AU-rich element (ARE) restored the hypoxia-suppressed DNMT1 3'-untranslated region (3'-UTR) reporter activity (P < 0.05 versus control). Levels of proteins of three hypermethylated genes in endometrial stroma cells, GATA6, HOXA3 and SLC16A5, were elevated after 72 h of hypoxia treatment (P < 0.05 versus control). Finally, a transplant-induced model of endometriosis demonstrated the down-regulation of DNMT1 and a decrease in 5-methylcytosine in the endometriotic tissues (P < 0.05, eutopic versus ectopic). LIMITATIONS, REASONS FOR CAUTION: Primary human cell cultures and a murine model were used in this study, and thus the results may not fully represent the situation in vivo. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to elucidate how microenvironmental hypoxia links to the epigenetic effects of DNA methylation in the endometriosis, and to delineate the molecular mechanism of hypoxia-coordinated AUF1/miR-148a interaction and recruitment to DNMT1 mRNA during the pathogenesis of endometriosis. The development of future therapeutics in endometriosis may aim at disrupting this specific interaction and eventually restore the epigenetic regulation. STUDY FUNDING AND COMPETING INTERESTS: This work was supported by the National Science Council of Taiwan (NSC101-2320-B-006-030-MY3). The author declares that there are no conflicts of interest.

Pathological functions of hypoxia in endometriosis.

Endometriosis is one of the most common gynecological diseases that significantly reduce the life quality of affected women. Research results from the past decade clearly demonstrated that aberrant production of estrogen and cyclooxygenase-2-derived prostaglandin E2 play indispensable roles in the pathogenesis of this disease. However, the etiology of endometriosis remains obscure. Recent evidence reveals a new facet of endometriotic pathogenesis by showing that hypoxia induces the expression of many important downstream genes to regulate the implantation, survival, and maintenance of ectopic endometriotic lesions. These new findings shed lights on future investigations of delineating the etiology of endometriosis and designing new therapeutic strategy for endometriosis.

Inhibition of dual specificity phosphatase-2 by hypoxia promotes interleukin-8-mediated angiogenesis in endometriosis.

STUDY QUESTION: How does hypoxia-mediated down-regulation of dual specificity phosphatase-2 (DUSP2) promote endometriotic lesion development? SUMMARY ANSWER: Inhibition of DUSP2 by hypoxia enhances endometriotic lesion growth via promoting interleukin-8 (IL-8)-dependent angiogenesis. WHAT IS KNOWN ALREADY: Angiogenesis is a prerequisite for the development of endometriosis. DUSP2 is down-regulated in endometriotic stromal cells in a hypoxia inducible factor-1α-dependent manner. Down-regulation of DUSP2 contributes to the pathological process of endometriosis. STUDY DESIGN, SIZE, DURATION: A laboratory study recruiting 20 patients of reproductive age with endometriosis and normal menstrual cycles, and an autoimplant-induced mouse model of endometriosis using 13 mice in a 28-day treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: IL-8 mRNA levels were assayed in endometrial stromal cells maintained in normoxic or hypoxic (1% O2) conditions, with or without DUSP2 knockdown. Promoter activity and chromatin immunoprecipitation (ChIP) assays were conducted to characterize the regulation of IL-8 by DUSP2. Conditioned media from cells maintained in normoxic or hypoxic conditions, and cells with/without DUSP2 knockdown were collected to investigate the angiogenic capacity using an in vitro tube formation assay. Reparixin, an IL-8 receptor blocker, was administered to investigate the role of IL-8 in hypoxia-mediated angiogenesis and the development of endometriotic-like lesions in an autotransplanted mouse model. MAIN RESULTS AND THE ROLE OF CHANCE: IL-8 mRNA was increased by both hypoxia and DUSP2 knockdown in endometrial stromal cells in an extracellular signal-regulated protein kinase-dependent manner (P < 0.05 versus control). Promoter activity and ChIP assays demonstrated that expression of IL-8 was regulated by CCAAT/enhancer binding protein α (P < 0.05 versus control). Furthermore, conditioned media collected from hypoxia-exposed or DUSP2 knockdown endometrial stromal cells promoted tube formation, which was abolished by co-treatment with reparixin (P < 0.05 versus control). Results from the autotransplanted mouse model demonstrated that number of blood vessels and size of endometriotic-like lesions were markedly reduced in recipient mice treated with reparixin (P < 0.05 versus control). LIMITATIONS, REASONS FOR CAUTION: This study was conducted in primary human cell cultures and a mouse model, therefore may not fully reflect the situation in vivo. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to highlight the potential application of an IL-8 receptor blocker as a therapeutic target to treat endometriosis. This study demonstrates IL-8 as a key angiogenic factor regulated by hypoxia/DUSP2, which suggests an alternative mechanism through which hypoxia may promote angiogenesis. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the National Science Council of Taiwan (NSC101-2314-B-006-043-MY2). The author declares that there is no conflict of interest.

