research article McKie et al. Research Article

The OPCML Tumor Suppressor Functions as a Cell Surface Repressor –Adaptor, Negatively Regulating Receptor Tyrosine Kinases in Epithelial Ovarian Cancer

Arthur B. McKie1, Sebastian Vaughan1, Elisa Zanini1, Imoh S. Okon1, Louay Louis1, Camila de Sousa1, Mark I. Greene4, Qiang Wang4, Roshan Agarwal1, Dmitry Shaposhnikov1, Joshua L. C. Wong1, Hatice Gungor1, Szymon Janczar1, Mona El-Bahrawy2, Eric W-F. Lam1, Naomi E. Chayen3, and Hani Gabra1

Abstract Epithelial ovarian cancer is the leading cause of death from gynecologic malignancy, and its molecular basis is poorly understood. We previously demonstrated that opioid bind- ing protein cell adhesion molecule (OPCML) was frequently epigenetically inactivated in epithelial ovarian cancers, with tumor suppressor function in vitro and in vivo. Here, we further show the clinical relevance of OPCML and demonstrate that OPCML functions by a novel mechanism in epithelial ovarian cancer cell lines and normal ovarian surface epithelial cells by regulating a ­specific repertoire of receptor tyrosine kinases: EPHA2, FGFR1, FGFR3, HER2, and HER4. OPCML negatively regulates receptor tyrosine kinases by binding their extracellular domains, altering ­trafficking via nonclathrin-dependent endocytosis, and promoting their degradation via a polyubiquitination-associated proteasomal mechanism leading to signaling and growth inhi- bition. Exogenous recombinant OPCML domain 1–3 protein inhibited the cell growth of epithelial ovarian can- cers cell in vitro and in vivo in 2 murine ovarian cancer intraperitoneal models that used an identical mechanism. These findings demonstrate a novel mechanism of OPCML-mediated tumor suppression and provide a proof- of-concept for recombinant OPCML protein therapy in epithelial ovarian cancers.

SIGNIFICANCE: The OPCML tumor suppressor negatively ­regulates a specific spectrum of receptor tyro- sine kinases in ovarian cancer cells by binding to their extracellular domain and altering trafficking to a nonclathrin, caveolin-1–associated endosomal ­pathway that results in receptor tyrosine kinase polyubiq- uitination and proteasomal degradation. Recombinant OPCML domain 1-3 recapitulates this mechanism and may allow for the implementation of an extracellular tumor-suppressor replacement strategy. Cancer Discovery; 2(2); 156–71. ©2012 AACR.

Authors’ Affiliations: 1Ovarian Cancer Action Research Centre, Division of Corresponding Authors: Arthur B. McKie and Hani Gabra, Ovarian Cancer Cancer, 2Department of Histopathology, Centre for Pathology, Imperial Action Research Centre, Department of Surgery and Cancer, Imperial College College London Hammersmith Campus, and 3Section of Biomolecular London, Du Cane Road, London W12 0NN, United Kingdom. Phone: 44–208– Medicine, Division of Surgery, Imperial College London, London, United 383–5828; Fax: 44–208–383–5830; E-mail: [email protected]; and Kingdom; 4Department of Pathology and Laboratory Medicine, University of Hani Gabra, [email protected] Pennsylvania School of Medicine, Philadelphia, Pennsylvania doi: 10.1158/2159-8290.CD-11-0256 Note: Supplementary data for this article are available at Cancer Discovery Online (http://www.cancerdiscovery.aacrjournals.org). © 2012 American Association for Cancer Research.

156 | CANCER DISCOVERY FEBRUARY 2012 www.aacrjournals.org

Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2012 American Association for Cancer Research. The BATTLE Trial: Personalizing Therapy for Lung Cancer research article

Introduction Ovarian cancer is the leading cause of death OPCML is a glycosyl phospha- from gynecologic malignancy (1). The molecular basis of tidylinositol (GPI)-anchored cell adhesion-like ovarian carcinogenesis is poorly understood but frequently molecule and a member of the IgLON family, denoting the involves lesions affecting p53 (2); BRCA1 and 2 (3); the immunoglobulin domain protein family that includes limbic phosphoinositide 3-kinase pathway, including dysregulation system-associated membrane protein (20, 21), OPCML, neurotr- of AKT (4); growth factor signaling pathways, including the imin (22), and more recently neuronal growth regulator 1 (23). epidermal growth factor (EGF) and fibroblast growth The IgLONs are medium-sized proteins (~55 kDa) compris- factor (FGF) pathways (5–9); and neoangiogenesis (10). ing 3 conserved extracellular I-type immunoglobulin domains We previously identified that opioid binding protein cell that share common molecular recognition properties enabling adhesion molecule (OPCML) was inactivated by LOH and homo- and heterodimerization between family members (24). epigenetic silencing in more than 80% of human epithelial GPI-anchored proteins are trafficked to the plasma membrane ovarian cancers (11). We demonstrated that OPCML expres- and often are associated with detergent-insoluble fractions sion inhibited ovarian cancer cell growth, enhanced inter- termed “lipid rafts” that mainly consist of sphingolipids and cellular attachment, and abrogated both subcutaneous and cholesterol (25). Lipid raft domains have also been shown to in- intraperitoneal tumorigenicity in vivo (11). In recent publica- fluence the distribution and signaling of many receptors from tions, others have also confirmed OPCML to be frequently tyrosine kinases to integrins (26–28), although there is still epigenetically inactivated in epithelial ovarian cancers (12– some debate about the definition and existence of physiologi- 14), brain tumors (15), non–small cell lung carcinoma (16), cally relevant lipid rafts (29). bladder cancer (17), cholangiocarcinoma (18), primary na- Here, we describe the mechanism underlying the in vitro sopharyngeal, esophageal, gastric, hepatocellular, colorectal, and in vivo tumor-suppression phenotype previously de- breast, and cervical cancers, as well as lymphomas (19), indi- scribed for OPCML (11). Our results reveal that OPCML cating that OPCML has broad tumor suppressor activity in negatively regulates a specific spectrum of receptor tyrosine common cancers. In many tumor types, OPCML was ubiq- kinases (RTK) through physical interactions with their extra- uitously nonexpressed. In several of these studies, investiga- cellular domains and promotion of their proteasomal degra- tors demonstrated a significant correlation between OPCML dation via trafficking redistribution, which in turn leads to hypermethylation and loss of expression in cancer cell lines altered RTK signaling. We also demonstrate that exogenous (11, 17, 19) and primary tumors (12, 14, 18), and OPCML recombinant OPCML is a potent RTK suppressor in most methylation and loss of expression were associated with poor ovarian cancer cell lines tested and provide a proof-of-con- survival for the patient (17). cept of its therapeutic potential in vivo.

FEBRUARY 2012 CANCER DISCOVERY | 157

Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2012 American Association for Cancer Research. research article McKie et al.

Results OPCML transfection in SKOV-3 (Fig. 1A), or transient, poly- clonal OPCML transfection in PEO1 ovarian cancer cells In Silico Analysis of Publicly Available (Fig. 1B; ref. 31). Microarray Datasets Confirms the Clinical These same RTKs were found to be reciprocally upregulated Relevance of OPCML when physiologic OPCML was knocked down by transient With several investigators demonstrating a correlation transfection with an siRNA pool in OSE-C2 (Fig. 1C; ref. 32). between OPCML methylation and loss of OPCML expres- Endogenous protein expression of OPCML and a variety of RTKs sion in cancer cell lines and primary tumors, we used the is shown in Figure 1. The first (left) lanes for SKOV3, PEO1, and online database of the Cancer Genome Atlas (TCGA) OSE-C2 (Fig. 1A–C) demonstrate that OPCML does not read- HumanMethylation27 (Illumina) assay to confirm the ily coexpress with the specific spectrum of RTKs identified previ- frequency of OPCML methylation in human cancer. We ously while not affecting unassociated RTKs. Deconvolution of found that 678 of 1537 (44%) of cancer patients with avail- the siRNA duplexes in this pool demonstrated their specificity able methylation data (representing breast, ovarian, brain, (Supplementary Fig. S4). Conversely, a group of other RTKs were leukemia, colon, renal, lung, and endometrial cancers) had unaltered by either OPCML overexpression or siRNA knockdown, OPCML-methylated tumors (with site-specific methylation including EPHA10, FGFR2, FGFR4, EGFR, HER3, VEGFR1, and rates ranging from 31% for breast cancer to 73% for colonic VEGFR3 (Fig. 1 and summarized in Supplementary Table S1). We adenocarcinoma; Supplementary Fig. S1). The ovarian cancer confirmed by quantification of immunofluorescence microscopy TCGA dataset (30) also indicated loss of OPCML expression (IFM) that the expression of OPCML dramatically reduced the in 92% of serous high-grade ovarian cancers, suggesting that levels of EPHA2, FGFR1, and HER2 but not the levels of FGFR2 mechanisms other than somatic methylation (such as LOH) or EGFR in BKS-2.1 cells compared with SKOBS-V1.2 (Fig. may also result in loss of OPCML expression. We then used 1D and E; Supplementary Fig. S5). Expression of the OPCML- the KMPlotter online ovarian and breast cancer meta-analysis regulated RTKs EphA2 and FGFR1 was analyzed in SKOV-3 and demonstrated that high OPCML expression (defined as (OPCML–) and BKS-2.1 (OPCML+) cells stimulated with EGF above median expression for breast cancer and above lowest and FGF1. No significant change in overall RTK mRNA levels quartile expression for ovarian cancer) is a highly significant was observed between the OPCML–/+ lines for these RTKs that favorable prognostic factor for progression in 1,090 ovarian show dramatic, OPCML-dependent changes in protein level, sug- −05 (HR 0.71, P = 4.3e ) and for relapse in 2,324 breast can- gesting that OPCML regulates these RTKs posttranscriptionally −16 cer (HR 0.57, P = 2.2e ) patients (Supplementary Fig. S2). (Supplementary Fig. S3D). These findings underscore the clinical relevance of OPCML Cell proliferation assays in FBS-supplemented media inactivation. that used stably transfected lower (SKOBS-3.5) and higher (BKS2.1) OPCML-expressing SKOV-3 clones showed signifi- Specific Downregulation of RTK Signaling by cant growth inhibition compared with SKOBS-V1.2 (vector OPCML Results in Inhibition of Ovarian Cancer control) and displayed similar growth kinetics as the nor- Cell Growth mal ovarian surface epithelial cell line OSE-C2 (Fig. 1F). To OPCML is a nontransmembrane, external lipid leaflet- evaluate whether this growth inhibition was attributable to anchored protein with no direct route to affect proliferative OPCML-mediated negative regulation of RTK phosphoacti- intracellular signaling. We therefore hypothesized that it vation and relevant downstream signaling pathways, we fur- may mediate its tumor suppressor properties via transmem- ther investigated the effects of acute ligand stimulation with brane signaling proteins and analyzed the effect of RTK EGF and FGF1. OPCML expression led to profound abroga- growth factor stimulation on OPCML gene expression. tion of phospho-HER2-Y1248, phospho-EGFR-Y1173, and SKOV-3 cells treated with EGF or FGF 1/2 rapidly induced phospho-FGFR1-Y766 (Fig. 2A and B). Although no EGFR OPCML RNA and protein expression (Supplementary Fig. loss was observed, loss of the activating dimerization part- S3A and S3B). This observation was expanded to a range of ners of EGFR (i.e., HER2 and HER4), coupled with the con- cell lines, including the transformed normal ovarian surface tinuing availability of the inactivating HER3 family member, epithelial cell line OSE-C2. Although maximal induction manifests as downregulation of EGFR signaling. Analysis of of OPCML in SKOV-3 and PEO-1 occurs at 30 minutes, downstream signaling revealed abrogation of phospho-ERK the OSE-C2 and OVCAR5 lines achieve this at 90 min- 1 and 2 (T202 and T204) and phospho-AKT-S473 (Fig. 2A), utes (Supplementary Fig. S3C).The previously described suggesting that both progrowth and prosurvival pathways stable OPCML-expressing SKOV-3 cell lines (BKS-2.1 and are inhibited by OPCML re-expression. SKOBS-3.5) and nonexpressing vector-only transfection control cells (SKOBS-V1.2) (11) were used to explore any Stable shRNA Knockdown of OPCML Results in interaction between OPCML and RTKs that were selected RTK Upregulation and Accelerated Growth of on the basis of being likely responders to EGF, FGF1, and Normal Ovarian Surface Epithelium FGF2. We also selected RTKs with potential importance in To expand upon our siRNA observations (Fig. 1C), we devel- ovarian cancer [vascular endothelial growth factor receptors oped stable short hairpin RNA (shRNA) OPCML knockdown (VEGFR) and ephrin type A receptors (EPHA)]. BKS-2.1 and lines in OSE-C2 (see Methods). OSE-C2 lines with 60% and SKOBS-3.5 demonstrated a profound downregulation of a 95% OPCML knockdown were generated (Fig. 2D). The 95% specific repertoire of RTKs in both the basal unstimulated OPCML knockdown line demonstrated HER2 and EPHA2 or ligand-stimulated state. EPHA2, FGFR1, FGFR3, HER2, upregulation (Fig. 2E). We analyzed the level of HER2 and and HER4 were reproducibly downregulated by either stable EPHA2 proteins in the empty-vector control line PLKO-2 and

158 | CANCER DISCOVERY FEBRUARY 2012 www.aacrjournals.org

Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2012 American Association for Cancer Research. OPCMLOPCMLOPCML HER2HER2HER2HER2 OPCMLOPCMLOPCML EPHA2EPHA2EPHA2EPHA2 OPCML Downregulates a Spectrum of RTKs research article

