Expanding Actin Rings Zipper the Mouse Embryo for Blastocyst Formation

Friday, 23 Mar 2018

Authors
Jennifer Zenker^1,6, Melanie D. White^1,6, Maxime Gasnier^1,6, Yanina D. Alvarez^1,2,6, Hui Yi Grace Lim¹, Stephanie Bissiere¹, Maté Biro^3,4,*, Nicolas Plachta^1,5,*

Author Affiliations
¹ Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR),   Singapore. 
² Institute for Molecular Biosciences and Centre for Microscopy and Microanalysis, University of   Queensland, Brisbane, Queensland, Australia. 
³ Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-University Munich, Munich,   Germany. 
⁴ Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore,   Singapore.

^* Corresponding author. Email: plachtan@imcb.a-star.edu.sg

Published in Cell on 19 April 2018.

Abstract 
Transformation from morula to blastocyst is a defining event of preimplantation embryo development. During this transition, the embryo must establish a paracellular permeability barrier to enable expansion of the blastocyst cavity. Here, using live imaging of mouse embryos, we reveal an actin zippering mechanism driving this embryo sealing. Preceding blastocyst stage, a cortical F-actin ring assembles at the apical pole of the embryo’s outer cells. The ring structure forms when cortical actin flows encounter a network of polar microtubules that exclude F-actin. Unlike stereotypical actin rings, the actin rings of the mouse embryo are not contractile, but instead expand to the cell-cell junctions. Here they couple to the junctions by recruiting and stabilizing adherens and tight junction components. Coupling of the actin rings triggers localized myosin II accumulation and initiates a tension–dependent zippering mechanism along the junctions, which is required to seal the embryo for blastocyst formation.

Figure

Legend for image: Actin rings in early mouse embryo

For more information on Nicolas PLACHTA's lab, please click here.

Single-cell analyses of human islet cells reveal de-differentiation signatures

Monday, 12 Feb 2018

Authors
Adrian Keong Kee Teo^1,2,3,4, Chang Siang Lim¹, Lih Feng Cheow⁵, Tatsuya Kin⁶, James A. Shapiro⁶, Nam-Young Kang⁷, William Burkholder⁵ and Hwee Hui Lau¹

Author Affiliations
¹ Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology,
  Proteos, Singapore, Singapore.
² School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
³ Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore,
  Singapore.
⁴ Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore.
⁵ Microfluidics Systems Biology Laboratory, Institute of Molecular and Cell Biology, Proteos, Singapore,   Singapore.
⁶ Department of Biomedical Engineering, National University of Singapore, Singapore, Singapore.
⁷ Clinical Islet Laboratory, University of Alberta Hospital, Edmonton, AB, Canada.
⁸ Laboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium, Helios, Singapore,   Singapore.
* Correspondence: Adrian Keong Kee Teo: ateo@imcb.a-star.edu.sg

Published online in Cell Death Discovery on 9 February 2018.

Abstract
Human pancreatic islets containing insulin-secreting β-cells are notoriously heterogeneous in cell composition. Since β-cell failure is the root cause of diabetes, understanding this heterogeneity is of paramount importance. Recent reports have cataloged human islet transcriptome but not compared single β-cells in detail. Here, we scrutinized ex vivo human islet cells from healthy donors and show that they exhibit de-differentiation signatures. Using single cell gene expression and immunostaining analyses, we found healthy islet cells to contain polyhormonal transcripts, and INS+ cells to express decreased levels of β-cell genes but high levels of progenitor markers. Rare cells that are doubly positive for progenitor markers/exocrine signatures, and endocrine/exocrine hormones were also present. We conclude that ex vivo human islet cells are plastic and can possibly de-/trans-differentiate across pancreatic cell fates, partly accounting for β-cell functional decline once isolated. Therefore, stabilizing β-cell identity upon isolation may improve its functionality.

