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PTPN12/PTP-PEST Regulates Phosphorylation- Dependent Ubiquitination and Stability of Focal Adhesion Substrates in Invasive Glioblastoma Cells Zhihua Chen1, John E

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PTPN12/PTP-PEST Regulates Phosphorylation- Dependent Ubiquitination and Stability of Focal Adhesion Substrates in Invasive Glioblastoma Cells Zhihua Chen1, John E Published OnlineFirst May 9, 2018; DOI: 10.1158/0008-5472.CAN-18-0085 Cancer Molecular Cell Biology Research PTPN12/PTP-PEST Regulates Phosphorylation- Dependent Ubiquitination and Stability of Focal Adhesion Substrates in Invasive Glioblastoma Cells Zhihua Chen1, John E. Morales1, Paola A. Guerrero1, Huandong Sun2, and Joseph H. McCarty1 Abstract Glioblastoma (GBM) is an invasive brain cancer with tumor cells that disperse from the primary N PTPase PEST-rich C PTP-PEST mass, escaping surgical resection and invariably GBM cell 1 +/– pY degrons giving rise to lethal recurrent lesions. Here we report focal adhesion that PTP-PEST, a cytoplasmic protein tyrosine N SH3 pY..Y...pY C-term C CAS phosphatase, controls GBM cell invasion by phys- ically bridging the focal adhesion protein Crk-asso- 23 ciated substrate (Cas) to valosin-containing protein (Vcp), an ATP-dependent protein segregase that Y805 Fem1C Ube2r2 + Ubiquitin Segregation selectively extracts ubiquitinated proteins from E3 ligase CNPBS D2 D1 SH2 multiprotein complexes and targets them for deg- RBX VCIP135 4 Degradation VCP Trim9 radation via the ubiquitin proteasome system. Cullin? via UPS Both Cas and Vcp are substrates for PTP-PEST, with the phosphorylation status of tyrosine 805 (Y805) Cullin RING Elongins in Vcp impacting affinity for Cas in focal adhesions ligase and controlling ubiquitination levels and protein stability. Perturbing PTP-PEST–mediated phos- phorylation of Cas and Vcp led to alterations in GBM cell-invasive growth in vitro and in preclinical A four-part model illustrates how PTP-PEST regulates phosphorylation-dependent ubiquitination mouse models. Collectively, these data reveal a of focal adhesion proteins to control GBM cell polarity and invasion. novel regulatory mechanism involving PTP-PEST, © 2018 American Association for Cancer Research Vcp, and Cas that dynamically balances phosphor- ylation-dependent ubiquitination of key focal pro- teins involved in GBM cell invasion. Significance: PTP-PEST balances GBM cell growth and invasion by interacting with the ATP-dependent ubiquitin segregase Vcp/p97 and regulating phosphorylation and stability of the focal adhesion protein p130Cas. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3809/F1.large.jpg. Cancer Res; 78(14); 3809–22. Ó2018 AACR. Introduction Patients with the malignant cancer glioblastoma (GBM) 1Department of Neurosurgery, University of Texas MD Anderson Cancer Center, have a median survival time of less than two years after 2 Houston, Texas. Institute for Applied Cancer Sciences, University of Texas MD diagnosis (1). This poor prognosis is largely due to invasive Anderson Cancer Center, Houston, Texas. GBM cells that escape surgical resection and give rise to recur- Note: Supplementary data for this article are available at Cancer Research rent lesions that are resistant to chemotherapy such as temo- Online (http://cancerres.aacrjournals.org/). zolomide. Targeted therapies such as the anti-VEGF blocking Corresponding Author: Joseph H. McCarty, Department of Neurosurgery, antibody bevacizumab have yielded disappointing results in University of Texas M.D. Anderson Cancer Center, Unit 1004, 1515 Holcombe GBM clinical trials, with no improvements in overall patient Boulevard, Houston, TX 77030. Phone: 713-792-0429; Fax: 713-834-6257; survival. Many patients treated with bevacizumab develop E-mail: [email protected] acquired resistance, leading to lethal recurrent lesions associ- doi: 10.1158/0008-5472.CAN-18-0085 ated with robust tumor cell invasion (2). While a great deal is Ó2018 American Association for Cancer Research. known about genes and pathways that promote GBM growth www.aacrjournals.org 3809 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst May 9, 2018; DOI: 10.1158/0008-5472.CAN-18-0085 Chen et al. and neovascularization, relatively little is understood about low levels of PTP-PEST display enhanced invasion due to mechanisms that drive GBM cell invasion during progression increased Cas phosphorylation and more dynamic focal adhesion who follow antiangiogenic therapy. disassembly. Collectively, these results not only elucidate novel PTP-PEST is a 110 kDa cytosolic phosphatase that contains a signaling pathways that link the phosphorylation and ubiquiti- 30 kDa N-terminal catalytic domain and a C-terminus with nation pathways. several proline, glutamate, serine, and threonine-rich (PEST) sequences. PTP-PEST plays important roles in promoting tissue Materials and Methods morphogenesis, with deletion of the murine PTP-PEST