SKOBS-V1.2SKOBS-V1.2SKOBS-V1.2SKOBS-V1.2 Transfection:Transfection:Transfection:Transfection: A StableStableStableStable clones clonesclones clones B PolyclonalPolyclonalPolyclonalPolyclonal transient transienttransient transient C PolyclonalPolyclonalPolyclonalPolyclonal transient transienttransient transient D OPCML HER2 OPCML EPHA2 -OPCML-OPCML-OPCML-OPCML-OPCML-OPCML inininininin PEO-1PEO-1PEO-1PEO-1PEO-1PEO-1in PEO-1 siRNAsiRNAsiRNAsiRNA ininin OSE-C2 OSE-C2OSE-C2in OSE-C2

OPCML HER2 OPCML EPHA2 SKOBS-V1.2 Transfection: Stable clones Polyclonal transient Polyclonal transient -OPCML in PEO-1 – + siRNA in OSE-C2 SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-V1.2 SKOBS-3.5 SKOBS-3.5 BKS-2.1 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-V1.2 SKOBS-3.5 SKOBS-3.5 BKS-2.1 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA – + OSE-C2 control non-si pool siRNA SKOBS-V1.2 SKOBS-3.5 BKS-2.1 OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 OSE-C2 control non-si control non-si pool siRNA pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 OSE-C2 control non-si control non-si pool siRNA pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA OSE-C2 control non-si pool siRNA BKS-2.1BKS-2.1BKS-2.1BKS-2.1 +OPCML+OPCML+OPCML+OPCML SKOBS-V1.2 +OPCML+OPCML+OPCML Transfection: Stable clones Polyclonal transient Polyclonal transientOPCMLOPCMLOPCML OPCML -OPCML OPCML HER2 OPCML EPHA2 in PEO-1 siRNA in OSE-C2 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 OSE-C2 control non-si pool siRNA BKS-2.1 EPHA2EPHA2EPHA2 +OPCML EPHA2EPHA2EPHA2 SKOBS-V1.2 Transfection: OPCMLStable clones Polyclonal transient Polyclonal transient -OPCML 200200200200 PP ==P 0.0041 0.00410.0041= 0.0041 – + in PEO-1 siRNA in OSE-C2 200 PPP = = 0.00410.00410.0041 SKOBS-V1.2 SKOBS-3.5 BKS-2.1 PP P0.0023 0.00230.00230.0023 OSE-C2 control non-si pool siRNA BKS-2.1 PPP = = 0.00230.00230.0023= FGFR1FGFR1FGFR1FGFR1 +OPCML OPCML OPCML HER2 OPCMLOPCML EPHA2HER2 OPCML EPHA2 EPHA2 150150150150 FGFR3FGFR3FGFR3FGFR3 150 FGFR3FGFR3FGFR3 PPP = =P 0.0401 0.04010.0401= 0.0401 – + 200 P = 0.0041 SKOBS-V1.2 SKOBS-3.5 BKS-2.1 OSE-C2 control non-si pool siRNA BKS-2.1 P = 0.0023 FGFR1 SKOBS-V1.2SKOBS-V1.2SKOBS-V1.2 EPHA2 HER2HER2HER2HER2 +OPCML SKOBS-V1.2SKOBS-V1.2SKOBS-V1.2 SKOBS-V1.2 SKOBS-V1.2 100100100100 Transfection: Stable clonesOPCML Polyclonal transient Polyclonal transient 100100 Transfection: Stable clones Polyclonal transient -OPCMLPolyclonal transient BKS-2.1BKS-2.1BKS-2.1BKS-2.1 in PEO-1 siRNA in OSE-C2200 P = 0.0041 -OPCML150 BKS-2.1 FGFR3 in PEO-1 siRNAP in OSE-C20.0023 P 0.0401 HER4HER4HER4HER4 = = (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary

HER4 units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary FGFR1 units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary OPCML HER2 OPCML EPHA2 units) (arbitrary (arbitrary units) (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary (arbitrary units) (arbitrary Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean Mean pixel intensity pixel Mean 50505050 505050 SKOBS-V1.2 EPHA2 HER2 150 FGFR3 100 P = 0.0401 BKS-2.1 – +EPHA10EPHA10EPHA10EPHA10 200 P = 0.0041 SKOBS-V1.2 SKOBS-3.5 BKS-2.1 – + SKOBS-V1.2 SKOBS-3.5 BKS-2.1 SKOBS-V1.2 OSE-C2 control non-si pool siRNA P 0.0023 BKS-2.1OSE-C2 control non-si pool siRNA = Transfection: Stable clones Polyclonal transient Polyclonal transient FGFR1 HER4 BKS-2.1 -OPCML units) (arbitrary SKOBS-V1.2000 0 HER2 OPCML HER2 +OPCMLOPCML EPHA2 +OPCML in PEO-1 siRNA in OSE-C2 intensity pixel Mean OPCML 100 50 OPCMLOPCMLOPCMLFGFR1FGFR1FGFR1FGFR1EPHA2EPHA2EPHA2EPHA2HER2HER2HER2HER2 EGFREGFREGFREGFRFGFR2FGFR2FGFR2FGFR2 OPCML FGFR2FGFR2FGFR2FGFR2 OPCML FGFR1 EPHA2 HER2 EGFR FGFR2 FGFR2 150 BKS-2.1 FGFR3 HER4 P = 0.0401

EPHA10 units) (arbitrary

EGFREGFREGFREGFR intensity pixel Mean SKOBS-V1.2EGFR 50 0 SKOBS-V1.2 Transfection: Stable clones– + Polyclonal transientEPHA2 Polyclonal transientHER2 EPHA2 SKOBS-V1.2 SKOBS-3.5 BKS-2.1 -OPCML 100 OSE-C2 control non-si pool siRNA OPCML0.30.30.30.3FGFR1 EPHA2 HER2 EGFR FGFR2 in PEO-1 siRNA in OSE-C2BKS-2.1 FGFR2 0.3 BKS-2.1 EPHA10 200 P = 0.0041 200 P = 0.0041 +OPCML HER3HER3HER3HER3 P = 0.0023 OPCML FGFR1 HER4 P = 0.0023

FGFR1 0 units) (arbitrary

EGFR OPCML FGFR1 EPHA2 HER2 EGFR intensity pixel Mean FGFR250 VEGFR1VEGFR1VEGFR1 FGFR2 VEGFR1VEGFR1VEGFR1 150 FGFR3 1500.3 0.20.20.20.2 – + FGFR3 P = 0.0401 0.20.20.2P = 0.0401 BKS-2.1BKS-2.1BKS-2.1 SKOBS-V1.2 SKOBS-3.5 BKS-2.1 BKS-2.1BKS-2.1BKS-2.1 EPHA2 OSE-C2 control non-si EPHA10pool siRNA HER3 BKS-2.1 EGFR VEGFR3VEGFR3VEGFR3+OPCMLVEGFR3 SKOBS-3.5SKOBS-3.5SKOBS-3.5SKOBS-3.5 HER2 200 P 0.0041 0 SKOBS-V1.2 SKOBS-V1.2 OPCML HER2 = SKOBS-V1.2SKOBS-V1.2SKOBS-V1.2SKOBS-V1.2 A570 A570 A570 A570 A570 A570 A570 A570 A570

100 A570 A570 A570 A570 A570 A570 A570 A570 A570 A570 A570 A570 A570 A570 A570 A570 A570 A570 A570 A570 A570

A570 SKOBS-V1.2 A570 A570 A570 A570 A570 A570 100 A570 P = 0.0023 0.3 OPCML FGFR1A570 EPHA2 HER2 EGFR FGFR2 FGFR1 FGFR2 VEGFR1 BKS-2.1 BKS-2.1 OSE-C2OSE-C2OSE-C2 HER3 βββ-Tubulin-Tubulin-Tubulin-Tubulin-Tubulinβ-Tubulin 0.2 0.10.10.10.1 HER4 HER4 BKS-2.1 (arbitrary units) (arbitrary 150 units) (arbitrary MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT Mean pixel intensity pixel Mean MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth assay cell MTT growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT SKOBS-3.5 Mean pixel intensity pixel Mean FGFR3 EPHA2 EGFR VEGFR3 P = 0.0401 50 assay growth cell MTT VEGFR1 50 SKOBS-V1.2 E 200 P =0.2 0.0041 F 0.3 A570 BKS-2.1 HER2 EPHA10 EPHA10 P = 0.0023 SKOBS-V1.2 OSE-C2 FGFR1 HER3 β-Tubulin 0.1 0.00.00.00.0 VEGFR3 100 SKOBS-3.50.00.00.0 BKS-2.10 0 24242424 48484848 72727272 96969696 144144144144 MTT cell growth assay growth cell MTT SKOBS-V1.2 150 A570 OPCML FGFR1 EPHA2 HER2 EGFROPCMLFGFR2FGFR1 EPHA2 HER2 EGFR FGFR2 HER4 FGFR2 VEGFR1 FGFR2 FGFR3 units) (arbitrary P = 0.0401 OSE-C2 TimeTimeTimeTime periodperiodperiod period (h)(h)(h) (h) -Tubulin Time period (h) β intensity pixel Mean 0.1 0.2 50 BKS-2.1 SKOBS-V1.2 0.0 HER2 EGFR VEGFR3 EGFR assay growth cell MTT SKOBS-3.5 EPHA10 100 24 48 72 96 144 BKS-2.1 SKOBS-V1.2 0.3 A570 0.3 0 Time period (h) OSE-C2 HER4 HER3 -Tubulin HER3 β units) (arbitrary OPCML FGFR1 EPHA2 HER2 EGFR0.0 FGFR2 0.1

FGFR2 intensity pixel Mean 50 24 48 72 96 144 VEGFR1 assay growth cell MTT VEGFR1 Time period (h) EPHA10 0.2 0.2 EGFR BKS-2.1 BKS-2.1 0 0.0 VEGFR3 0.3VEGFR3 SKOBS-3.5 SKOBS-3.5 OPCML FGFR1 EPHA2 HER2 EGFR FGFR2 FGFR2 24 48 72 96SKOBS-V1.2144 HER3 A570 SKOBS-V1.2 A570 Time period (h) OSE-C2 OSE-C2 β-Tubulin β-Tubulin 0.1 0.1 VEGFR1 EGFR MTT cell growth assay growth cell MTT

0.2 assay growth cell MTT Figure 1. 1 OPCML negatively regulates a specific repertoireBKS-2.1 of RTKs. Western blots demonstrating that OPCML negatively regulates EPHA2, FGFR1, FGFR3, 0.3HER2, and HER4 but not EPHA10, FGFR2, EGFR, HER3, VEGFR1, and VEGFR3; shown in stably transfected SKOV-3 cells (A), containing pcDNA3.1 SKOBS-3.5 VEGFR3 HER3 vector only (–) and pcDNA3.1-OPCML with lower (SKOBS-3.5) and higher (BKS2.1) OPCML expression; polyclonal transiently transfected PEO1 cells (B) SKOBS-V1.20.0 A570 with vector only (–) and OPCML (+). OSE-C2 (normal ovarian surface epithelial) cells (c), physiologically expressing0.0 OPCML which were untreated (–), or transiently transfected with nonsilencing duplex (Non-si), orOSE-C2 OPCML-directed24 siRNA48 duplexes (si)72 demonstrated96 reciprocal24 144 upregulation48 of72 the same 96 144 β-Tubulin VEGFR1 0.1 specific repertoire of RTKs. -tubulin control for loading is shown in bottom panel. d, immunofluorescence microscopy showing HER2 and EPHA2 0.2 β Time period (h) expression in vector control SKOBS-V1.2 and stable transfection OPCML expressingBKS-2.1 BKS2.1 cells. Abundant HER2 and EPHA2 expressionTime period seen in(h) MTT cell growth assay growth cell MTT VEGFR3 SKOBS-V1.2 (green) is abrogated in BKS2.1 associated with expression of OPCMLSKOBS-3.5 (red). Scale bars, 10 µm. E, comparison of vector (white bars) and OPCML (black bars) transfected SKOV-3 cells showing quantification of the ImmunofluorescentSKOBS-V1.2 pixel intensity for OPCML, FGFR1, EPHA2, HER2, EGFR, and FGFR2A570 demonstrates the same OPCML-associated RTK spectrum of downregulation by a second method. F, a 144-hour in vitro growth assay 0.0 OSE-C2 β-Tubulin compares0.1 vector control SKOBS V1.2 (OPCML deficient), BKS2.1 (strongly expressing OPCML), SKOBS-3.5 (lower OPCML expressor), and OSE-C2 24 (normal48 ovarian surface72 epithelial96 cell line).144 Cell proliferation was quantified by MTT assay showing OPCML expression is strongly growth suppressive.