Figure


Figure legend: Summary figure highlighting de-differentiation signatures in human pancreatic cells. Single-cell gene expression analyses performed on six batches of isolated human islets ex vivo reveal instances of polyhormonal expression, decreased expression of genes representative of β-cell identity and increased expression of pancreatic progenitor genes. Altogether, these data suggest that the pancreatic cells are undergoing cell fate transitions as opposed to being in fixed stable states. Red cells represent INS+ β-cells. Green and blue cells represent the other endocrine cells. Yellow cells represent rare polyhormonal cells.

For more information on Adrian TEO's lab, please click here.

 

Phosphoproteomics reveals network rewiring to a pro-adhesion state in annexin-1-deficient mammary epithelial cells

Monday, 08 Jan 2018

Authors
Asfa Alli-Shaik^1,†, Sheena Wee^1,† Lina H. K. Lim², Jayantha Gunaratne^*1,3

Author Affiliations
¹ Translational Biomedical Proteomics, Institute of Molecular and Cell Biology, Agency for Science,   Technology and Research, 61 Biopolis Drive, Singapore 138673
² Department of Physiology, Immunology Programme, Centre for Life Sciences, Yong Loo Lin School of   Medicine, 28 Medical Drive, National University of Singapore, Singapore 117456
³ Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, 10 Medical   Drive, Singapore 117597
† These authors contributed equally to this work.
*Corresponding author: jayanthag@imcb.a-star.edu.sg

Published in Breast Cancer Research on 12 December 2017.
https://breast-cancer-research.biomedcentral.com/articles/10.1186/s13058-017-0924-4

Abstract
Background: Annexin-1 (ANXA1) plays pivotal roles in regulating various physiological processes including inflammation, proliferation and apoptosis, and deregulation of ANXA1 functions has been associated with tumorigenesis and metastasis events in several types of cancer. Though ANXA1 levels correlate with breast cancer disease status and outcome, its distinct functional involvement in breast cancer initiation and progression remains unclear. We hypothesized that ANXA1-responsive kinase signaling alteration and associated phosphorylation signaling underlie early events in breast cancer initiation events and hence profiled ANXA1-dependent phosphorylation changes in mammary gland epithelial cells.

Methods: Quantitative phosphoproteomics analysis of mammary gland epithelial cells derived from ANXA1-heterozygous and ANXA1-deficient mice was carried out using stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry. Kinase and signaling changes underlying ANXA1 perturbations were derived by upstream kinase prediction and integrated network analysis of altered proteins and phosphoproteins.

Results: We identified a total of 8110 unique phosphorylation sites, of which 582 phosphorylation sites on 372 proteins had ANXA1-responsive changes. A majority of these phosphorylation changes occurred on proteins associated with cytoskeletal reorganization spanning the focal adhesion, stress fibers, and also the microtubule network proposing new roles for ANXA1 in regulating microtubule dynamics. Comparative analysis of regulated global proteome and phosphoproteome highlighted key differences in translational and post-translational effects of ANXA1, and suggested closely coordinated rewiring of the cell adhesion network. Kinase prediction analysis suggested activity modulation of calmodulin-dependent protein kinase II (CAMK2), P21-activated kinase (PAK), extracellular signal-regulated kinase (ERK), and IκB kinase (IKK) upon loss of ANXA1. Integrative analysis revealed regulation of the WNT and Hippo signaling pathways in ANXA1-deficient mammary epithelial cells, wherein there is downregulation of transcriptional effects of TEA domain family (TEAD) suggestive of ANXA1-responsive transcriptional rewiring.

Conclusions: The phosphoproteome landscape uncovered several novel perspectives for ANXA1 in mammary gland biology and highlighted its involvement in key signaling pathways modulating cell adhesion and migration that could contribute to breast cancer initiation.

