DNA fragility at topologically associated domain boundaries is promoted by alternative DNA secondary structure and topoisomerase II activity.

CCCTC-binding factor (CTCF) binding sites are hotspots of genome instability. Although many factors have been associated with CTCF binding site fragility, no study has integrated all fragility-related factors to understand the mechanism(s) of how they work together. Using an unbiased, genome-wide approach, we found that DNA double-strand breaks (DSBs) are enriched at strong, but not weak, CTCF binding sites in five human cell types. Energetically favorable alternative DNA secondary structures underlie strong CTCF binding sites. These structures coincided with the location of topoisomerase II (TOP2) cleavage complex, suggesting that DNA secondary structure acts as a recognition sequence for TOP2 binding and cleavage at CTCF binding sites. Furthermore, CTCF knockdown significantly increased DSBs at strong CTCF binding sites and at CTCF sites that are located at topologically associated domain (TAD) boundaries. TAD boundary-associated CTCF sites that lost CTCF upon knockdown displayed increased DSBs when compared to the gained sites, and those lost sites are overrepresented with G-quadruplexes, suggesting that the structures act as boundary insulators in the absence of CTCF, and contribute to increased DSBs. These results model how alternative DNA secondary structures facilitate recruitment of TOP2 to CTCF binding sites, providing mechanistic insight into DNA fragility at CTCF binding sites.

KIX domain determines a selective tumor-promoting role for EP300 and its vulnerability in small cell lung cancer.

EP300, a transcription coactivator important in proliferation and differentiation, is frequently mutated in diverse cancer types, including small cell lung cancer (SCLC). While these mutations are thought to result in loss of EP300 function, the impact on tumorigenesis remains largely unknown. Here, we demonstrate that EP300 mutants lacking acetyltransferase domain accelerate tumor development in mouse models of SCLC. However, unexpectedly, complete Ep300 knockout suppresses SCLC development and proliferation. Dissection of EP300 domains identified kinase inducible domain-interacting (KIX) domain, specifically its interaction with transcription factors including MYB, as the determinant of protumorigenic activity. Ala627 in EP300 KIX results in a higher protein-binding affinity than Asp647 at the equivalent position in CREBBP KIX, underlying the selectivity of KIX-binding partners for EP300. Blockade of KIX-mediated interactions inhibits SCLC development in mice and cell growth. This study unravels domain-specific roles for EP300 in SCLC and unique vulnerability of the EP300 KIX domain for therapeutic intervention.

YAP1 overexpression contributes to the development of enzalutamide resistance by induction of cancer stemness and lipid metabolism in prostate cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is a malignant and lethal disease caused by relapse after androgen-deprivation (ADT) therapy. Since enzalutamide is innovated and approved by US FDA as a new treatment option for mCRPC patients, drug resistance for enzalutamide is a critical issue during clinical usage. Although several underlying mechanisms causing enzalutamide resistance were previously identified, most of them revealed that drug resistant cells are still highly addicted to androgen and AR functions. Due to the numerous physical functions of AR in men, innovated AR-independent therapy might alleviate enzalutamide resistance and prevent production of adverse side effects. Here, we have identified that yes-associated protein 1 (YAP1) is overexpressed in enzalutamide-resistant (EnzaR) cells. Furthermore, enzalutamide-induced YAP1 expression is mediated through the function of chicken ovalbumin upstream promoter transcription factor 2 (COUP-TFII) at the transcriptional and the post-transcriptional levels. Functional analyses reveal that YAP1 positively regulates numerous genes related to cancer stemness and lipid metabolism and interacts with COUP-TFII to form a transcriptional complex. More importantly, YAP1 inhibitor attenuates the growth and cancer stemness of EnzaR cells in vitro and in vivo. Finally, YAP1, COUP-TFII, and miR-21 are detected in the extracellular vesicles (EVs) isolated from EnzaR cells and sera of patients. In addition, treatment with EnzaR-EVs induces the abilities of cancer stemness, lipid metabolism and enzalutamide resistance in its parental cells. Taken together, these results suggest that YAP1 might be a crucial factor involved in the development of enzalutamide resistance and can be an alternative therapeutic target in prostate cancer.

DUSP2 regulates extracellular vesicle-VEGF-C secretion and pancreatic cancer early dissemination.