MTT cell growth assay growth cell MTT Time period (h) FEBRUARY 2012 CANCER DISCOVERY | 159 0.0 Downloaded24 from cancerdiscovery.aacrjournals.org48 72 96 144 on September 24, 2021. © 2012 American Association for Time period (h) Cancer Research. research article McKie et al. SKOBS-V1.2 BKS-2.1 SKOBS-V1.2 SKOBS-3.5

– + + – + + – + + – + + EGF (50 ng/mL) A C OPCML knockdown in stable shRNA clones p pp 0 30 60 0 30 60 0 30 60 0 30 60 Minutes SKOBS-V1.2SKOBS-V1.2SKOBS-V1.2SKOBS-V1.2 BKS-2.1BKS-2.1BKS-2.1BKS-2.1 SKOBS-V1.2SKOBS-V1.2SKOBS-V1.2SKOBS-V1.2 SKOBS-3.5SKOBS-3.5SKOBS-3.5 0.3

–– ++ ++ –– ++ ++ –– ++ + –– ++ ++ EGFEGFEGFEGF (50 (50 (50(50 ng/mL) ng/mL) ng/mL)ng/mL) 0.25 PLKO-2 sh464-23 sh339-24 p pp

– + + – + + – + + – + + p pp p pp EGF (50 ng/mL) p pp – + + – + + – + + – + + p pp – + + – + + – + + – + + p pp p pp p pp p pp p pp – + + – + + – + + – + tHER2+ p pp OPCMLOPCML knockdown knockdown in stablein stable shRNA shRNA clones clones p pp 0.2 OPCMLOPCMLOPCMLOPCML knockdown knockdownknockdown knockdown in inin stable instablestable stable shRNA shRNAshRNA shRNA clones clonesclones clones 0 000 30303030 60606060 0000 30303030 60606060 0000 303030 606060 000 303030 606060 MinutesMinutesMinutes HER2 0.15 pp 0.30.30.30.3 0.1

SKOBS-V1.2 BKS-2.1 SKOBS-V1.2 SKOBS-3.5 pp PLKO-2 sh464-23 sh339-24

0.25 PLKO-2 sh464-23 sh339-24 PLKO-2 sh464-23 sh339-24

0.25 PLKO-2 sh464-23 sh339-24 PLKO-2 sh464-23 sh339-24 PLKO-2 sh464-23 sh339-24 PLKO-2 sh464-23 sh339-24 PLKO-2 sh464-23 sh339-24 PLKO-2 sh464-23 sh339-24

0.25 PLKO-2 sh464-23 sh339-24 pHER2 0.250.250.250.25 PLKO-2 sh464-23 sh339-24 tHER2tHER2tHER2tHER2tHER2tHER2tHER2 0.25 PLKO-2 sh464-23 sh339-24

OPCML/HPRT 0.05 SKOBS-V1.2 BKS-2.1 SKOBS-V1.2 SKOBS-3.5 0.20.20.20.2 EphA2 – + + – + + – + + – + + EGF (50 ng/mL) HER2HER2HER2HER2

0 pp p pp 0.150.150.150.15 pppppp – + + – + + – + + – + + EGF (50 ng/mL) OPCML knockdown0.150.15 in stable shRNA clones 0 30 60 0 30 60 0 30 60 0 30 60 p pp Minutes tEGFR 0.10.10.10.1 pp OPCML pHER2pHER2pHER2pHER2 OPCML knockdown in stable shRNA clones pppppp

0 30 60 0 30 60 0 30 60 0 30 60 OPCML/HPRT OPCML/HPRT PLKO-1 PLKO-2 Minutes 0.3 OPCML/HPRT 0.05 OPCML/HPRT OPCML/HPRT OPCML/HPRT OPCML/HPRT OPCML/HPRT 0.050.050.05 OPCML/HPRT OPCML/HPRT OPCML/HPRT OPCML/HPRT 0.050.05 sh464-23 sh339-24 EphA2EphA2EphA2 SKOBS-V1.2 BKS-2.1 SKOBS-V1.2 SKOBS-3.5 Scrambled EphA2EphA2 tHER2 0.3 0.25 0000 PLKO-2 sh464-23 sh339-24 α/β-Tubulin 0.2 shRNA clones pEGFRtEGFRtEGFR0.25 PLKO-2 sh464-23 sh339-24 – + + – + + – + + – + + EGF (50 ng/mL) tHER2 tEGFRtEGFRtEGFRtEGFRtEGFRtEGFR HER2 OPCMLOPCMLOPCML p pp pp OPCML

0.15 OPCML knockdown in stable shRNA clones0.2 PLKO-1PLKO-1PLKO-1PLKO-1 PLKO-2PLKO-2PLKO-2PLKO-2 PLKO-1PLKO-1 PLKO-2PLKO-2 sh464-23sh464-23sh464-23sh464-23 sh339-24sh339-24sh339-24sh339-24 HER2 0 30 60 0 30 60 0 30 60 0 30 60 Minutes pp ScrambledScrambledScrambledScrambled sh464-23sh464-23sh464-23 sh339-24sh339-24sh339-24 0.15 0.1 D ScrambledScrambledpp α//β-Tubulin-Tubulin pHER2 ααα////β/ββ-Tubulin-Tubulin-Tubulin-Tubulinα//β-Tubulin-Tubulin 64 -23 39 -24

0.3 OPCML/HPRT 0.05 KO-2 shRNAshRNA clonesclones pEGFRpEGFRpEGFR0.1 ppshRNAshRNAshRNAshRNA clones clonesclones clones EphA2 pHER2 β-TubulinpEGFR P L s h4 0.25 0 s h3 tHER2 OPCML/HPRT 0.05 EphA2 0.2 SFM 10%FBS EGF tEGFR p 0 HER2 OPCML 0.15 PLKO-1 PLKO-2 tEGFR sh464-23 sh339-24 OPCML 0.1 βββ-Tubulin-Tubulin-Tubulin-Tubulin-Tubulin Scrambled pHER2 tERK β-Tubulin-Tubulin PLKO-1 PLKO-2 α/β-Tubulin sh464-23 sh339-24SFMSFMSFM 10%FBS10%FBS10%FBS EGFEGFEGF OPCML/HPRT 0.05 pEGFR ScrambledshRNA clones SFM 10%FBS EGF EphA2 SFM 10%FBS EGFα/β-Tubulin PLKO-2 sh339-24 0 shRNA clones PLKO-2 sh339-24 PLKO-2 sh339-24 pEGFR d 3 4 tEGFR pERK (T202/T204)tERK-tERKtERKtERK2tERK -2 OPCML mble ptERKtERK PLKO-1 PLKO-2 ra h464 h339 HER2 β-Tubulin s s PLKO-2 sh339-24 Sc PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 pp α/β-TuPLKO-2 bulin sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 SFM 10%FBSPLKO-2 sh339-24 EGF PLKO-2 sh339-24 β-Tubulin shRNA clonesβ-TubulinpERKpERKpERK (T202/T204) (T202/T204)(T202/T204) pEGFR pERK (T202/T204) EphA2HER2HER2HER2 E SFM 10%FBS EGF HER2 tERK βββ-Tubulin-Tubulin-Tubulin-Tubulin-Tubulin tERK β-Tubulin-Tubulin β-TubulinEphA2EphA2EphA2 PLKO-2 sh339-24 -Tubulin tAkt PLKO-2 sh339-24 PLKO-2 sh339-24 EphA2 β EphA2 pERKSFM (T202/T204)10%FBS EGF PLKO-2 sh339-24 PLKO-2 sh339-24 PLKO-2 sh339-24 HER2 ββ-Tubulin-Tubulin-Tubulin-Tubulin-Tubulin pERK (T202/T204) pAkt S473tAkttAkttAkttAkttAkt ββ-Tubulin tAkt β-Tubulin-Tubulin 24 24 24 2 tERK β2 -Tubulin 2 tAkttAkt HER2 9 - 9 - 9 - 0.9 3 3 3 EphA2 pAktpAktpAkt S473 S473S473 Scram s h PLKO - s h s h β-Tubulin PLKO - β-TubulinPLKO - ppp pAkt S473 0.8 pERK (T202/T204) PLKO 21 EphA2 0.90.90.9 β-Tubulin tAkt HER2 0.7 464-23ScramScramScram βββ-Tubulin-Tubulin-Tubulin-Tubulin-Tubulin 0.9 pppppp 0.80.8 Scram -Tubulin-Tubulin 0.80.8 PLKOPLKOPLKO 21 2121 -Tubulin β 0.6 339-24 β-Tubulin ppp β tAkt 0.8 PLKO 21 SKOBS-V1.2 BKS-2.1 SKOBS-V1.2 SKOBS-3.5pAkt S473 EphA2 0.70.70.7 464-23464-23464-23 pp 0.5 0.7 339-24339-24464-23 – + + – + + – + + – + + EGF (10 ng/mL) F 0.9 0.60.60.6 339-24339-24339-24 p B SKOBS-V1.2 BKS-2.1 SKOBS-V1.2pAkt S473 SKOBS-3.5 SKOBS-V1.2SKOBS-V1.2SKOBS-V1.2 BKS-2.1BKS-2.1BKS-2.1 SKOBS-V1.2SKOBS-V1.2SKOBS-V1.2 SKOBS-3.5SKOBS-3.5SKOBS-3.5 0.4Scram 339-24 pppp 0 30 60 0 30 60 0 30 60 0 30β-Tubulin60 Minutes β-Tubulin 0.6 ppp tAkt 0.8 0.50.50.5 SKOBS-V1.2 BKS-2.1 SKOBS-V1.2 SKOBS-3.5 Absorbance PLKO 21 pp – + + – + + – + + –– + + EGFEGFEGF (10 (10(10 ng/mL) ng/mL)ng/mL)0.9 p –– ++ ++ –– ++ ++ –– ++ ++ –– ++ ++ EGF (10 ng/mL) Scram0.3 0.5 pp β-Tubulin EGF (10 ng/mL) 0.7 464-230.40.40.4 ppp – 000 +303030 +606060 –000 +303030 +606060 –000 30+3030 6060+60 0–00 303030+ tFGFR1606060+ MinutesMinutesEGF (10 ng/mL)0.8 p

PLKO 21Absorbance Absorbance Absorbance Absorbance Absorbance Absorbance Absorbance pAkt S473 0.2 Absorbance Absorbance 0.4 0 30 60 0 30 60 0 30 60 0 30 60 Minutes 0.6 339-240.30.30.3 Absorbance Absorbance SKOBS-V1.2 BKS-2.1 SKOBS-V1.2 SKOBS-3.5 0.7 464-23 Absorbance pp 0.9 tFGFR1tFGFR1tFGFR1tFGFR1tFGFR1 0.1 0.3 ScrampFGFR1-Y766 0.5 0.20.20.2 -Tubulin 339-24 – + + – +β + – + + – + + EGF (10 ng/mL) tFGFR1tFGFR1 0.6 ppp 0.0 p SKOBS-V1.2 BKS-2.1 SKOBS-V1.2 SKOBS-3.5 0.8 PLKO 21 0.2 0.4 0.10.10.1 pp 0 30 60 0 30 60 0 30 60 0 30 60 Minutes pFGFR1-Y766pFGFR1-Y766pFGFR1-Y766 0.5 0 20 40 60 80 100 – + + – + + – + + – + + 0EGF.7 (10 ng/mL)464-23α/β-Tubulin Absorbance 0.00.00.00.1 pFGFR1-Y766 0.3 0.0p Time (h) 0.4 0 20 40 60 80 100 0 30 60 0 30 60 0 30 60 0 30 60 0Minutes.6 tFGFR1339-24 0.0000 20 20 20 404040 606060 808080 100100100 Absorbance

e 0.2 SKOBS-V1.2 BKS-2.1 SKOBS-V1.2 SKOBS-3.5 ααα////β/ββ-Tubulin-Tubulin-Tubulin-Tubulin-Tubulin pp0.3 0 20 40TimeTimeTime (h) (h)(h)60 80 100 0.5 tFGFR1 α//β-Tubulin-Tubulin 0.1

EGF (10 ng/mL) rb anc – + + – + + – + + – + + pFGFR1-Y766 p 0.2 Time (h)

s o 0.4 0 30 60 0 30 60 0 30 60 0 30 60 Minutes 0.0

A b 0.1 0pFGFR1-Y766.3 0 20 40 60 80 100 α/β-Tubulin tFGFR1 0.0 Time (h) 0.2 0 20 40 60 80 100 α/β-Tubulin Figure 2. OPCML-associated RTK modulation affects 0downstream.1 signaling. a, Western blots of total and phospho HER2 and EGFRTime protein (h) from SKOBS-V1.2 (vector control),pF BKS-2.1GFR1-Y7 cells,66 and SKOBS-3.5 were subjected to a 60-minute EGF (50 ng/mL) time course demonstrating profound abrogation of tHER2, HER2, and EGFR phosphorylation (Y-1248 and Y-1173 , respectively)0 .in0 BKS2.1 and SKOBS-3.5. OPCML transfection was also associated with abrogation of EGF phospho- activation of ERK 1 and 2 (T-202/T-204) and Akt (S-473), more0 profoundly20 in BKS-2.140 line. Representative60 80 Western1 0blots0 were run in parallel with equivalent loadings of the same lysateα/β-T preparationubulin for each of the 3 cell lines and are defined by their -tubulin bands. BKS2.1 tERK/pERK (left) is from the same blot as tERK/pERK for β Time (h) SKOBS-V1.2/SKOBS3.5 on the right, and therefore the same SKOBSV1.2 empty vector–transfected control data are shown alongside for ease of interpretation (original blot shown in Supplementary Fig. S9). BKS2.1 and SKOBS V1.2 tAkt/pAkt are from the same blot: removal of irrelevant additional sample lanes, for ease of interpretation, is indicated by black boxes. SKOBS V1.2 and SKOBS 3.5 tAkt/pAkt (bottom right) are a separate representative blot. b, the same cells subjected to a 60-minute FGF1 (10 ng/mL) time course again demonstrate loss of total and phospho (Y-766) FGFR1. c, analysis of shRNA clones by quantitative reverse-transcription PCR for OPCML showing 2 clones exhibiting reduced OPCML mRNA levels relative to the PLKO-1 and PLKO-2 (empty–vector clones) and shSCRAMbled controls. We focused on 2 clones; *sh464–23, which exhibited 60% knockdown, and **sh339–24, which exhibited 95% knockdown of OPCML. D, Western blot analysis demonstrated that total HER2 and EphA2 are strongly upregulated in the shRNA lines with 95% OPCML knockdown. E, Western blots showing the upregulation of HER2 and EphA2 upon 95% knockdown of OPCML selectively on serum and on exposure to ligand stimulation of cells. F, cell proliferation assay shows that OSE- C2-sh339–24 line with 95% knockdown of OPCML exhibits greater rates of proliferation at 48, 72, and 96 hours compared with PLKO-2 control (*P = 0.002, **P = 0.009, and ***P = 0.003, respectively) compared with shRNA scrambled and empty–vector PLKO controls in 10% FBS. No such growth rate difference was demonstrated in 0.5% FBS, underscoring the RTK ligand dependency of OPCML effect (data not shown).