Figure

Figure legend: Annexin-1 (ANXA1)-regulated phosphoproteome network. Schematic network of the ANXA1-regulated phosphoproteome interacting with the predicted transcription factors. Proteins were grouped into their respective functional process or localizations for visualization purpose. The stress fibers are depicted as pink lines and the focal adhesions are marked as blue ovals. Phosphoproteins with increased site abundance upon loss of ANXA1 are shown with brown-filled nodes and those with decreased site abundance are represented by green-filled nodes. Diamond-shaped nodes represent kinases. Nodes with only border colors denote predicted upregulated (brown) and downregulated (green) transcription factors.

For more information on Jayantha GUNARATNE's lab, please click here.

 

A Novel Human Systemic Lupus Erythematosus Model in Humanised Micea

Wednesday, 27 Dec 2017

Authors
Merry Gunawan¹, Zhisheng Her¹, Min Liu¹, Sue Yee Tan¹, Xue Ying Chan¹, Wilson Wei Sheng Tan¹, Shubasree Dharmaraaja¹, Yong Fan², Chee Bing Ong³, Eva Loh⁴, Kenneth Tou En Chang⁴, Thiam Chye Tan⁵, Jerry Kok Yen Chan^6,7, Qingfeng Chen^1,2,8*

Author Affiliations
¹ Humanized mouse unit, Institute of Molecular and Cell Biology, Agency for Science, Technology and   Research (A*STAR), Singapore
² Key Laboratory for Major Obstetric Diseases of Guangdong Province, The Third Affiliated Hospital of   Guangzhou Medical University, Guangzhou, 510150, China
³ Advanced Molecular Pathology Laboratory, Institute of Molecular and Cell Biology, Agency for Science,   Technology and Research (A*STAR), Singapore
⁴ Department of Pathology and Laboratory Medicine, KK Women's and Children's Hospital, Singapore
⁵ Department of Obstetrics & Gynaecology, KK Women's and Children's Hospital, Singapore
⁶ Department of Reproductive Medicine, KK Women's and Children's Hospital, Singapore
⁷ Experimental Fetal Medicine Group, Yong Loo Lin School of Medicine, National University of Singapore
⁸ Department of Microbiology and immunology, Yong Loo Lin School of Medicine, National University of   Singapore, Singapore

Published in Scientific Reports on 30 November 2017.

*Corresponding author: Qingfeng Chen. E-mail: qchen@imcb.a-star.edu.sg
https://www.nature.com/articles/s41598-017-16999-7

Abstract
Mouse models have contributed to the bulk of knowledge on Systemic Lupus Erythematosus (SLE). Nevertheless, substantial differences exist between human and mouse immune system. We aimed to establish and characterise a SLE model mediated by human immune system.
Injection of pristane into immunodeficient mice reconstituted with human immune system (humanised mice) recapitulated key SLE features, including: production of human anti-nuclear autoantibodies, lupus nephritis, and pulmonary serositis. There was a reduction in the number of human lymphocytes in peripheral blood, resembling lymphopenia in SLE patients. Concurrently, B cells and T cells were systemically hyperactivated, with a relative expansion of CD27+ and CD27-IgD- memory B cells, increased number of plasmablasts/plasma cells, and accumulation of effector memory T cells. There was also an increased production of human pro-inflammatory cytokines, including: IFN-g, IL-8, IL-18, MCP-1, and IL-6, suggesting their role in SLE pathogenesis. Increased expression of type I IFN signature genes was also found in human hepatocytes.

Altogether, we showed an SLE model that was mediated by human immune system, and which recapitulated key clinical and immunological SLE features. The advancements of humanised mice SLE model would provide an in vivo platform to facilitate translational studies and pre-clinical evaluations of human-specific mechanisms and immunotherapies.

