PTPRN 2 and PLC β1 promote metastatic breast cancer cell migration through PI (4,5)P 2 ‐dependent actin remodeling

• DOI: 10.15252/embj.201591973
• Publication Date: 2015-12-01T03:18:47Z
• Authors: Caitlin A Sengelaub; Kristina Navrazhina; Jason B Ross; Nils Halberg; Sohail F Tavazoie
• Keywords: Phosphatidylinositol 4,5-Diphosphate; 0301 basic medicine; Phospholipase C beta; Breast Neoplasms; Articles; Mice, SCID; Actins; 3. Good health; 03 medical and health sciences; Actin Depolymerizing Factors; Cell Movement; Cell Line, Tumor; Animals; Humans; Receptor-Like Protein Tyrosine Phosphatases, Class 8

Article30 November 2015Open Access PTPRN2 and PLCβ1 promote metastatic breast cancer cell migration through PI(4,5)P2-dependent actin remodeling Caitlin A Sengelaub Laboratory of Systems Cancer Biology, Rockefeller University, New York, NY, USA Search for more papers by this author Kristina Navrazhina Jason B Ross Nils Halberg Sohail F Tavazoie Corresponding Author Information Sengelaub1, Navrazhina1, Ross1, Halberg1 1 1Laboratory *Corresponding author. Tel: [phone]; Fax: 7209; E-mail: [email protected] The EMBO Journal (2016)35:62-76https://doi.org/10.15252/embj.201591973 PDFDownload PDF article text main figures. Peer ReviewDownload a summary the editorial decision process including letters, reviewer comments responses to feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Altered abundance phosphatidyl inositides (PIs) is feature cancer. Various PIs mark identity diverse membranes in normal malignant cells. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) resides predominantly plasma membrane, where it regulates cellular processes recruiting, activating, or inhibiting proteins at membrane. We find that enzymatically reduce membrane PI(4,5)P2 levels cells two independent mechanisms. These genes are upregulated highly cells, their increased expression associates with human relapse. Reduction these enzymes releases PI(4,5)P2-binding protein cofilin from its inactive membrane-associated state into cytoplasm mediates turnover dynamics, thereby enhancing capacity. Our findings reveal an enzymatic network lipid-dependent sequestration actin-remodeling factor. Synopsis phosphatidylinositol phosphatase activity harboring tyrosine phospholipase overexpressed Both levels, cofilin-mediated metastasis. Increased clinically correlates migration. modulate remodeling. Introduction major determinants regulators trafficking both disease states (Vicinanza et al, 2008; Balla, 2013). functions differ greatly depending on phosphorylation hydroxyl groups inositol ring head group, which can be metabolically interconverted different phosphorylated PI kinases phosphatases, reduced second messengers hydrolyzers. Through use biochemical cell-biological methods, roles various secretion, endocytosis, intracellular signaling have begun elucidated. One function serve as docking lipids recruitment specific compartments, results enhancement inhibition activity. has previously been shown regulate migration, key progression (Clark 2000; Sahai Marshall, 2002; Condeelis Segall, 2003; 2005; Ling 2006; Luga 2012). Enhanced migratory capacity required phenotype must move surrounding tissue, enter vasculature, ultimately arrive colonize distal organs (Bissell Radisky, 2001; Chiang Massague, Hanahan Weinberg, 2011). Identifying lipid could provide additional insights mechanisms potential targets development against progression. Here, we identify enzymes, PLCβ1, toward metastasis was initially identified auto-antigen type I diabetes present neuroendocrine (Lan 1996; Lu Wasmeier Hutton, 1996). As transmembrane protein, shuttles between secretory vesicles Due presence neurosecretory vesicles, implicated insulin neurotransmitter exocytosis; however, precise role pathway unknown (Cai belongs family, but does not exhibit phosphoprotein substrates due several critical amino acid variations PTP domain (Magistrelli Recently, found (PIP) and, lesser extent, PI3P (Caromile 2010). family PLC hydrolyze generate diacylglycerol (DAG) triphosphate (IP3) (Rhee, 2001). localizes mainly inner leaflet activated Gaq G proteins, although subset nucleus (Smrcka 1991; Taylor 1991). Nuclear regulating proliferation differentiation (Manzoli 1997). best-characterized member PLCγ1, oncogenesis effects motility adhesion mediated IP3 DAG downstream events (Rebecchi Pentyala, Jones 2005). another promotes reducing PI(4,5)P2. convergently complimentary experiments, targeted reduction membrane-bound cofilin, increasing novel co-modulators drivers Results Given importance multiple processes, were intrigued finding transcriptomic profiling MDA-MB-231 vivo-selected derivative LM2 subline revealed genes, PLCB1, possess known transcript (Figs 1A B, EV1A B) (Minn 2008). validated upregulation line, CN34, exhibited markedly CNLM1a relative parental population B). To functionally test metastasis, depleted performed tail vein colonization assays. Knockdown lung (Fig 1C D, Appendix Fig S1A Depletion either CnLM1a also decreased (Appendix S1C D). Cells knockdown significantly signal