160 | CANCER DISCOVERY FEBRUARY 2012 www.aacrjournals.org

Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2012 American Association for Cancer Research. OPCML Downregulates a Spectrum of RTKs research article

95% knockdown shRNA339–24 line after serum starvation the His tag), whereas the 95-kD ∆-5 exhibited little change followed by 1 hour of exposure to serum-free medium, 10% in band intensity between SKOBS-V1.2 and BKS-2.1 (Fig. 3D; FBS-supplemented medium, or 50 ng/mL EGF stimulation on left quadrants). Densitometric quantification revealed a 50% Western blots (Fig. 2E). The levels of HER2 and EPHA2 in- reduction in exogenous 185-kD Neu, with no reduction seen creased upon OPCML knockdown in a serum- and ligand-de- in the 95-kD ∆-5 species (Fig. 3E; upper chart). pendent fashion. Reciprocally to the overexpression of OPCML To understand this interaction more clearly, we transiently in cancer lines, the stable knockdown of OPCML in OSE-C2 cotransfected HER2/OPCML-null Cos cells with OPCML and resulted in increased proliferation. The empty-vector control the Neu constructs, which revealed a clear OPCML-induced line PLKO-2, the stably expressing sh339–24 and sh464–23 downregulation of 185kD-Neu with unaltered expression of knockdown cell lines (exhibiting 95% and 60% knockdown, 95-kD ∆-5 (Fig. 3C; right quadrants). Quantitative densito- respectively), and an shRNA scrambled control line were di- metric analysis illustrated a significant (75%) downregula- rectly compared in 10% FBS-supplemented medium, and a sig- tion of 185-kD Neu and no alteration in 95-kD ∆-5 expression nificant increase in proliferative rate was associated with the in OPCML cotransfected Cos cells (Fig. 3E; lower chart). Cell 95% knockdown of OPCML (P = 0.003, Fig. 2F). Thus, stably proliferation assays of the Cos cotransfections clearly demon- knocking down OPCML results in upregulation of RTKs from strated that 185-kD Neu transfection accelerated cell growth physiologically normal levels in ovarian surface epithelial cells and that OPCML cotransfection significantly and completely to levels equivalent to those observed in SKOV-3 cancer cells abrogated this acceleration (Fig. 3F; upper graph). In the same (15-fold in the case of EPHA2) and is associated with acceler- assay, 95-kD ∆-5 transient transfection demonstrated similar ated growth in 10% serum. growth acceleration that was unaffected by OPCML cotrans- fection (Fig. 3F; lower graph). These findings reveal that nega- OPCML Directly Interacts with Extracellular tive regulation of RTKs by OPCML is functionally mediated Domains of Specific RTKs through extracellular domain protein binding. We selected EPHA2, FGFR1, and HER2 as examples of OPCML-regulated RTKs and EGFR as an example of an RTK Alteration of RTK Membrane Distribution not negatively regulated by OPCML. Immunoprecipitation of by OPCML BKS-2.1 cell lysates with antibodies to EPHA2, FGFR1, and To confirm the predicted “lipid-raft” localization of the HER2 coprecipitated OPCML, suggesting an interaction with GPI-anchored protein OPCML, we prepared detergent-solu- these 3 RTKs. A reciprocal immunoprecipitation, in which ble and -insoluble fractions from SKOBS-V1.2 and BKS-2.1 α-OPCML was used as the first antibody, confirmed that these cells (see Methods), which revealed that OPCML was local- RTKs all bound to OPCML. However, no interaction was seen ized within the cholesterol-rich, detergent-insoluble fraction between OPCML and EGFR, whose expression levels are not along with caveolin-1, a marker of a distinct form of lipid negatively regulated by OPCML (Fig. 3A). To confirm our ini- raft domain known as caveolae; Fig. 4A). We analyzed HER2 tial immunoprecipitation studies, we constructed a recombi- as an example of an OPCML-regulated RTK in this analy- nant GST-OPCML fusion protein consisting of Ig domains sis. The OPCML-expressing cell line exhibited a reduced level 1–3. Pull-down experiments with the use of SKOV-3 cell lysates of HER2 with sequestration of the remaining HER2 in the revealed that FGFR1 and HER2 interacted with GST-OPCML detergent-insoluble membrane fraction. However, in the D1–3 (Fig. 3B). We were able to confirm the specificity of these OPCML nonexpressing line, HER2 is equally distrib- interactions by demonstrating again that EGFR was not pulled uted between the 2 fractions. The total level of EGFR, a down in this assay, in agreement with our immunoprecipita- non–OPCML-regulated RTK, was not affected by OPCML tion data in Figure 3A. We further determined that the interac- expression, although there was a shift to the detergent-insol- tion with GST-OPCML D1–3 was mediated by the extracellular uble membrane fraction (Fig. 4A). These data indicate that ­domain of the RTKs for FGFR1 and HER2 (Fig. 3C). OPCML expression leads not only to loss of HER2 expression Because this finding suggested that OPCML-mediated but also HER2 redistribution on the plasma membrane. negative regulation of a specific repertoire of RTKs is defined IFM was used to examine the trafficking of OPCML in by a physical binding event with the RTK extracellular do- cells by the use of EEA-1 (a marker of the early endosome) main, we sought to further demonstrate the functional con- and caveolin-1 (a marker of the raft-caveolar pathway) to in- sequences of OPCML binding to these RTKs by using HER2, vestigate this apparent redistribution. OPCML was seen to an RTK highly expressed in SKOV-3 cells, as a paradigm. colocalize with caveolin-1 rather than EEA1 (Fig. 4B). IFM To understand the mechanism of OPCML-mediated down- colocalization of HER2 with EEA-1 or caveolin-1 (Fig. 4C; regulation of HER2, we transiently transfected BKS-2.1 or left) demonstrated a quantitative shift away from EEA-1/ SKOBS-V1.2 cells with either a full-length, 185-kD rat HER2/ clathrin colocalization of HER2 to colocalization with the ca- Neu construct (Neu) or the previously reported 95-kD (∆-5) veolin-1 detergent-insoluble membrane fraction in OPCML- Neu construct with a deleted extracellular domain sequence expressing BKS2.1 cells. These observations suggest a shift (33) as shown in Supplementary Figure S6 and Figure 3D to from clathrin/EEA-1–associated HER2 trafficking in OPCML F. In SKOBS-V1.2 (OPCML–), transfection of full-length Neu nonexpressing cells to a pathway mediating a different form and ∆-5 resulted in a clear increase in the amount of intact of caveolin-1-associated trafficking (Fig. 4C; right). 185-kD protein and 95-kD truncated species compared with empty-vector control transfection. The Relative Abundance of Cell-Surface In OPCML-expressing BKS-2.1 cells, however, both endog- HER2 Is Modulated by OPCML enous HER2 and the transfected full-length 185-kD Neu ex- The turnover of HER2 on the surface of cells was mea- hibited a depleted signal (visualized by immunoblotting for sured by pulse-chase biotinylation of reactive free amino or