gene (Ptpn12) in all cells leading to embryonic lethality (3). Struc- Ethics statement tural studies of the PTP-PEST catalytic domain reveal that it Approval for the use of human specimens was obtained from recognizes phosphotyrosine (pY) motifs in diverse substrates the Institutional Review Board (IRB) at the University of Texas MD (4), including Rho GEFs, GAPs, and focal adhesion proteins Anderson Cancer Center. The IRB waived the requirement for such as paxillin and focal adhesion kinase (FAK). Cultured informed consent for previously collected residual tissues from À À fi PTP-PEST / cells show defective polarity and migration due, surgical procedures stripped of unique patient identi ers accord- in part, to abnormal activation of Rho GTPase signaling and ing to the Declaration of Helsinki guidelines. All animal proce- imbalances in cell–ECM adhesion (5, 6). dures and experiments conducted in this study were reviewed and Focal adhesions are multiprotein complexes that connect the approved by the University of Texas MD Anderson Cancer Center cytoskeleton to the extracellular matrix (ECM) via integrins (7). Institutional Animal Care and Use Committee (IACUC). Integrin–ECM adhesions continually develop and disassemble as a cell moves, with intermediate structures (nascent adhesions) GBM cell culturing and analysis forming and growing into larger focal adhesions at the leading Primary human GBM cells from patient samples were cultured edge, and subsequently disassembling under the cell body (8). A in the following growth media: DMEM-F12 (Mediatech), 20 key regulatory event in the formation and disassembly of focal ng/mL EGF and bFGF (Gibco), B27 supplement (Life Technolo- adhesions is posttranslational tyrosine phosphorylation, related gies) and 1 U/mL penicillin–streptomycin (Gibco). After 7 to 10 to activities of tyrosine kinases such as Src and FAK (9). Crk- days, spheroids were passaged by accutase (Sigma #A 6964) treat- associated substrate (Cas) is a 130-kDa protein that was originally ment and mechanical disruption using a 1-mL syringe and a 23- identified as a substrate of Src (10). There are several members of gauge needle, and dissociated cells were replated in fresh growth the Cas protein family: Cas, also known as breast cancer anti- media. LN229 GBM cells and HEK 293T cells were purchased from estrogen resistance (Bcar1), Nedd9, Cass4, and embryonal Fyn the ATCC. GBM cell lines were cultured in DMEM (Mediatech) substrate (Efs; ref. 11). Cas is a core component of focal adhesions supplemented with 10% FBS (Atlanta Biologics) and antibiotics. where it bridges multiple signaling proteins to modulate adhesion All cells were authenticated by DNA short tandem repeat profiling and motility (12). Cas-deficient cells show normal focal adhesion in an institutional Characterized Cell Line Core Facility. In addi- assembly, but dramatically impaired disassembly, leading to tion, all cultured cells were routinely tested for mycoplasma using defective migration and invasion (13). commercially available kits (Thermo Fisher Scientific), and only Phosphorylation and ubiquitination are tightly coupled pro- those cells deemed mycoplasma free were used for experiments. cesses, with "phosphodegron" sequences in target proteins PTPN12-dependent cell viability was quantified using the recruiting E3 ubiquitin ligases and other proteins involved in CellTiter-Glo luminescent assay kit (Promega) according to the degradation by the ubiquitin proteasome system (14). Proteins manufacturer's protocol. Briefly, control or PTPN12 KO spheres are covalently tagged with ubiquitin via the activities of three were dissociated as detailed above and 5 Â 103 stem-like GBM enzymes, termed E1, E2, and E3 (15). Ubiquitinated proteins cells (GSC) were added per well in a 96-well format. At 48, 72, and within multicellular complexes are selectively removed via chap- 96 hours after plating CellTiter-Glo reagent was added to each erone activities associated with Vcp, a 97-kDa evolutionarily well. Plates were incubated at room temperature for 10 minutes conserved protein (16). Vcp catalyzes the segregation of ubiqui- and the luminescence intensity was measured with a microplate tinated proteins from organelles, chromatin, and multiprotein reader. complexes, and promotes destruction by the proteasome (17). To analyze adherent cells by immunofluorescence, spheroids Vcp protein contains two AAAþ adenosine triphosphatase were dissociated as detailed above and GSCs were plated on glass (ATPase) domains and an N-domain, which interacts with lipids coverslips coated with laminin for 24 hours (10 mg/mL) prior to in the plasma membrane and other proteins, including E2 and E3 fixation and permeabilization. For Matrigel invasion assays, 5 Â enzymes (18). A Peptide:N-glycanase/UBA or UBX (PUB) 104 GBM cells (control or PTPN12 KO) were added in serum-free domain-interaction sequence (PBS) in the
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