Early dissemination is a unique characteristic and a detrimental process of pancreatic ductal adenocarcinoma (PDAC); however, the underlying mechanism remains largely unknown. Here, we investigate the role of dual-specificity phosphatase-2 (DUSP2)-vascular endothelial growth factor-C (VEGF-C) axis in mediating PDAC lymphangiogenesis and lymphovascular invasion. Expression of DUSP2 is greatly suppressed in PDAC, which results in increased aberrant expression of extracellular vesicle (EV)-associated VEGF-C secretion. EV-VEGF-C exerts paracrine effects on lymphatic endothelial cells and autocrine effects on cancer cells, resulting in the lymphovascular invasion of cancer cells. Tissue-specific knockout of Dusp2 in mouse pancreas recapitulates PDAC phenotype and lymphovascular invasion. Mechanistically, loss-of-DUSP2 enhances proprotein convertase activity and vesicle trafficking to promote the release of the mature form of EV-VEGF-C. Collectively, these findings represent a conceptual advance in understanding pancreatic cancer lymphovascular invasion and suggest that loss-of-DUSP2-mediated VEGF-C processing may play important roles in early dissemination of pancreatic cancer. Abbreviations: DUSP2: dual-specificity phosphatase-2; VEGF-C: vascular endothelial growth factor-C; EV: extracellular vesicles; PDAC: pancreatic ductal adenocarcinoma; KD: knockdown.

Sepsis-associated acute respiratory distress syndrome in individuals of European ancestry: a genome-wide association study.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a lung inflammatory process caused mainly by sepsis. Most previous studies that identified genetic risks for ARDS focused on candidates with biological relevance. We aimed to identify novel genetic variants associated with ARDS susceptibility and to provide complementary functional evidence of their effect in gene regulation. METHODS: We did a case-control genome-wide association study (GWAS) of 1935 European individuals, using patients with sepsis-associated ARDS as cases and patients with sepsis without ARDS as controls. The discovery stage included 672 patients admitted into a network of Spanish intensive care units between January, 2002, and January, 2017. The replication stage comprised 1345 individuals from two independent datasets from the MESSI cohort study (Sep 22, 2008-Nov 30, 2017; USA) and the VISEP (April 1, 2003-June 30, 2005) and MAXSEP (Oct 1, 2007-March 31, 2010) trials of the SepNet study (Germany). Results from discovery and replication stages were meta-analysed to identify association signals. We then used RNA sequencing data from lung biopsies, in-silico analyses, and luciferase reporter assays to assess the functionallity of associated variants. FINDINGS: We identified a novel genome-wide significant association with sepsis-associated ARDS susceptibility (rs9508032, odds ratio [OR] 0·61, 95% CI 0·41-0·91, p=5·18 × 10-8) located within the Fms-related tyrosine kinase 1 (FLT1) gene, which encodes vascular endothelial growth factor receptor 1 (VEGFR-1). The region containing the sentinel variant and its best proxies acted as a silencer for the FLT1 promoter, and alleles with protective effects in ARDS further reduced promoter activity (p=0·0047). A literature mining of all previously described ARDS genes validated the association of vascular endothelial growth factor A (VEGFA; OR 0·55, 95% CI 0·41-0·73; p=4·69 × 10-5). INTERPRETATION: A common variant within the FLT1 gene is associated with sepsis-associated ARDS. Our findings support a role for the vascular endothelial growth factor signalling pathway in ARDS pathogenesis and identify VEGFR-1 as a potential therapeutic target. FUNDING: Instituto de Salud Carlos III, European Regional Development Funds, Instituto Tecnológico y de Energías Renovables.

Novel idiopathic pulmonary fibrosis susceptibility variants revealed by deep sequencing.

BACKGROUND: Specific common and rare single nucleotide variants (SNVs) increase the likelihood of developing sporadic idiopathic pulmonary fibrosis (IPF). We performed target-enriched sequencing on three loci previously identified by a genome-wide association study to gain a deeper understanding of the full spectrum of IPF genetic risk and performed a two-stage case-control association study. METHODS: A total of 1.7 Mb of DNA from 181 IPF patients was deep sequenced (>100×) across 11p15.5, 14q21.3 and 17q21.31 loci. Comparisons were performed against 501 unrelated controls and replication studies were assessed in 3968 subjects. RESULTS: 36 SNVs were associated with IPF susceptibility in the discovery stage (p<5.0×10-8). After meta-analysis, the strongest association corresponded to rs35705950 (p=9.27×10-57) located upstream from the mucin 5B gene (MUC5B). Additionally, a novel association was found for two co-inherited low-frequency SNVs (<5%) in MUC5AC, predicting a missense amino acid change in mucin 5AC (lowest p=2.27×10-22). Conditional and haplotype analyses in 11p15.5 supported the existence of an additional contribution of MUC5AC variants to IPF risk. CONCLUSIONS: This study reinforces the significant IPF associations of these loci and implicates MUC5AC as another key player in IPF susceptibility.