lungs 24 h post-injection compared control indicating impacts early stages EV1C). investigate clinical significance quantified primary tumor cDNA samples derived patients diagnosed Interestingly, tumors advanced (stage IV) 1E F). Furthermore, PLCB1 associated worse overall survival 1G) metastasis-free 1H) large patient cohorts (Gyorffy 2010; Genome Atlas Network, establish relevant functional promoters Figure 1. A, B. (A) (B) determined qRT–PCR. N = 5. C. Bioluminescence imaging quantification 40,000 transduced shRNAs targeting hairpin. For shCntrl, sh1PTPRN2: 5 mice/group. sh2PTPRN2: 6 mice. Right, H&E staining representative sections. D. transfected siRNA siRNA. siCntrl: si1PLCβ1, si2PLCβ1: E, F. (E) (F) analyzed cancers (stages I-IV) tissue TissueScan qPCR Array Breast Panels II III (Origene, 97). Expression normalized each gene. G. Kaplan–Meier curve representing cohort (N 528) tumor's (data TCGA Research Patients whose tumors' higher lower than median classified low (blue) high (red) expression. H. 1,609) KMPlot, Gyorffy Patients' Data information: Error bars represent SEM. *P < 0.05, **P 0.01, ***P 0.001. Download figure PowerPoint Click here expand figure. EV1. Western blot analysis MCF 10A, MDA-MB-231, LM2, CNLM1a1 lysate using anti-PTPRN2 anti-PLCβ1 (B). GAPDH used loading control. Densitometry below blots adjusted values. day after injection PTPRN2, si1PLCb1, si2PLCb1: mechanism(s) mediate investigated phenotypes. diminished ability invade Matrigel migrate porous trans-well insert 2A EV2A However, depletion neither gene affected rates, viability, cytotoxicity, caspase 3/7 EV2C–E S2A–C). further tested scratch assay. over 24-h period 2C). greater extent single S2D). four other lines (BT-549, CNLM1a, HCC-1806, MDA-MB-468) S2E–L). 2. drive invasion A. 50,000 inserts/group. Migration assay 100,000 images Scale bar, 100 μm. Quantification area covered made confluent wells/group. E. PTPRN2C945A, vector subjected (D) assays (E). overexpressing vector. G, PLCβ1H331Q, (G) (H). I. Cntrl PLCβ1H331Q OE: EV2. time points post-transfection. siCntrl values C, Proliferation 20,000 (C), (D), 3 hairpins F, (F), PTPRN2C945A (G), (G). less MDA EV2F G). Overexpression sufficient increase invasive least 50% 2D–H), without affecting rates EV2H). overexpression alone S2M). demonstrated activities substrate Caromile whether necessary phenotypes, mutated catalytic versions. contains C(X)5R domain, common phosphatases (Wasmeier Barford 1998). Mutation cysteine residue structurally similar serine abrogates PTPRN2's dephosphorylate 2010); mutation generates non-hydrolyzable phospho-serine intermediate trapping rendering enzyme nonfunctional. dissect influence enzyme's substrate, instead alanine PTPRN2C945A. while wild-type invasion, mice, dependent equivalent failed enhance phenotypes 2D–F). conserved histidine residue. PLCβ1's H331 abrogate (Ramazzotti catalytically 2G–I). pro-metastatic next (Martin, Vicinanza localization addition some cytoplasmic EV3A). act dephosphorylates PI(4,5)P2, hydrolyzes diacyclglycerol (DAG). first immunocytochemical techniques accurately reflect changes mass (Hammond 2009, population, consistent 3A). relationship Addition exogenous (Ozaki 2000) prior intravenous mice treated carrier 3B EV3B). would expected only transiently thus impact colonization. Consistent this, S3A). EV3. Representative retrovirally PTPRN2-FLAG, PLCβ1-FLAG, immunostained anti-FLAG DAPI (blue). 10 Mean fluorescence intensity incubated alone. anti-PI(4,5)P2 antibody microscopy. 50 cells/group. 3. then immediately injected. (C) quantified. Left, immunofluorescence stained 4′,6-diamidino-2-phenylindole (DAPI, blue). 3C EV3C). Conversely, depleting 3D). confirmed our data quantifying enzyme-linked immunosorbent (ELISA). fractions amounts lipid, quantity S3B). modulation lines. MDA-MB-468 S3C indicate manipulated phosphoinositide methods. sought determine defect PTPRN2- PLCβ1-depleted rescued decreasing selectively deplete rapamycin-induced dimerization system developed purpose (Heo Varnai 2006). In system, polyphosphate-5-phosphatase E (INPP5E), depletes dephosphorylating it, fused FKBP recruited constitutively membrane-inserted Lyn11 FRB. constructs rapamycin showed DMSO S4A). Adding PTPRN2-depleted displayed enhanced cells—effectively rescuing 4A 4. (A), Lyn11-FRB INPP5E-FKBP, nM mean PIP5K1A, PIP5KB, PIP5KC combined PIP5K retroviral PIP5K1A set adding 25% EV3B) 2000). Increasing abrogated 4C since PI4P, (D'Angelo 2008), had no effect S4B). PTPRN2/PLCβ1-mediated decrease capacity, upstream PIP5K. PI4P (van den Bout Divecha, 2009). expressed three isoforms (PIP5K1A, PIP5K1B, PIP5K1C) experienced 4E) focused isoform poorly S4C 4F S4E Taken together, negatively govern Plasma binding effector recruit Several involved dynamics inhibited gelsolin, profilin, twinfilin, capping (Saarikangas Of correlate 5A S5A–E) progression, well oral squamous carcinoma, renal ovarian (Martoglio Unwin Wang 2004, 2007; Turhani Cofilin binds prevents bind (Yonezawa Ojala Gorbatyuk When hydrolyzed, released acts severing (Ghosh 2004; Andrianantoandro Pol