FEBRUARY 2012 CANCER DISCOVERY | 161

Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2012 American Association for Cancer Research. P = 0.009 L L 1.6 P = 0.009 L L 1.6 A2 FR1 A2 FR1 1.4 IP: Eph OPCM IP: FG OPCM 1.4 P = 0.80 IP: Eph OPCM IP: FG OPCM 1.2 = 0.80 1.2 P 1 IB:EphA2 IB:FGFR1 1 IB:EphA2 IB:FGFR1 0.8 0.8 0.6 PP == 0.0090.009 0.6 LL LL 1.61.6 PPPP = = == 0.009 0.009 0.0090.009 0.4 LLLLLL LLLLLL IB:OPCML IB:OPCML 1.61.61.61.6 A2A2 0.4 A2A2A2A2A2 FR1FR1 IB:OPCML IB:OPCM1.4L A2 FR1FR1FR1FR1FR1 1.41.41.41.4 0.2 IIP:P: EEphph OPCOPCMM IIP:P: FFGG OPCOPCMM 1.41.4 IIIIP:IP:IP:IP: EEEEphEphphphphph OPCOPCOPCOPCMMMM IIIIP:IP:IP:IP: FFFFGGFGG OPCOPCOPCOPCMMMM PP == 0.800.80 0.2 IP: Eph OPCM IP: FG OPCM 1.21.2 PPPP = = == 0.80 0.80 0.800.80 0.0 1.21.21.21.2 0.0 111 Mean V1.2 Mean BKS Mean V1.2 Mean BKS IBIB::EpEphhA2A2 IIB:B:FGFGFR1FR1 111 Mean V1.2 Mean BKS Mean V1.2 Mean BKS IBIBIBIBIBIBIB::::Ep:Ep:Ep:EphhhA2hA2A2A2 IIIIB:IB:IB:IB:FGFGFGFGFR1FR1FR1FR1 0.80.8 + Neu + Neu + D5 + D5 0.80.80.80.8 + Neu + Neu + D5 + D5 IP: HER2 OPCML IP: 0.60.6EGFR OPCML IP: HER2 OPCML 0.6I0.60.6P:0.6 EGFR OPCML 8 P = 0.041 P = 0.71 IIB:B:OOPCPCMMLL IIBB:O:OPCPCMMLL 0.40.40.40.4 8 = 0.041 = 0.71 IIIIB:IB:IB:IB:OOOPCPCPCPCMMMLLLL IIIIBIBIBIB:O:O:O:O:O:O:OPCPCPCPCMMMLLLL 0.40.4 P P IB: HER2 IB: EGFR 0.20.2 IB: HER2 IB: EGF0.20.20.20.2R 0.00.0 6 0.00.00.00.0 6 MeanMean V1.2V1.2MeanMean BKSBKS MeanMean V1.2V1.2MeanMean BKSBKS MeanMeanMean V1.2V1.2 V1.2MeanMeanMean BKSBKS BKS MeanMeanMean V1.2V1.2 V1.2MeanMeanMean BKSBKS BKS IB: OPCML IB: OPCML ++ NeuNeu ++ NeuNeu ++ D5D5 ++ D5D5 IB: OPCML IB: OPCML ++++ Neu Neu NeuNeu ++++ Neu Neu NeuNeu McKie et al.++++ D5 D5 D5D5 ++++ D5 D5 D5D5 4 researchIIP:P: HER2HER2 articleOPCMLOPCML IIP:P: EGFREGFR OPCMLOPCML 4 IIIIP:IP:IP:IP: HER2HER2HER2HER2 OPCMLOPCMLOPCMLOPCML IIIIP:IP:IP:IP: EGFREGFREGFREGFREGFR OPCMLOPCMLOPCMLOPCML 8 8888 PPP = === 0.041 0.0410.0410.041 PPP = === 0.71 0.710.710.71 8 PPP == = == 0.0410.041 0.041 0.0410.041 PPP == = == 0.710.71 0.71 0.710.71 2 IIIB:IB:B:IB: HER2 HER2HER2 HER2 IIIB:IB:B:IB: EG EGEG EGFFFRFRRR 2 IIIB:B:IIB: HER2HER2 HER2 IIIB:B:IIB: EGEG EGFFFRRR PPP = == 0.009 0.0090.009 LLLL LLLL 3 1.61.61.6 PP == 0.0090.009 LLL LLL 3 1.61.61.666 A A2A2A2 B te 1- C 6666 A2A2A2 FR1FR1FR1FR1 1- FR1FR1 sa te 1.41.41.4 3 0 IIIP:P: EEphphph OPCOPCMM IIIP:P: FFFGG OPCOPCMM ly 3 0 IIB:B: OPCOPCIIIP:IP:P:MMLL EEEphphph OPCOPCOPCMMM IIIB:IB:B:IB: O OOO OPCPCPCIPCIIP:IP:P:MMMLLLL FFFGGG OPCOPCOPCMMM sa PPP = == 0.800.800.80 Cos Cos Cos Cos IIIIB:IB:IB:IB: OPC OPC OPCMMMLLLL IIIB:IB:IB: OO O OOPCPCPCMMMLLL 3 ly PCML 1.21.21.244 Cos Cos Cos Cos 3 -O PCML 4444 + Neu + Neu + D5 + D5 -O 111 PCML 1- + Neu + Neu + D5 + D5 IBIBIBIBIBIB:::::Ep:EpEphhhA2A2A2 IIIIIB:IB:B:FGFGFGFR1FR1FR1 IP GST: SKOV- GST GST -O PCML 1- - OPCML + OPCML - OPCML + OPCML IP GST: SKOV- GST GST 0.822 -O - OPCML + OPCML - OPCML + OPCML 0.80.80.82222 put IP GST: In putGST GST 33 33333 IB: EGFR 0.60.60.6 IP GST: In GST GST 1-3333 IB: EGFR 0.60.6 tete 1 1111- ---1- - IB: HER2-ECD tetetetetetetete 1 111 -1--1--- 33 0.400 ssaa 33333 I0.40.4B0.4000:0 HER2-ECD IIIIIB:IB:B:OOOPCPCPCMMMLLL lylyssssaasasaaa IIIIIBIBB:O:O:O:O:O:OPCPCPCMMMLLL 33333 000 lllylyllylyyly CosCos CosCos CosCos CosCos 33 llyyllyy PCMPCMLL IB: HER2 CosCosCosCos CosCosCosCos CosCosCosCos CosCosCosCos 3333 3 3 PCMPCMPCMPCMPCMLLLLLL 0.20.20.2 Cos Cos Cos Cos --OO IB: HER2 IB: FGFR1-ECD++ NeuNeu ++ NeuNeu ++ D5D5 ++ D5D5 -----O-OO--O IB: FGFR++1++ -ECDNeu Neu NeuNeu ++++ Neu Neu NeuNeu ++++ D5 D5 D5D5 ++++ D5 D5 D5D5 1 PCMLPCML 11-- 0.0 ++ Neu Neu ++ Neu Neu ++ D5 D5 ++ D5 D5 1 IPIP GSGSTT:: PCMLPCMLPCMLPCMLPCML 1 1 11 ---1----- 0.00.00.0 - OPCML + OPCML - OPCML + OPCML IPIPIPIPIPIPIP GS GS GSTTTT::::::: SKOVSKOV-- GSGSTT GSTGST --OO ---- OPCML -OPCMLOPCMLOPCML OPCML +++ + OPCML OPCMLOPCML OPCML ---- OPCML -OPCMLOPCMLOPCML OPCML +++ + OPCML OPCMLOPCML OPCML IPIPIP GS GSTT::: SKOVSKOVSKOVSKOVSKOV------GSGSGSGSTTTTT GSTGSTGSTGST -----O-OO--O Mean--- - -OPCMLOPCML OPCML OPCMLOPCML V1.2 Mean+++ OPCMLOPCML OPCML BKS --- - -OPCMLOPCML OPCML OPCMLOPCMLMean V1.2+++ OPCMLOPCML OPCMLMean BKS putIBput: FGFR1 MeanMean V1.2V1.2 MeanMean BKSBKS MeanMean V1.2V1.2 MeanMean BKSBKS putputputputput IB: FGFR1 p p IIPIPP GS GSGSTTT::: IInnputputputput GSGSTT GSTGST + Neu + Neu + D5 + D5 0.8 IBIB:: EEGGFRFR IIIIPPIIP GSGS GSTTT:::::: IIIInIInnnIInn GSGSGSGSTTTTT GSTGSTGSTGST +++ NeuNeuNeu +++ NeuNeuNeu +++ D5D5D5 +++ D5D5D5 p p IBIBIBIBIBIBIB:::: : E: E:EE E EEGGGFRFRFRFR 0.8 p IIIIIP:IP:P: HER2HER2HER2 OPCMLOPCMLOPCML IIIIIP:IP:P: IIBIBB:: : HER2 HER2HER2EGFREGFREGFR---ECDECDECDOPCMLOPCMLOPCML IB: OPCML Cos+EV IP: HER2 OPCML IP: IIIIBBIIB:::: : HER2:HER2HER2 HER2 HER2HER2EGFR---ECDECD--ECDOPCML IB: OPCML p Cos+EV 888 P === 0.0410.0410.041 P === 0.710.710.71 IIIIB:IB:IB:IB: HER2 HER2 HER2 HER2 IB: FGFR1-ECD 88 PPP = ==== 0.041 0.0410.0410.0410.041 PPP = ==== 0.71 0.710.710.710.71 0.6 IIB:IB: HER2 HER2 IIIIBIBIBIB:::: : F: :F F FGGGGFRFRFRFR111-ECD1-ECD-ECD-ECD-ECD-ECD-ECD 11 0.6 Cos+Neu IIIIIB:IB:B: HER2 HER2HER2 IIIIIB:IB:B: EG EGEGFFFRRRIB: FGFR1-ECD 1111 Cos+Neu IBIB:: FFGGFR1FR1 P = 0.009 Cos+Neu+OP L L IBIBIBIBIBIBIB:::: : F: F:FF F FGFG1.6GFR1FR1FR1FR1 666 pppp 0.4 Cos+Neu+OP 00..88 pppppppp 0.4 A2 FR1 SKOBS-V1.2 BKS-2.1 SKOBS-V1.20 00BKS-2.1.0...8.88..8 Cos transient transfections 1.4 SKOBS-V1.2 BKS-2.1 SKOBS-V1.20.8 BKS-2.1 Cospp transient transfections Cos+OPCML IP: Eph OPCM IP: FG OPCM IIIIB:IB:B: OPC IBOPCOPCIB:: OPCOPCMMMLLLMMLL IIIIIB:IB:B: O OOOOOPCPCPCMMMLLL P = 0.80 pppp CoCos+s+EVEV Cos+OPCML IIB:B: IBIBOPCIBIBIBIBIB:::: : OPC: OPC:OPCOPC OPC OPCOPCM1.2LLMMMLLLL 444 CoCoCos+s+s+s+EVEVEV 0.2 0.6 44 0.2 Neu + + – 000–.0...6.66..6 + + – – CoCos+s+NNeeuu 1 Neu + + – 0.6 – + + – – CoCoCoCos+s+s+s+NNNNeeeueuuu IB:EphA2 IB:FGFR1 OPCML – + – + – + – + 0.8 OPCML – + – 222 + – + – + Cos+Neu+OP 0.0 00..44 22 CoCoCoCos+s+s+s+NNNNeeeueuu+u+++OPOPOPOP 0.0 000.0...4.44..4 Cos+Neu+OP 24 48 72 D 0.6 SKOBS-V1.2SKOBS-V1.2P = 0.009 BKS-2.1BKS-2.13333 SKOBS-V1.2SKOBS-V1.2 BKS-2.1BKS-2.1 CosCos transienttransient transfectionstransfections 24 48 72 L L 1.6 SKOBS-V1.2SKOBS-V1.2SKOBS-V1.2SKOBS-V1.2 BKS-2.1 BKS-2.1 BKS-2.1BKS-2.1 SKOBS-V1.2SKOBS-V1.2SKOBS-V1.2SKOBS-V1.2 BKS-2.1 BKS-2.1 BKS-2.1BKS-2.1 NCosCosCosCoseu- 1transient transient transienttransient85kD transfections transfections transfectionstransfections E CoCos+Os+OPCPCMMLL SKOBS-V1.2tetetetete BKS-2.1 11 1 1111------SKOBS-V1.2 BKS-2.1 Cos transient transfections CoCoCoCos+Os+Os+Os+OPCPCPCPCMMMLLLL A2 tetetete Neu-185kD3 0 1 FR1 0.4 sssaaa 333 P = 0.0090.2 000 1 IB:OPCML IB:OPCML 1.4 L lysssaaa L 1.6 000.0..2.22.2 NNeeuu lllllyyllyy++ ++ –– –– ++ ++ –– –– 000...22..2 CosCosCos CosCosCos CosCosCos CosCosCos pppp IP: Eph OPCM IP: FG OPCM A2NNNNeeeueuuu 0.2 3333 +++ PCMPCMPCM++LLLL+ –––– P––– –= 0.80 +++ +++ –––– –––– CosCos CosCos CosCos CosCos pppp NNeeuu 1.2 333 ++ PCMPCM+LL+ FR1 –– –– His++ ++ –– –– OOPCMLPCML – ----O--OO + – + – + – + 1.4 +++ Neu NeuNeu +++ Neu NeuNeu +++ D5 D5D5 +++ D5 D5D5 IP: Eph OOOPCMLPCMLPCMLPCMLOPC0.0M –––– IP:++++ FG OPC––––M ++++ –––– His+++PCML+ 1- –––– ++++ 0.0 ++ NeuNeu ++ NeuNeu ++ D5D5 ++ D5D5 0.8 ––– +++ ––– +++ ––– ++PCML+PCMLPCML 1 11------––– +++ 000..0..0.00.0 P = 0.80 0.8 IPIPIPIPIPIP GS GSGSTTT:::::: 1 SKOVSKOV--- GSGSTTT GSTGST -O 1.2 00..0..0 ---- OPCML OPCMLOPCMLOPCML +++ OPCML OPCMLOPCML ---- OPCML OPCMLOPCMLOPCML +++ OPCML OPCMLOPCML IB:EphA2 IB:FGFR1 SKOVSKOVSKOVMean------V1.2GSGSGSTTTMeanGST GSTGSTBKS Mean V1.2 Mean BKS ----O--OO -- 24 24OPCML24OPCML ++ 4 4OPCML4OPCML888 -- OPCML7OPCML77222 ++ OPCMLOPCML put 242424 444888 777222 ppp Cos+EV NNeeuu--110.88855kkDD IP GST: putputputα/β-Tub 1 Cos+EV NNNeeeeuuuu----11-1-188885555kkkDkDD + Neu + Neu + D5IIIIPIPP GS GSGSTTT:::::+ D5 IIIIInInInn GSGSαGS/TTβTT-TubGSTGSTGST 11 0.6 ppp IB:EphA2 IBIBIBIBIBIB::::: : E EEEEEGGGFRFRFR IB:FGFR1 1111 0.6 Cos+OPCML IP: HER2 OPCML IP: EGFR OPCML P = 0.009 0.6 IB: HER2-ECD 0.8 pppppppp Cos+OPCML L L 1.6 IIIIBIBB:::: : HER2 HER2HER2HER2HER2----ECDECDECD pppppppppppppppp A2 HisHis 0.48 P = 0.041 P = 0.71 0.6 IB:OPCML FR1 IB:OPCML HisHisHis 0.80.8 0.4 Cos+D5 1.4 IIIIIB:IB:B: HER2 HER2HER2 IB: FGFR1-ECD ∆-5 + + – 0.80.80.8–0.8 + + – – 0.4 Cos+D5 IP: Eph OPCM IB: HER2IP: FG OPCM IB: EGFR P =0.2 0.80 IIIIBIBB:::: : F FFGGFRFRFR111-ECD-ECD-ECD-ECD ∆-5 + +0.4 – 111 – + + – – 1.2 IB:OPCML IB:OPCML OPCML – + 111 ppp – + CCosos+EV+EV Cos+D5+OP αα//ββ-T-Tubub0.0 OPCML – + – + – pppppppppppp+ – + CCCCosososos+EV+EV+EV+EV Cos+D5+OP IB: FαGααα/////βFR1β/β/β-T-T-T-T-T-Tubububub6 – +0.2 0.60.6 ppp– + 1 IBIBIBIBIB:::: : F FFFFGGFR1FR1FR1 Mean V1.2 Mean BKS Mean V1.2 Mean BKS 0.60.60.60.6 CCosos+OPCML+OPCML 0.2 IB:EphA2 IB:FGFR1 0.0 pppppp CCCCosososos+OPCML+OPCML+OPCML+OPCML 0.2 0.8 Neu-95kD 000.....8.88 IB: OPCML IB: OPCML + Neu + Neu + D5 + D5 Neu-95kD Mean V1.2 Mean BKS Mean V1.2p Mean BKS Cos+D5 0.6 IBIBIB::: OPCOPCOPC--55MMLL 4 –– –– –– –– 0.40.4 ppp CCCCosososos+D5+D5Co+D5CoCo+D5s+s+s+EVEVEV IP: HER2 OPCML IP: EGFR OPCML IBIBIBIBIB:::: : OPC OPCOPCOPCOPC∆∆∆∆-----55-5-M5MLLL ++++ ++++ –––– –––– ++++ ++++ –––– –––– 0.40.40.40.4 CosCo+D5Cos+s+s+EVEV 0.0 ∆∆ ++ ++ ++ ++ + Neu + Neu + D5 + D5 0.0 24 48 72 0.4 P = 0.009 OOPCMLPCMLPCML8 –– ++ –– ++ –– ++ –– ++ 000.....6.66 CCosos+D+D55++OOPP IB:OPCML L IB:OPCML L 1.6 IP: HER2 OOOPCMLPCMLPCMLOPCML –––– P I=P: 0.041+++ EGFR OPCML––– ++P+ = 0.71 O–––PCML– +++ ––– +++ 0.6 CCCCosososos+D+DCo+DCoCo+D55s+5s+s++5+++OONNNOPPeePePuuu 24 48 72 A2 2 OPCML 0.2 IB: HER2 FR1 IB: EGFR 0.2 0.20.20.20.2 1.4 8 P 0.2= 0.041 P = 0.71 Cos+Neu+OP IP: Eph OPCM IP: FG OPCM 3 0.0 NNeeuu--995=5kD kD0.80 CoCoCos+s+s+NNNeeeuuu+++OPOP 1.2 NNNNeeeeuuuu---P--99-9- 95555kDkDkDkD 000.....4.44 1- Mean V1.2 MeanIB: BKSHER2 Mean V1.26Mean BKSIB: EGFR α/β-Tub te SKOBS-V1.2SKOBS-V1.2SKOBS-V1.2 BKS-2.1 BKS-2.1BKS-2.1 SKOBS-V1.2SKOBS-V1.2SKOBS-V1.2 BKS-2.1 BKS-2.1BKS-2.1 CosCosCos transient transienttransientα/β-Tub transfections transfectionstransfections 0.00.00.00.0 Cos+OPCML sa 1 3 0 0.00.0 2244 4488 7272 CoCoCos+Os+Os+OPCPCPCMMLLL IB:EphA2 IB:FGFR1 ly + Neu + Neu + D5 + D5 Cos Cos Cos Cos 6 22242444 44484888 72727272 IB: OPCML 3 PCML IB: OPCML OOPCMLPCMLPCML 0.2 IP: HER2 OPCML IP: EGFR OPCML 0.8 OOOPCMLPCMLPCML4 000....2.22 -O NNNeeeuuu ++++ Neu +++ + Neu ––– + D5 ––– + D5 +++ +++ ––– ––– PCML0.68 1- PI B:= 0.041OPCML P = 0.71 IB: OPCML IP GST: SKOV- GST GST -O OOOPCMLPCMLPCML –- OPCML + + OPCML – - OPCML+ + OPCML – + – + 4 0.4 ααα/////βββ/-T-T-T-T-T-Tubububub ––– ++ ––– ++ ––– ++ ––– ++ 00....00 IIB:B: OHER2PCML IIB:B:O EGPCFMRL put ααα//ββ//β-T-T-T-Tubub2 000...0.00 IP GST: In GST GST 2424 4488 7722 IB: EGFR 0.26 242424 444888 777222 3 Neu-185kD IB: HER2-ECD NNeeeuuu----111888555kkkDD 2 1 te 1- 0.0 111 IB: OPCML IB: OPCML sa 3Mean V1.2 Mean BKS Mean V1.20Mean3 BKS pppppppppppp IB: HER2 ly 4 1- pppppppp PCML IB: FGFR1-ECD te Cos Cos Cos Cos 3 + Neu + Neu HisHisHis + D5F 1 + D5 3 0 -O sa 0.80.80.8 IP: HER2 OPCML IP: EGFR OPCML ly + Neu + Neu + D5 + D5 Cos 0.80.8 Cos Cos Cos IB:IP FG GSFR1T: PCML2 1- 3 PCML - OPCML + OPCML - OPCML + OPCML SKOV- GST GST -O 8 P = 0.041 -O P = 0.71 p p + Neu + Neu + D5 pppppp + D5 CCCososos+EV+EV+EV ααα/////β/ββ-T-T-T-Tububub 0.8 PCML 1- ppppppppp 3 put IP GST: αα ββ - OPCML 0.60.60.6+ OPCML - OPCML + OPCML IB: HER2 1- IB: EGFR IP GST: In GST GST SKOV- GST GST -O 0.60.6 Cos+OPCML te IBIB: OPC: EGMFRL p Cos+EV CCCososos+OPCML+OPCML+OPCML sa 3 06 put ly IB: HER2-ECD Cos Cos Cos Cos IP GST: In GST GST 3 PCML IB: EGFR 0.6 0.4 CCCososos+D5+D5+D5 -O + Neu + Neu ∆∆∆----5+55 D5 ++ ++D5 +++ ––– ––– Co+++s+Neu +++ ––– ––– 0.40.40.4 IB: OPCML IIB:B: HER2OPCML ∆∆-5 ++ ++IB: HER2-ECD– – ++ ++ – – PCML 1I-B: FGFR1-ECD 4 Figure 3. OPCML directly interacts with the extracellular domain of EPHA2, FGFR1, and HER2, IP GST: SKOV- GST GST -O - OPCML + OPCML O-OO OPCMLPCMLPCMLPCML 1+ OPCML– + ––– +++ – + ––– +++ CCCososos+D+D+D555+++OOOPPP IB: HER2 ––– ++ –– ++ Co––s+– Neu++OP+ –– ++ Cos+D5+OP put 0.4 IB: FGFR1-ECD a prerequisite for downregulation. a, immunoprecipitation for0.2 OPCML showing binding for IB: FGFR1 IP GST: In GST GST 1 0.20.20.2 IB: EGFR SKOBS-V1.2 BKS-2.1 SKOBS-V1.2 BKS-2.1 Cos transient transfections2 p p EPHA2,Co FGFR1,s+OPCM andL HER2. Similarly, immunoprecipitation for each of these proteins in turn IB: HER2-ECD NNNeeeuuu----999555kDkDkD0.8 IB: FGFR1 showed reciprocal binding for OPCML. We were not able to demonstrate coimmunoprecipitation 3 0.2 p 0.00.00.0 p p IB: HER2 1- NIBe:u OPCML + + – – + + – – Cos+EV 0.8 0.00.0 te IB: FGFR1-ECD with EGFR and OPCML after numerous attempts. b, GST pull-down assay222444 with SKOV-3444888 cell lysate 727272 sa 3 1 0 OOPCMLPCML p 24 48 72 ly OPCML – + – + – + – IB+: OPCML OOPCMLPCMLPCML 0.6 Cos+EV 3 PCML Cos Cos Cos 0.0 Cos showingCo s+bindingNeu of HER2 and FGFR1 to GST-OPCML 1–3 and no binding of EGFR to GST-OPCML IB: FGFR1 -O + Neu + Neu + D5p p + D5 24 48 72 0.6 PCML 1- 0.8 1–3 protein.Cos+Ne Theu+OP presence of OPCML fusion proteins in these assays was verified by Cos+Neu IP GST: SKOV- GST GST - OPCML + OPCML α- ////OPCMLβ-T-T-Tubub + OPCML Neu-185kD -O p ααα///β/ββ-T-T-Tububub 0.41 immunoblotting (bottom). c, confirmatory in vitro interaction studies were undertaken verifying IB: OPCML SKOBS-V1.2 BKS-2.1put SKOBS-V1.2 BKS-2.1 Cos transient transfections Cos+EV Cos+Neu+OP IP GST: In GST GST pppp Cos+OPCML 0.4 IB: EGFR 0.6 binding of HER2 and FGFR1 extracellular domain protein, generated from in vitro translation kit His IB: HER2-ECD SKOBS-V1.2 BKS-2.10.2 SKOBS-V1.2Cos+Neu BKS-2.1 Cos transient(TnT; transfections HER2 extracellular domain) and in Escherichia coli (FGF receptor-extracellular domain) to Cos+OPCML Neu + + – – + + – – 0.8 IB: HER2 Cos+Neu+OP GST-OPCML 1–3 fusion protein. Both HER20.2 and FGFR1 extracellular domains were seen to bind OPCML IB: FGFR–1-ECD + – + – + 0.41 – Ne+u + + – – + + Cos– +EV – SKOBS-V1.2 BKS-2.1 α/βSKOBS-V1.2-Tub BKS-2.1 Cos transient transfections 0.0 ppp to GST-OPCML 1–3. d, to demonstrate a mechanistic link between the RTK extracellular domain OPCML 0.6 Cos+OPCML IB: FGFR1 – + 24 – +48 7–2 + Cos– +OPCML+ Neu-185kD 0.2 p p and OPCML, we undertook transient transfection0.0 of His-tagged full-length Neu/HER2 Neu + + – – + + – – 0.8 1 extracellular domain or extracellular domain-less Neu24 (Δ5-P95), and48 we demonstrated72 Neu-185kD p 0.4 Cos+D5 OIBPCML: OPCML – + ∆-5 – ++ + – + – – – + + + – – Cos+EV pppp downregulation of the transfected full-length1 185-kD extracellular domain containing the Neu in His 0.0 OPCML – + – + – + 0.6 – 24 + 48 72 0.8 Cos+Neu eitherC theos+D OPCML5+OP BKS2.1 or OPCML transiently cotransfected Cos-1 (Cos-Neu pppp+ OPCML); in Neu-185kD 1 His 0.2 Cos+Neu+OP ppp contrast,Cos +EVthere was no change in expression0.8 of the transiently transfected extracellular domain- Nα/eβu-T-9ub5kD 0.4 pppp less Neu ( 5-P95) in the presence of OPCML. e, the relative mean band intensity from 5 SKOBS-V1.2 BKS-2.1 SKOBS-V1.2 BKS-2.1 Cos transient transfections 0.6 Δ Cos+EV His α/β-Tub 0.0 Cos+OPCML Cos+OPCML ppp 0.8 separate Neu construct transfections in BKS2.1/SKOBS-V1.20.6 and 3 separate cotransfections in 0.2 24 48 72 Cos+OPCML Neu + + OPCML– – + + – – Cos+EV Cos1 cellsCos+D5 shows that in both cell systems, the level of full-length Neu protein is significantly α/β-Tub ∆-5 + + – – + + – – ppp 0.4 OPCML – + – + – + – + 0.6 reduced by 50% to 80% when coexpressed with OPCML (P = 0.009 and P = 0.041, respectively),C os+D5 OPCML – + – + – + 0.0 – ∆-+5 + + Cos–+OPCML – + + C–os+D5+OP– 0.4 α/β-Tub 24 48 72 whereas the level of the extracellular domain-less 95-kD Neu species is unchanged in both OPCML +0.2 Cos–+D5 + + – + Cos+D5+OP ∆N-e5u-185kD + + – – + + – – 0.4 – – systems regardless of OPCML status. f, growth assays in Neu and OPCML nonexpresing Cos-1 Neu-95kD 1 0.2 OPCML – + – + – + – + pppp Cos+D5+OP cells conclusively demonstrate that the significant growth acceleration (P = 0.003) induced by Neu-95kD 0.0 His 0.2 24 48 72 transiently transfected p185 Neu (Cos+ Neu) is abrogated by transient cotransfection (dotted OPCML 0.8 0.0 Neu-95kD line) with OPCML (Cos + Neu + OP, *P = 0.004 at 48 hours, **P = 0.016 at 72 hours), reducing Cos+EV 24 48 72 α/β-Tub 0.0 OPCML ppp the growth rate to that of the empty vector (Cos + EV) and OPCML (Cos + OPCML) controls. In 0.6 24 48 72 OPCML α/β-Tub Cos+OPCML contrast, the similarly significantly growth accelerated extracellular domain-less Δ5-P95 (P = α/β-Tub Cos+D5 0.015) was not inhibited by OPCML cotransfection (Cos + Neu + OP, ***P = 0.56 at 48 hours, ∆-5 + + – – + + – – 0.4 α/β-Tub ****P = 0.092 at 72 hours), and this demonstrates the functional necessity of the extracellular OPCML – + – + – + – + Cos+D5+OP 0.2 domain for regulation of the RTK by OPCML. IB, immunoblotting; IP, immunoprecipitation. Neu-95kD 0.0 24 48 72 162 | CANCER DISCOVERY FEBRUARY 2012 www.aacrjournals.org OPCML