Hypoxia-Induced Downregulation of DUSP-2 Phosphatase Drives Colon Cancer Stemness.

Cancer stem-like cells (CSC) evolve to overcome the pressures of reduced oxygen, nutrients or chemically induced cell death, but the mechanisms driving this evolution are incompletely understood. Here, we report that hypoxia-mediated downregulation of the dual specificity phosphatase 2 (DUSP2) is critical for the accumulation of CSC in colorectal cancer. Reduced expression of DUSP2 led to overproduction of COX-2-derived prostaglandin E2, which promoted cancer stemness via the EP2/EP4 signaling pathways. Genetic and pharmacological inhibition of PGE2 biosynthesis or signal transduction ameliorated loss-of-DUSP2-induced tumor growth and cancer stemness. Genome-wide profile analysis revealed that genes regulated by DUSP2 were similar to those controlled by histone deacetylase. Indeed, treatment with novel histone deacetylase inhibitors abolished hypoxia-induced DUSP2 downregulation, COX-2 overexpression, cancer stemness, tumor growth, and drug resistance. Our findings illuminate mechanisms of cancer stemness and suggest new cancer therapy regimens. Cancer Res; 77(16); 4305-16. ©2017 AACR.

Targeting hypoxia-mediated YAP1 nuclear translocation ameliorates pathogenesis of endometriosis without compromising maternal fertility.

Endometriosis is a highly prevalent gynaecological disease that severely reduces women's health and quality of life. Ectopic endometriotic lesions have evolved mechanisms to survive in the hypoxic peritoneal microenvironment by regulating the expression of a significant subset of genes. However, the master regulator controlling these genes remains to be characterized. Herein, by using bioinformatics analysis and experimental verification, we identified yes-associated protein 1 (YAP1) as a master regulator of endometriosis. Nuclear localization and transcriptional activity of YAP1 were up-regulated by hypoxia via down-regulation of LATS1, a kinase that inactivates YAP1. Disruption of hypoxia-induced YAP1 signalling by siRNA knockdown or inhibitor treatment abolished critical biological processes involved in endometriosis development such as steroidogenesis, angiogenesis, inflammation, migration, innervation, and cell proliferation. Treatment with a YAP1 inhibitor caused the regression of endometriotic lesions without affecting maternal fertility or the growth rate of offspring in the mouse model of endometriosis. Taken together, we identify hypoxia/LATS1/YAP1 as a novel pathway for the pathogenesis of endometriosis and demonstrate that targeting YAP1 might be an alternative approach to treat endometriosis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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.

Targeted methylation of two tumor suppressor genes is sufficient to transform mesenchymal stem cells into cancer stem/initiating cells.

Although DNA hypermethylation within promoter CpG islands is highly correlated with tumorigenesis, it has not been established whether DNA hypermethylation within a specific tumor suppressor gene (TSG) is sufficient to fully transform a somatic stem cell. In this study, we addressed this question using a novel targeted DNA methylation technique to methylate the promoters of HIC1 and RassF1A, two well-established TSGs, along with a two-component reporter system to visualize successful targeting of human bone marrow-derived mesenchymal stem cells (MSC) as a model cell system. MSCs harboring targeted promoter methylations of HIC1/RassF1A displayed several features of cancer stem/initiating cells including loss of anchorage dependence, increased colony formation capability, drug resistance, and pluripotency. Notably, inoculation of immunodeficient mice with low numbers of targeted MSC resulted in tumor formation, and subsequent serial xenotransplantation and immunohistochemistry confirmed the presence of stem cell markers and MSC lineage in tumor xenografts. Consistent with the expected mechanism of TSG hypermethylation, treatment of the targeted MSC with a DNA methyltransferase inhibitor reversed their tumorigenic phenotype. To our knowledge, this is the first direct demonstration that aberrant TSG hypermethylation is sufficient to transform a somatic stem cell into a fully malignant cell with cancer stem/initiating properties.