Figure


Figure legend: Lupus nephritis and pulmonary inflammation. (a) (Left) Kidney sections from hu-mice and NSG with or without pristane injection were H&E stained and pathologically evaluated. Scale bar represents 50µm and images are representative from two independent experiments (control NSG n = 3; pristane NSG n = 3; control hu-mice n = 8; pristane hu-mice n = 12). (Right) Mean glomeruli area was measured from 50 random glomeruli from kidneys of each experimental animal (control NSG n = 3; pristane NSG n = 3; control hu-mice n = 5; pristane hu-mice n = 6). (b) Immunohistochemistry of human IgG and IgM on kidney sections from control and pristane-injected hu-mice Scale bar represents 50µm and images are representative from two independent experiments (control n = 8; pristane n = 12). (c) Protein content in the urine of control and pristane-injected NSG, control and pristane-injected hu-mice at 10 weeks post-pristane injection was measured using Uristix reagent strips. Figure is from two independent experiments (control NSG n = 8; pristane NSG n = 3; control hu-mice n = 8; pristane hu-mice n = 7). (d) Lung sections were H&E stained and pathologically evaluated. Scale bar represents 200µm and images are representative from two independent experiments (control n = 6; pristane n = 5). ** P<0.01.

For more information on Qingfeng CHEN's lab, please click here.

 

Induced-Decay of Glycine Decarboxylase Transcripts as an Anticancer Therapeutic Strategy for Non-Small-Cell Lung Carcinoma

Wednesday, 20 Dec 2017

Authors
Jing Lin^1,2, Jia Hui Jane Lee³, Kathirvel Paramasivam⁴, Elina Pathak³, Zhenxun Wang³,
Zacharias Aloysius Dwi Pramono⁵, Bing Lim³, Keng Boon Wee^1,2 and Uttam Surana^4,6,7

Author Affiliations
¹ Bioinformatics Institute, A*STAR, 30 Biopolis Street, Singapore 138671, Singapore
² Institute of High Performance Computing, A*STAR, 1 Fusionopolis Way, Singapore 138632, Singapore
³ Genome Institute of Singapore, A*STAR, 60 Biopolis Street, Singapore 138672, Singapore
⁴ Department of Pharmacology, National University of Singapore, 16 Medical Drive, Singapore 117660,   Singapore
⁵ Department of Research, National Skin Centre, 1 Mandalay Road, Singapore 308205, Singapore
⁶ Bioprocessing Technology Institute, A*STAR, 20 Biopolis Way, Singapore 138668, Singapore
⁷ Institute of Molecular and Cellular Biology, A*STAR, 61 Biopolis Drive, Singapore 138673, Singapore

Published in Molecular Therapy Nucleic acids 9:263–273, 15 December 2017

Abstract
Self-renewing tumor-initiating cells (TICs) are thought to be responsible for tumor recurrence and chemo-resistance. Glycine decarboxylase, encoded by the GLDC gene, is reported to be overexpressed in TIC-enriched primary non-small-cell lung carcinoma (NSCLC). GLDC is a component of the mitochondrial glycine cleavage system, and its high expression is required for growth and tumorigenic capacity. Currently, there are no therapeutic agents against GLDC. As a therapeutic strategy, we have designed and tested splicing-modulating steric hindrance antisense oligonucleotides (shAONs) that efficiently induce exon skipping (half maximal inhibitory concentration [IC₅₀] at 3.5–7 nM), disrupt the open reading frame (ORF) of GLDC transcript (predisposing it for nonsense-mediated decay), halt cell proliferation, and prevent colony formation in both A549 cells and TIC-enriched NSCLC tumor sphere cells (TS32). One candidate shAON causes 60% inhibition of tumor growth in mice transplanted with TS32. Thus, our shAONs candidates can effectively inhibit the expression of NSCLCassociated metabolic enzyme GLDC and may have promising therapeutic implications.