α/β-Tub Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2012 American Association for Cancer Research. SKOBS-V1.2 BKS-2.1 SKOBS-V1.2 BKS-2.1

NR DRM NR DRM NR DRM NR DRM

tEGFR tEGFR

tHER2 tHER2

OPCML OPCML

OPCML Downregulates a Spectrum of RTKs Caveolin-1 Caveolin-1 research article

A B

SKOBS-V1.2 BKS-2.1 SKOBS-V1.2SKOBS-V1.2 BKS-2.1BKS-2.1 SKOBS-V1.2 BKS-2.1 8000 8000 NR DRM NR DRM NRNR DRM DRM NRNR DRM DRM NR DRM NR DRM OPCML OPCML CAV-1 6000 CAV-1 6000 tEGFRtEGFR tEGFRtEGFR

4000 4000 tHER2tHER2 tHER2tHER2 OPCML 2000 OPCML 2000 EEA-1 OPCML EEA-1 OPCMLOPCML OPCML OPCML with Colocalization Colocalization with OPCML with Colocalization 0 0 Caveolin-1 Cav-1 EEA-1 Cav-1 EEA-1 Caveolin-1Caveolin-1 Number of colocalized pixels Number of colocalized pixels

C HER2/EEA-1 HER2/CAV-1 HER2/EEA-1 HER2/CAV-1

80008000 80008000 40 (P 40= 0.0160) EEA-1 CAV-1 (P = 0.0160) BKS2.1 BKS2.1 EEA-1 CAV-1 OPCML+ OPCML OPCMLOPCML OPCML+6000 CAV-1CAV-1 60006000 CAV-1CAV-1 6000 30 (P = 0.0383) 30 (P = 0.0383)

40004000 40004000 20 20 SKOBS-V1.2 SKOBS-V1.2 OPCML- OPCMLOPCML 20002000 OPCMLEEA-1 OPCML-20002000 EEA-1EEA-1 markers endocytic EEA-1EEA-1 10 markers endocytic 10 Colocalization with OPCML with Colocalization Colocalization with OPCML with Colocalization Colocalization with OPCML with Colocalization Colocalization with OPCML with Colocalization Colocalization with OPCML with Colocalization Colocalization with OPCML with Colocalization Colocalization with OPCML with Colocalization Colocalization with OPCML with Colocalization Colocalization with OPCML with Colocalization % HER2 colocalization with colocalization HER2 %

00 with colocalization HER2 % 00 Cav-1 EEA-1 Cav-1Cav-1 0 EEA-1EEA-1 HER2 HER2 Cav-1HER2 EEA-1 HER2 0 EEA-1 CAV-1 NumberEEA-1 of colocalized pixelsBKS-2.1 SKOBS-V1.2 NumberNumber ofof colocalizedcolocalized pixelspixels CAV-1 BKS-2.1 SKOBS-V1.2 Cell line Cell line HER2/EEA-1 HER2/CAV-1 HER2/EEA-1HER2/EEA-1 HER2/CAV-1 HER2/CAV-1