Histone H4 deacetylation facilitates 53BP1 DNA damage signaling and double-strand break repair.

53BP1 and other DNA damage response (DDR) proteins form foci at double-strand breaks (DSBs) which promote their repair by nonhomologous end joining (NHEJ). Focal accumulation of 53BP1 depends on the specific interaction of its tandem Tudor domain with dimethylated lysine 20 in histone H4 (H4K20me2). How 53BP1 foci dynamics are regulated is unclear since H4K20me2 is highly abundant, established largely in the absence of DNA damage, and uncertainty exists about the roles of candidate H4K20 methyltransferases in 53BP1 foci formation. Here, we show that 53BP1 foci assemble primarily on H4K20me2 established prior to DNA damage by the SETD8 and SUV420 methyltransferases rather than de novo H4K20 methylation mediated by MMSET/WHSC1. Moreover, we define a novel role for H4K16 acetylation in regulating 53BP1 foci dynamics. Concurrent acetylation at H4K16 antagonizes 53BP1 binding to extant H4K20me2 until DSBs elicit transient, localized H4 deacetylation that facilitates 53BP1 foci formation and NHEJ, and is associated with global repression of gene transcription. Our findings demonstrate that rapid induction of H4 deacetylation by DSBs affects multiple aspects of the DDR, and also suggest that antagonism of 53BP1 binding to H4K20me2 by H4K16 hyperacetylation may contribute to the efficacy of histone deacetylase inhibitors for cancer therapy.

Hypoxia-inhibited dual-specificity phosphatase-2 expression in endometriotic cells regulates cyclooxygenase-2 expression.

Endometriosis is one of the most common gynaecological diseases that significantly reduces the life qualify of affected women and their families. Aberrant expression of cyclooxygenase-2 (COX-2), and thus over-production of prostaglandin E(2) (PGE(2) ) has been shown to play critical roles in the development of this disease. However, the mechanism responsible for COX-2 over-expression remains obscure. Here, we provide evidence for what we believe is a novel mechanism in regulating COX-2 expression in endometriotic stromal cells. Dual-specificity phosphatase-2 (DUSP2), a nuclear phosphatase that inactivates mitogen-activated protein kinase (MAPK), is markedly down-regulated in stromal cells of ectopic endometriotic tissues, which results in prolonged activation of extracellular signal-regulated kinase (ERK) and p38 MAPK and increased COX-2 expression. Expression of DUSP2 is inhibited by hypoxia inducible factor-1α (HIF-1α) at the transcriptional level. Treatment of normal endometrial stromal cells with hypoxia, or chemicals that cause HIF-1α accumulation, results in DUSP2 down-regulation, prolonged ERK phosphorylation and COX-2 over-expression. In contrast, forced expression of DUSP2 under hypoxia abolishes HIF-1α-induced ERK phosphorylation and COX-2 expression. Furthermore, suppression of DUSP2 by HIF-1α in eutopic endometrial stromal cells increases sensitivity of cox-2 gene to interleukin-1ß stimulation, a phenomenon resembling endometriotic stromal cell characteristics. Taken together, these data suggest that DUSP2 is an important molecule in endometrial physiology and that hypoxia-inhibited DUSP2 expression is a critical factor for the development of endometriosis.