Figure


Figure legend:Transfection of shAONs at 100 nM Leads to Efficient and Specific Skipping of GLDC Target Exon in A549 Cells (A) The number of PTCs generated and the percentage of amino acid residues removed when each of the 14 out-of-frame exons are skipped individually. GLDC has a total of
25 exons. (B) Images of agarose gel electrophoresis of the PCR products demonstrating specific exon skipping induced by shAON transfection in A549 cells. Cells were harvested 24 hr post-transfection. 100 mg/mL cycloheximide was added 5 hr after shAON transfection to inhibit the skipped transcripts from undergoing NMD. (C) The exonskipping efficiency induced by each shAON as determined by densitometry analysis of the PCR products. The y axis shows the skipping efficiency, which is the percentage of the amplicons with exon skipping relative to the total amplicons (skipped + non-skipped). Data are presented as means ± SEM. (D) DNA sequencing of the bands corresponds to the skipped transcript excised from agarose gel (as indicated in B) to confirm skipping of the specific target exon.

For more information on Uttam SURANA 's lab, please click here.

 

Organelle Specific O-Glycosylation Drives MMP14 Activation, Tumor Growth, and Metastasis

Friday, 24 Nov 2017

Authors
Anh Tuan NGUYEN^1,6, Joanne CHIA^1,6, Manon ROS¹, Kam Man HUI^1,2,3,4, Frederic Saltel⁵ and Frederic BARD^1,2

Author Affiliations
¹ Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673
² Department of Biochemistry, National University of Singapore, 21 Lower Kent Ridge Road, Singapore   119077
³ Division of Cellular and Molecular Research, National Cancer Centre Singapore, 11 Hospital Drive,   Singapore 169610
⁴ Duke-NUS Graduate Medical School, Singapore, 8 College Road, Singapore 169857
⁵ INSERM, U1053 Bordeaux Research In Translational Oncology, BaRITOn, F-33000 Bordeaux, France;   Univ. Bordeaux, U1053 Bordeaux Research In Translational Oncology, BaRITOn, F-33000 Bordeaux,   France
⁶ These authors contributed equally

Published in Cancer Cell on 13 November 2017.

Abstract
The Sweet Jaws of Cancer:
In the human body, cell coordination relies heavily on complex sugars attached to cell surface proteins. These sugars regulate how cells maintain tissue integrity. How this sugar-based coordination system is regulated is still poorly understood and it is not known whether and how cancer cells subvert it. In the Fred Bard lab, we are interested in the mechanisms of regulation of cell surface sugars. These sugars are added in a series of intracellular compartments that ressemble an assembly line. We found that this assembly line is dynamic and can be re-modelled upon activation of signalling cascades. In this study, we show liver cancer cells re-organise their O-GalNAc sugar assembly line, resulting in a massive up-regulation of GalNac addition. This process allows liver tumors to grow within the normal tissue. Sugar coupling to the molecular scissor protein called MMP14 strongly activates it. This confers cancer cells the ability to chew through the ExtraCellular Matrix of normal tissue, ultimately allowing tumors to gain space at the expense of normal cells. This sugar-based activation process, which we coined the GALA pathway, occurs in nine out of ten of human liver cancers and appears to be involved in other solid tumors, opening a new angle of attack against cancer.

Figure

Figure legend: Top panel: In normal cells, O-GalNAc glycosylation on proteins occurs in the Golgi apparatus. The initiating step of this process is catalysed by the GALNTs glycosyltransferases that adds a GalNAc sugar to a serine/theonine residue on the protein. The O-glycan is sequentially extended by other glycosyltransferases as the glycoprotein traverses through the Golgi. Bottom panel: Relocation of GALNT to the ER, also known as GALA, augments O-glycosylation on multiple proteins including matrix metalloproteinase MMP14 and also glycosylates ER resident proteins that are normally not O-glycosylated. Increased glycosylation on MMP14 enhances its activity and thus potentiating extracellular matrix degradation, tumor growth and invasiveness.

For more information on Frederic BARD 's lab, please click here.