Figure 4. Location and colocalization of OPCML with HER2 to a detergent-resistant/caveolin-1–specific compartment. A, OPCML sequesters 4040 ((PP == 0.0160)0.0160) RTKs into detergent-resistant membrane (DRM) fraction;4040 SKOBS-V1.2EEA-1EEA-1 and BKS2.1 cells CAV-1 CAV-1 were subjected((PP == 0.0160)0.0160) to membrane fractionation to separate BKS2.1BKS2.1 EEA-1EEA-1 CAV-1 CAV-1 BKS2.1BKS2.1 DRM fraction/lipid raft fraction from the detergent-soluble/bulk membrane phase. We analyzed an equal volume from each fraction with SDS-PAGE OPCML+ OPCML+OPCML+ followed by Western blotting with anti-EGFR, anti-HER2, anti-OPCML, and anti–caveolin-1 antibodies. OPCML was found to be strictly located in 3030 ((PP == 0.0383)0.0383) the DRM fraction with caveolin-1. Transfection of OPCML3030 was associated((PP == 0.0383)0.0383) with shift of HER2 and EGFR to the DRM fraction. NR, non-raft. b, confocal coimmunofluorescence demonstrates increased colocalization of caveolin-1 with OPCML and concurrently shows decreased colocalization with EEA-1, suggesting a redistribution of HER2 to lipid-raft domains. Scale bars, 10 µm. The bar chart shows quantification of the total number of 2020 OPCML pixels colocalized with caveolin-1 and EEA-1, implying2020 that OPCML localizes mainly with lipid-raft domains. c, confocal coimmuno- SKOBS-V1.2SKOBS-V1.2 fluorescence of HER2 colocalization with EEA-1 and caveolin-1 (left) with quantification as a percentage of the total number of HER2 pixels SKOBS-V1.2SKOBS-V1.2 OPCML- detected within the cells. These studies demonstrate that OPCML-expressing cells are associated with a switch of HER2 trafficking from clathrin- OPCML-OPCML- endocytic markers endocytic endocytic markers endocytic endocytic markers endocytic endocytic markers endocytic endocytic markers endocytic endocytic markers endocytic mediated internalization of early endosomes to lipid-raftmarkers endocytic 10 domain mechanisms of internalization. Scale bars, 10 µm; bottom right panel, 50 µm. endocytic markers endocytic 1010 endocytic markers endocytic 1010 % HER2 colocalization with colocalization HER2 % % HER2 colocalization with colocalization HER2 % % HER2 colocalization with colocalization HER2 % % HER2 colocalization with colocalization HER2 % % HER2 colocalization with colocalization HER2 % % HER2 colocalization with colocalization HER2 % % HER2 colocalization with colocalization HER2 % % HER2 colocalization with colocalization HER2 % % HER2 colocalization with colocalization HER2 % HER2 00 HER2HER2 HER2HER2 00 EEA-1EEA-1 CAV-1HER2 BKS-2.1BKS-2.1 SKOBS-V1.2SKOBS-V1.2 sulfhydrEEA-1EEA-1yl groups on surface-accessibleCAV-1CAV-1 proteins of BKS-2.1BKS-2.1BKS-2.1 The substantial HER2SKOBS-V1.2SKOBS-V1.2 ubiquitination observed in the control Cell line and SKOBS-V1.2 cell lines. Biotinylated fractions were then OSE-C2CellCell line linePLKO cell line was profoundly suppressed in the captured on a NeutrAvidin (Thermo Scientific) agarose resin, OPCML stable knockdown line sh339–24 (Fig. 5B; top) and immunoblotted for HER2, and compared with the total in- was associated with upregulation of total HER2 (Fig. 2E). We put fraction. The biotinylated fractions demonstrated accel- witnessed a reciprocal result upon transfection of the same erated loss of surface HER2 protein, with an 85% decrease hemagglutinin construct into SKOBS-V1.2 (OPCML–) and in the level of surface-labeled HER2 at 4 hours in OPCML– BKS-2.1 (OPCML+) cells with and without EGF stimulation. expressing BKS-2.1 cells with only a 23% reduction in surface OPCML restoration in BKS-2.1 was associated with increased HER2 in OPCML– ­negative SKOBS-V1.2 cells (Fig. 5A; right). ubiquitination of HER2 (Fig. 5C; top). We treated the normal OSE-C2 PLKO (tightly con- trolled/repressed HER2 expression) and OPCML nonex- OPCML Negatively Regulates RTK Levels via pressing OSE-C2 stable knockdown line sh339–24 with Polyubiquitination and Proteasomal Degradation MG-132, a potent inhibitor of the proteasomal 26S pro- We transfected the OPCML-expressing and knockdown teinase. MG-132 treatment of PLKO resulted in strong up- normal ovarian surface epithelial cells (OSE-C2) discussed regulation of HER2 (Fig. 5B; bottom). In sh339–24, HER2 previously with a hemagglutinin-tagged ubiquitin construct. was strongly upregulated by OPCML knockdown, and

FEBRUARY 2012 CANCER DISCOVERY | 163

Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2012 American Association for Cancer Research. research article McKie et al.

A BKS-2.1 SKOBS-V1.2 l 120 HER2 + OPCML t a l l l l BKS-2.1 SKOBS-V1.2 l 120 HER2 + OPCML t o t a t a t a l t a BKS-2.1 SKOBS-V1.2 t a 120 . HER2 -+OOPPCCMMLL t o t o t o t a t o 0 2 4 0 2 4 Hours t o 100 . . . . . HER2 - OPCML vs 100 t o HER2 - OPCML 0 2 4 0 2 4 Hours 100 HER2- . HER2 - OPCML vs vs vs vs vs 100 HER2- HER2- HER2- t HER2- HER2-

0 2 4 0 2 4 Hours of

z ed 80 vs HER2- t t t l i t t of of of of HER2of ty z ed z ed z ed 80 z ed bio z ed 80 t l i l i l i l i l i inp u of HER2 s i HER2 ty ty ty

HER2 z ed

ty 80 biio ty

l i 60 inp u inp u inp u inp u s i inp u s i s i s i HER2s i bio ty 60 te n

60 inp u s i norm a te n te n te n te n te n i n 60 HER2 norm a norm a

HER2 norm a norm a norm a total e d 40 te n i n i n i n i n i n v e HER2 HER2 HER2 HER2 HER2 HER2 HER2 norm a e d

e d 40 e d 40 e d total e d 40 total la t i n v e v e v e HER2 v e v e HER2 at i

total e d 40 la t la t la t la t la t n y v e 20 at i at i at i at i at i la t Re l n y n y n y n y n y 20 ot i

20at i Re l Re l Re l Re l Re l n y

b i 20 ot i ot i ot i ot i ot i 0 Re l b i b i b i ot i b i b i 0 0 2 4 b i 0 0 2 4 0 Time (h2 ours) 4 Time (hours) Time (hours) PLKO sh339-24 SKOBS-V1.2 BKS-2.1 PLKO +sh339-24 – B (OPCML ) (OPCML ) C SKOBS-V1.2(OPCML–) BKS-2.1 (OPCML+) + PLKO – sh339-24 (OPCML+) (OPCML ) –SKOBS-V1.2 + BKS-2.1 + – (OPCML ) (OPCML ) IP: HER2 Input(OPCMLIP) Input(OPCMLIP) (OPCML–) (OPCML+) IP:IP: HER2HER2 InputInput IPIP InputInput IPIP IP: HER2 Input IP Input IP IP: HER2 IP:IP: HER2HER2 Input -Serum +EGF Input -Serum +EGF IP: HER2InputInput -Serum-Serum +EGF InputInput -Serum-Serum +EGF Input -Serum +EGF Input -Serum +EGF IB:HA IB: HA IB:HAIB:HA IB:IB: HAHA IB:HA IB: HA

IB:HER2 IB:HER2IB:HER2 IB:HER2

SKOBS-V1.2 BKS-2.1 PLKO sh-339-24 SKOBS-V1.2 BKS-2.1 PLKO sh-339-24 CQ (0.1 mM): –SKOBS-V1.2+ – – BKS-2.1+ – PLKO CQ ((0.1 mM):: – + – – + – – + – sh-339-24+ 0.1 µM MG-132 MG-132 (0.1 µM): – – + – – + MG-132 (0.1 µM)CQ: (0.1 mM): – + – – + – – + – + 0..1 µM MG-132 MG-132 ((0.1.1 µM):: – – + – – + – + – + 0.1 µM MG-132 MG-132 (0.1tE GFµM)R: – – + – – + tHER2 tEtEGFR tHER2tHER2 tEGFR tHER2 tHER2 ttHER2 β–Tubulin tHER2 β–Tubulin β–Tubulin OPCML OPCML OPCML β−Tubulin β−Tubulin β−Tubulin (P = 0.0042) (P = 0..0042) (P =HE0R.0042 2) HER2 (P = 0.0412) (P = 0H..E0R4122) EGFR (P = 0.0412) EGFR EGFR densitometry % Change in densitometry % Change in densitometry % Change in densitometry % Change in densitometry % Change in densitometry % Change in densitometry % Change in densitometry % Change in densitometry % Change in densitometry % Change in densitometry % Change in densitometry - % Change in - densitometry % Change in densitometry CQ MG-132 % Change in CQ MG-132 - CQ MG-132 - CQ MG-132 - SKOCBQS-VM1G.2-132 - SKOCBQS-VM1G.2-132 SKOBS-V1.2 SKOBS-V1B..K2S-2.1 SKOBS-V1B..K2S-2.1 SKOBS-V1.2 SKOBS-V1.2 BKS-2..1 BKS-2..1 BKS-2.1 BKS-2.1

Figure 5. Mechanism of OPCML-mediated RTK downregulation. a, pulse-chase biotinylation followed by Western blotting demonstrates a more rapid loss of HER2 protein in OPCML-transfected BKS2.1 than OPCML-deficient SKOBS V1.2 (left). Quantitation of relative signal intensity of biotinylated HER2 normalized – against input total HER2 expressed as a percentage of the initial pulse. b, normal OSE-C2 cells PLKO (OPCML+) and sh339–24 (OPCML ) (top) were transiently transfected with a hemagglutinin-tagged ubiquitin, serum starved, and then treated with MG-132 for 2 hours. Ubiquitinated proteins were detected by immunoprecipitating with HER2 then immunoblotting with an antihemagglutinin antibody and show that OPCML knockdown dramatically reduces polyubiquitination of HER2. Western blot of PLKO (OPCML+) and sh339–24 (OPCML knockdown) treated with the proteasomal inhibitor MG-132 (0.1 μM) for 2 hours demonstrate clear upregulation of tHER2 by MG132 in OPCML containing but not OPCML-deficient OSE-C2 (bottom). c, immunoprecipitation of HER2 followed by antihemagglutinin antibody in SKOBS-V1.2 (OPCML–) and BKS-2.1 (OPCML+) (top), showing that OPCML expression in BKS2.1 is associated with polyubiquitination of HER2 and that conversely this polyubiquitination was diminished in OPCML-deficient SKOBS-V1.2. BKS-2.1 (OPCML+) and SKOBS-V1.2 were treated with the lysosomal inhibitor chloroquine (CQ; 0.1 mM) or the proteasomal inhibitor MG-132 (0.1 μM) for 2 hours (below). Immunoblotting for EGFR, HER2, OPCML, and β-tubulin is shown. Densitometric analysis of these Western blot data for HER2 (top) and EGFR (bottom) with β-tubulin as a control demonstrate that MG132 proteasomal inhibition selectively upregulates of HER2 but not EGFR in OPCML-expressing BKS2.1 but not OPCML-deficient SKOBS-V1.2.