Transglutaminase 2 Is a Direct Target Gene of YAP/TAZ-Letter

Friday, 17 Nov 2017

Authors
Chen-Ying Liu^1,2*, Ajaybabu V. Pobbati², Zhenyu Huang¹, Long Cui¹ and Wanjin Hong²

Author Affiliations
¹ Department of Colorectal and Anal Surgery, Xinhua Hospital, Shanghai Jiaotong University School of   Medicine, Shanghai 200092, China

² Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 61   Biopolis Drive, Proteos, Singapore 138673, Singapore

Published in Cancer Res. 2017 Sep 1;77(17):4734-4735.  Published first August 15, 2017.

Abstract
Transglutaminase 2 (TG2) is a multifunctional protein that is overexpressed in multiple cancers and its expression levels positively correlate with metastasis and poor prognosis. Interestingly, TG2 is an activator of NF-kB and is also a direct target of NF-kB. It has been shown that knockdown of TG2 inhibits YAP activity. Here, in various cell lines, we show that TG2 is a direct target of YAP/TAZ; by upregulating TG2 expression, YAP/TAZ probably also forms a positive-feedback loop and amplifies oncogenic programs. TG2 has been a promising therapeutic target for many diseases, including cancer. Our results suggest that inhibition of TG2 could be a potent therapeutic strategy for the YAP/TAZ-active cancers.

Figure

Figure legend: Analysis of Cancer Cell Line Encyclopedia (CCLE) microarray dataset reveals that the mRNA expression of TG2 correlates well with the known YAP/TAZ target genes and YAP/TAZ mRNA levels. The co-expression data was extracted from the cBioportal database. The values of Pearson’s correlation and Spearman’s correlation were generated and used for evaluating the significance of co-expression by the cBioportal.

For more information on Wanjin HONG's lab, please click here.

ETS (E26 transformation-specific) up-regulation of the transcriptional co-activator TAZ promotes cell migration and metastasis in prostate cancer

Friday, 10 Nov 2017

Authors
Chen-Ying Liu^‡,§, Tong Yu^‡, Yuji Huang^‡, Long Cui^‡ and Wanjin Hong^§

Author Affiliations
^‡ Department of Colorectal and Anal Surgery, Xinhua Hospital, Shanghai Jiaotong University School of   Medicine, Shanghai 200092, China

^§ Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 61   Biopolis Drive, Proteos, Singapore 138673, Singapore

Published in J Biol Chem, Jun 2;292(22):9420-9430. Epub 2017 Apr 13.

Abstract
Prostate cancer is a very common malignant disease and a leading cause of death for men in the Western world. Tumorigenesis and progression of prostate cancer involves multiple signaling pathways, including the Hippo pathway. Yes-associated protein (YAP) is the downstream transcriptional co-activator of the Hippo pathway, is overexpressed in prostate cancer, and plays a vital role in the tumorigenesis and progression of prostate cancer. However, the role of the YAP paralog and another downstream effector of the Hippo pathway, transcriptional co-activator with PDZ-binding motif (TAZ), in prostate cancer has not been fully elucidated. Here, we show that TAZ is a basal cell marker for the prostate epithelium. We found that overexpression of TAZ promotes the epithelial-mesenchymal transition (EMT), cell migration, and anchorage-independent growth in the RWPE1 prostate epithelial cells. Of note, knock down of TAZ in the DU145 prostate cancer cells inhibited cell migration and metastasis. We also found that SH3 domain binding protein 1 (SH3BP1), a RhoGAP protein that drives cell motility, is a direct target gene of TAZ in the prostate cancer cells, mediating TAZ function in enhancing cell migration. Moreover, the prostate cancer-related oncogenic E26 transformation-specific (ETS) transcription factors, ETV1, ETV4, and ETV5, were required for TAZ gene transcription in PC3 prostate cancer cells. MAPK inhibitor U0126 treatment decreased TAZ expression in RWPE1 cells, and ETV4 overexpression rescued TAZ expression in RWPE1 cells with U0126 treatment. Our results show a regulatory mechanism of TAZ transcription and suggest a significant role for TAZ in the progression of prostate cancer.

Figure

Figure legend: TAZ is a basal cell marker for prostate epithelium. (A) Immunohistochemical analysis of TAZ in normal prostate epithelium, hyperplasia and prostate cancer tissues. The representative images of TAZ in the normal prostate epithelium (a), TAZ in the hyperplasia tissue (b), no expression of TAZ in the prostate cancer tissue (c); strong expression of TAZ in some prostate cancer tissues (d) were shown. Scale bar: 50μm (a,c,d), 100μm (b). (B) The expression level of TAZ is positively associated with basal marker TP63 and negatively correlated with the luminal markers CK8 and CK18. Co-expression data in the prostate cancer TCGA dataset were extracted from the cbioportal database. The Pearson’s correlation coefficient and Spearman’s correlation coefficient values were generated by the cbioportal.

For more information on Wanjin HONG's lab, please click here.

Deubiquitinating Enzyme USP9X Suppresses Tumor Growth via LATS kinase and Core Components of the Hippo pathway

Friday, 03 Nov 2017

Authors
Aleksandra Toloczko^1,2,5, Fusheng Guo^1,5, Hiu-Fung Yuen¹, Qing Wen³, Stephen A. Wood⁴, Yan Shan Ong¹, Pei Yi Chan¹, Asfa Alli Shaik¹, Jayantha Gunaratne¹, Mark J. Dunne², Wanjin Hong^1,6 and Siew Wee Chan^1,6

Author Affiliations
¹  Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR),    Singapore, Republic of Singapore
²  ISchool of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, United    Kingdom
³  ICentre for Public Health and Centre for Cancer Research & Cell Biology, School of Medicine, Dentistry    and Biomedical Sciences, Queen’s University Belfast, Belfast, United Kingdom
⁴  Eskitis Institute for Drug Discovery, Griffith University, Queensland, Australia
⁵  These authors contributed equally to this work
⁶  Corresponding authors: mcbcsw@imcb.a-star.edu.sg (Siew Wee Chan), mcbhwj@imcb.a-star.edu.sg    (Wanjin Hong).

Published in Cancer Research, 77(18):4921-4933 on September 15, 2017

Abstract
The core LATS kinases of the Hippo tumor suppressor pathway phosphorylate and inhibit the downstream transcriptional co-activators YAP and TAZ, which are implicated in various cancers. Recent studies have identified various E3 ubiquitin ligases that negatively regulate the Hippo pathway via ubiquitination, yet few deubiquitinating enzymes (DUB) have been implicated. In this study, we report the DUB USP9X is an important regulator of the core kinases of this pathway. USP9X interacted strongly with LATS kinase and to a lesser extent with WW45, KIBRA, and Angiomotin, and LATS co-migrated exclusively with USP9X during gel filtration chromatography analysis. Knockdown of USP9X significantly downregulated and destabilized LATS and resulted in enhanced nuclear translocation of YAP and TAZ, accompanied with activation of their target genes. In the absence of USP9X, cells exhibited an epithelial-to-mesenchymal transition phenotype, acquired anchorage-independent growth in soft agar, and led to enlarged, disorganized, three-dimensional acini. YAP/TAZ target gene activation in response to USP9X knockdown was suppressed by knockdown of YAP, TAZ, and TEAD2. Deletion of USP9X in mouse embryonic fibroblasts resulted in significant downregulation of LATS. Furthermore, USP9X protein expression correlated positively with LATS but negatively with YAP/TAZ in pancreatic cancer tissues as well as pancreatic and breast cancer cell lines. Overall, these results strongly indicate that USP9X potentiates LATS kinase to suppress tumor growth.

Figure

Figure legend: (A) Knockdown of USP9X (USP9X-KD) in MCF10A cells resulted in epithelial-mesenchymal transition (EMT). (B) Three-dimensional culture of USP9X-KD cells in matrigel. USP9X-KD acini have enlarged and disorganized morphology. (C) Immunohistochemical (IHC) staining of USP9X and LATS in normal and pancreatic cancer tissue arrays. USP9X and LATS protein expression were predominantly negative in pancreatic cancer tissues. (D) USP9X and LATS were expressed weakly as opposed to strong YAP/TAZ expression in tissue sections derived from pancreatic cancer patients.

For more information on Wanjin HONG's lab, please click here.
For more information on Jayantha GUNARATNE's lab, please click here.

Cavin-2 regulates the activity and stability of endothelial nitric-oxide synthase (eNOS) in angiogenesis

Friday, 27 Oct 2017

Authors
Gandhi T. K. Boopathy^‡§1, Madhura Kulkarni^¶, Sze Yuan Ho^¶, Adrian Boey^‡, Edmond Wei Min Chua^‡,
Veluchamy A. Barathi^§**‡‡, Tom J. Carney^‡§¶, Xiaomeng Wang^‡§¶, and Wanjin Hong^‡§2

Author Affiliations
^‡Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Proteos, Singapore
^¶Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore
^Singapore Eye Research Institute (SERI), 20 College Road, 169856 Singapore
**Ophthalmology and Visual Sciences Academic Clinical Program, Duke-NUS Graduate Medical School, 8 College Rd., 169857 Singapore
^‡‡Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
^§SERI-IMCB Programme in Retinal Angiogenic Diseases (SIPRAD), SERI-IMCB, Singapore

Published online in The Journal of Biological Chemistry on September 14, 2017

Cavin-2 regulates the activity and stability of endothelial nitric-oxide synthase (eNOS) in angiogenesis
Gandhi T. K. Boopathy, Madhura Kulkarni, Sze Yuan Ho, Adrian Boey, Edmond Wei Min Chua, Veluchamy A. Barathi, Tom J. Carney, Xiaomeng Wang, and Wanjin Hong
J. Biol. Chem. (2017) 292(43) 17760–17776
https://doi.org/10.1074/jbc.M117.794743

 

Abstract
Angiogenesis is a highly regulated process for formation of new blood vessels from pre-existing ones. Angiogenesis is dysregulated in various pathologies, including age-related macular degeneration, arthritis, and cancer. Inhibiting pathological angiogenesis therefore represents a promising therapeutic strategy for treating these disorders, highlighting the need to study angiogenesis in more detail. To this end, identifying the genes essential for blood vessel formation and elucidating their function are crucial for a complete understanding of angiogenesis. Here, focusing on potential candidate genes for angiogenesis, we performed a morpholino-based genetic screen in zebrafish and identified Cavin-2, a membrane-bound phosphatidylserine-binding protein and critical organizer of caveolae (small microdomains in the plasma membrane), as a regulator of angiogenesis. Using endothelial cells, we show that Cavin-2 is required for in vitro angiogenesis and also for endothelial cell proliferation, migration, and invasion. We noted a high level of Cavin-2 expression in the neovascular tufts in the mouse model of oxygen-induced retinopathy, suggesting a role for Cavin-2 in pathogenic angiogenesis. Interestingly, we also found that Cavin-2 regulates the production of nitric oxide (NO) in endothelial cells by controlling the stability and activity of the endothelial nitric-oxide synthase (eNOS) and that Cavin-2 knockdown cells produce much less NO than WT cells. Also, mass spectrometry, flow cytometry, and electron microscopy analyses indicated that Cavin-2 is secreted in endothelial microparticles (EMPs) and is required for EMP biogenesis. Taken together, our results indicate that in addition to its function in caveolae biogenesis, Cavin-2 plays a critical role in endothelial cell maintenance and function by regulating eNOS activity.

Figure

Figure legend: Scheme of the regulation of nitric oxide (NO) production by Cavin-2 in endothelial cells. The presence of Cavin-2 positively helps in NO production by stabilizing and activating eNOS. The loss of Cavin-2 adversely effects NO production by destabilizing and inactivating eNOS.

For more information on Wanjin HONG's lab, please click here.