164 | CANCER DISCOVERY FEBRUARY 2012 www.aacrjournals.org

Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2012 American Association for Cancer Research. OPCML Downregulates a Spectrum of RTKs research article treatment with MG-132 had no further impact on HER2 transfection of OPCML would by definition select cells that level (Fig. 5B; bottom). have survived and therefore be less apoptosis-prone (indeed, We repeated this experiment in SKOV-3 cancer cells. OPCML- previous fluorescence-activated analysis demonstrated no evi- expressing BKS-2.1 cells were exposed to MG-132, and this pre- dence of apoptosis), we undertook annexin-V and caspase-glo vented OPCML-mediated HER2 degradation with consequent assays and demonstrated modest but significant evidence of HER2 upregulation. In contrast, chloroquine, a weak base that al- apoptosis in unselected SKOV-3 and A2780 (Supplementary ­ kalinizes the lysosome, demonstrated no such inhibition of HER2 ­Fig. S7A and S7B) cells. This finding suggests that both growth degradation (Fig. 5C; bottom). Furthermore, in SKOBS-V1.2 cells ­inhibition and apoptosis occur upon application of rOPCML. lacking OPCML expression (Fig. 5C; bottom), HER2 was strongly In view of these in vitro findings and the detailed in vivo exper- upregulated and MG132 had no additional impact on HER2 ex- iments we previously published (11) that clearly demonstrated pression in these cells, in an analogous fashion to the sh339–24 the in vivo tumor suppressor phenotype of stably transfected cell line in Figure 5B. Taken together, these data suggest that OPCML, we tested whether the rOPCML protein was effective RTK polyubiquitination followed by proteasomal degradation is as an in vivo anticancer therapy. Mice were injected intraperi- preferentially used for OPCML-mediated negative regulation of toneally with either SKOV-3 or A2780 ­cancer cells, and after 1 HER2 both under physiologic conditions and in cancer. week they received twice-weekly intraperitoneal injections of OPCML therefore negatively regulates the activity of a spe- either 1 mL (10 μM) bovine serum albumin (BSA) or 1 mL (10 cific repertoire of RTKs by binding the extracellular domains μM) rOPCML. The experiment was ­terminated ­after 3 weeks of those RTKs, inducing “lipid-raft” associated sequestration, because of obvious extensive intraperitoneal tumor growth mediating a switch away from clathrin internalization to cave- and deteriorating condition of BSA-treated control animals, olin-1–associated enhancement of RTK polyubquitination to whereas rOPCML-treated mice remained healthy throughout ultimately result in proteasomal degradation of those RTKs. the duration of the experiment (Supplementary Fig. S8A). Consideration of our data in the context of recent publica- rOPCML significantly and profoundly suppressed intraperito- tions (34) would suggest that clathrin-independent carriers/ neal tumor growth (Fig. 7B and E). In addition, the formation GPI-enriched early endosomal compartment-mediated inter- of ascites was profoundly and significantly inhibited by rOP- nalization is a possible pathway for the observed OPCML- CML in vivo in both immunoprecipitation models (Fig. 7C), mediated internalization and degradation of RTKs such as and in A2780 tumor-bearing mice, rOPCML significantly in- HER2 and this is linked to the observable strong tumor sup- hibited the number of intraperitoneal deposits compared with pressor phenotype of OPCML. the BSA control (Fig. 7D). Western blotting of the recovered SKOV-3 intraperitoneal Exogenous Recombinant OPCML Protein Inhibits xenograft from control and rOPCML-treated animals (there Ovarian Cancer Growth In Vitro and In Vivo was insufficient A2780 xenograft recovered for Western The extracellular membrane location and mechanism of blotting because of the suppression of tumorigenicity) con- action of OPCML raised the possibility of direct extracel- firmed the same spectrum of rOPCML-mediated RTK down- lular tumor suppressor protein therapy, which would avoid regulation as previously shown in vitro (Fig. 7F), including the complexities of gene therapy for intracellular tumor the lack of EGFR downregulation. Immunohistochemical suppressor replacement or intracellular delivery of protein staining of tumor sections from animals treated with rOP- therapies. We purified recombinant human OPCML domain CML by the use of an OPCML antibody showed peripheral 1–3 protein (rOPCML) by using a bacterial expression vector cell surface staining of OPCML in contrast to the weak or (pHis-Trx) subcloned with domains 1–3 of OPCML, exclud- absent cy­ toplasmic OPCML staining seen in tumor sections ing the signal peptide and GPI anchor sequences (Fig. 6A). from BSA-treated control animals (Supplementary Fig. The addition of rOPCML protein to cell culture supernatant S8B). demonstrated a specific, dose-dependent, highly significant inhibition of cell growth in OPCML nonexpressing SKOV-3 ovarian cancer cells without affecting normal OSE-C2 ovar- Discussion ian surface epithelial cells that express a physiologic level of OPCML is frequently inactivated by somatic methyl- OPCML (Fig. 6B). ation and LOH in more than 80% of epithelial ovarian We then confirmed that rOPCML profoundly and signifi- cancers (11) and in many other cancers (ref. 19; also see cantly inhibited cell growth in 6 of 7 non–OPCML-expressing Supplementary Figure S1 and TCGA http://tcga-portal. epithelial ovarian cancer cell lines (Fig. 6C). IFM demonstrated nci.nih.gov/tcga-­portal/AnomalySearch.jsp) with evi- that rOPCML treatment resulted in HER2 downregulation, dence of prognostic importance and near-ubiquitous loss mirroring the expression of OPCML in the same cell line (Fig. of expression in cell lines and clinical biopsies (ref. 17; 6D). Immunoblotting demonstrated that the addition of rOP- Supplementary Fig. S2 and Kaplan-Meier Plotter: http:// CML protein to culture media potently downregulated the kmplot.com/breast/index.php?p=1). We demonstrate here same spectrum of RTKs as OPCML transfection and abrogated the tumor suppressor mechanism of action of OPCML. pERK and pAKT in both SKOV-3 and A2780 (Fig. 7A) cells. OPCML negatively regulates a specific RTK repertoire con- Exogenous application of rOPCML thus uses the same mech- sisting of EPHA2, FGFR1, FGFR3, HER2, and HER4 recep- anism of action as transfection-induced re-expression of the tors and does not regulate EGFR, HER3, the remaining FGF normal GPI-anchored, glycosylated, OPCML protein. Noting receptors, VEGFR1/3, and many of the other EPHA receptors that pAKT was abrogated and understanding that stable (see Supplementary Table S1). Immunoprecipitation and

FEBRUARY 2012 CANCER DISCOVERY | 165

Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2012 American Association for Cancer Research. research article McKie et al.

A SignalSignal peptidepeptide GPI-anchor site

Met11 ThrThr2727 ProPro3939 SerSer126126 ProPro136136 ThrThr219219 ProPro223223 ThrThr310310 AspAsp322322

OPCML D1 D2 D3

ProPro3939 SerSer126126 ProPro136136 ThrThr219219 ProPro223223 ThrThr310310 HIS-TRX-OPCML HIS-TRX-OPCML His -TRX D1 D2 D3 (rOPCML)(rOPCML) His66-TRX-TRX D1 D2 D3 21 21

B 1.51.5 pp p pp pppp pp OSE-C2 Figure 6. Recombinant OPCML protein PP == NSNS SKOV-3SKOV-3 effect in vitro. a, schematic diagram (left) of OPCML and rOPCML, SDS–PAGE 1.01.0 analysis of rOPCML expressed in Escherichia coli. Lane 1, Coomassie Blue; lane 2, rOPCML Western blot. b, rOPCML targets cancer but not normal cells (left); OSE-C2 and SKOV-3 cells were subjected 0.50.5 to various concentrations of rOPCML (0.5, 1, 2, 5, and 10 μM). The MTT proliferation normalized to control to normalized normalized to control to normalized control to normalized assay is shown relative to control vehicle- MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT assay growth cell MTT treated (dotted line) at 48 hours. A dose- dependent inhibition of SKOV-3 cell 0.00.0 growth demonstrates the specificity of rOPCML for OPCML-deficient cells (NS, 0.50.5 11 22 55 1010 nonsignificant, *P = 0.004, **P  0.0001). rOPCMLrOPCML ((mmM) c, the OSE-C2 line and a panel of ovarian cancer lines (SKOV-3, IGROV, OVISE, OVCAR-5, A2780, PEA1, and PEA2) were C 1.51.5 exposed to 10 μM of rOPCML for 24 hours (white bar) and 48 hours (black bar) with MTT cell growth was normalized to vehicle-only controls (*P  0.0001, αP = β βα NS 0.0284, and P = 0.0079). These data 1.01.0 demonstrate that 6 of 7 (86%) of the p TimeTime (h)(h) ovarian cancer lines (but not OSE-C2) p p 2424 were significantly and profoundly growth suppressed after exposure to rOPCML. 4848 p d, confocal coimmunofluorescence N = 4 demonstrates the relative abundance 0.50.5 p

normalized to control to normalized of HER2 protein in SKOV-3 cells after normalized to control to normalized control to normalized p MTT cell growth assay growth cell MTT MTT cell growth assay growth cell MTT assay growth cell MTT p p p either PBS application (top) or rOPCML p p application (bottom) with punctate colocalization of OPCML with HER2 in the latter. 0.00.0 PEA1 PEA2 PEA1 PEA1 PEA2 PEA2 A2780 A2780 A2780 OVISE OVISE OVISE SKOV-3 OSE-C2 SKOV-3 SKOV-3 OSE-C2 OSE-C2 IGROV-1 IGROV-1 IGROV-1 OVCAR-5 OVCAR-5 OVCAR-5

D Dap1 HER2 OPCML MERGE

PBSPBS

++ rOPCMLrOPCML

166 | CANCER DISCOVERY FEBRUARY 2012 www.aacrjournals.org

Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2012 American Association for Cancer Research. Control r-OPCML Tumor burden RTKs P = 0.4286 SKOV-3 A2780 6000 P = 0.4286 + + + +

le mg) ic ( t Veh

e 4000 M) – + – + weig h µ o us or r m u m PCML(2 p e rO 2000 HER2 t n A2780 T e a M 48) Y12 0 – + – + rOPCML (10 µM) pHER2 ( R EGF A2780 SKOV-3 T

3) Y117 Ascites R ( SKOV-3 pEGF OPCML Downregulates a Spectrum of RTKs research article R1 2500 P = 0.0218 FGF Control r-OPCML T Tumor burden Control Controlr-OPCML r-OPCML RTKs 2000 Tumor burden Tumor burden ( m L) Control r-OPCML ) A RTKs RTKs B TumorP burden= 0.4286 F SKOV-3 66 SKOV-3 A2780 e s 6000 P = 0.4286 RTKs 6000 P = 0.4286 (Y7 SKOV-3 A2780SKOV-3 ci t 1A2780500 6000 P = 0.0286 P = 0.4286 P = 0.4286 + + SKOV-3 A2780 a s 6000 le ++ ++ P = 0.4286 P = 0.4286 ic GFR1 R1 + + + + pF ++ eal ++ P = 0.4286 Veh le mg) ic 1000 M) – + o n Vehicle ( t µ VEGF ++ ++ 4000 2 T Veh Vehicle * – + – + e 4000 4000 µM) – + – + – + Peri t – + AKT VehicleM) M) 4000 weig h µ o us T µ 500 PCML ( M) – + – + rO µ or r m

per mouse per tHER2 u m rOPCML(2PCML(2 p e ) 0 2000 mouse per rO HER2 rOPCML(2 2000 A2780

t n 2000 S473 THER2 HER2 mouse per rOPCML (10 µM) A2780 A2780 rOPCML(2T – + – + HER2) T e a 2000

Mean tumor weight (mg) weight tumor Mean p HER2 A2780 M pAKT ( T A2780 SKOV-3 (mg) weight tumor Mean 2 (Y1248

48) (mg) weight tumor Mean Y12 0 ERK 1/ 0 0 R T +– +– rOPCML (10 µM) pHER2 (Y1248) Number of deposits0 – + – + rOPCML (10 µM) rOPCML (10 µM) 2 R +– +– tEGF pHER2 ( EGFR pHER2 (Y1248) A2780 SKOV-3 rOPCML (10 µM) T +– +– pHER2 E(Y1248)GF EGFR A2780 SKOV-3 A2780 SKOV-3 pERK 1/ T T 20 R EGFR A2780 SKOV-3 ) T P = 0.0142 pEGF lin 3) s Ascites (Y1173 si t C Ascites Ascites Tubu Y117 15 SKOV-3 β- R ( Ascites pEGFR (Y1173) 2500 SKOV-3 SKOV-3 pEGF P = 0.0218 pEGFR (Y1173) depo 2500 P = 02500.0218 SKOV-3 R1 P = 0.0218 R1 pEGFRFGFR1 (Y1173) 2500 TFGF FGFR1 10 P = 0.0218 T T um or 2000 tFGF FGFR1 t 2000 2000 SKOV-3

T ( m L)

of SKOV-3 ) 2000 FR1 ) SKOV-3 66 e s

be r 1500 SKOV-3 (Y7 5 ci t 1500 P = 0.0286 pFG(Y766 ++ P = 0.0286 1500 + + a s P = 0.0286 le ++ ic N um 1500 pFGFR1GFR1 (Y766)R1 P = 0.0286 Vehicle – +++ pF pFGFR1 (Y766) eal 1000 lin VehµM) Vehicle VEGFR1 1000 1000 bu M) – + M) – + TVEGF 0 o n µ µ pFGFR1* (Y766) VEGFR1 -Tu 2Vehicle * T T 1000 rOPCML (10 µM) β M) – + Peritoneal ascites ( m L) ascites Peritoneal µ VEGFR1AKT * – + – +

T Peri t 500 * TAKT AKT 500 ( m L) ascites Peritoneal rOPCMLPCML (2( T T 500

A2780 SKOV ( m L) ascites Peritoneal -3 rO AKT 500 tHER2 rOPCML (2 T 0 rOPCMLtHER2 (2 tHER2 ) 0 +– +– 0 rOPCML (10 µM) tHER2 rOPCML (10 µM) rOPCML (10 M) pHER2 S473 0 – + – + +– +– µ HER2) p pHER2 pAKT (S473) A2780 SKOV-3 rOPCML (10 µM) AKT ( +– +– (Y1248) p 2 pAKT (S473) A2780 SKOV-3 A2780 SKOV-3 pHER2 (Y1248 (Y1248) pAKT (S473) A2780 SKOV-3 ERK 1/2 (Y1248) TERK 1/ ERK 1/2 T D Number of deposits R T Number of deposits Number of deposits tEGFR ERK 21/2 tEGF T Number of deposits tEGFR pERK 1/2 20 tEGFR ERK 1/ 20 R p pERK 1/2 20 P = 0.0142 pEGFR ) pERK 1/2 20 P = 0.0142 pEGF lin s P = 0.0142 (Y1173) pEGFR (Y1173 si t 15 P = 0.0142 pEGFR (Y1173) β-TubulinTubu 15 β- β-Tubulin 15 (Y1173) β-Tubulin 15 depo Control r-OPCML R1 Tumor burden 10 10 tFGFR1 RTKs um or 10 tFGF tFGFR1 t P = 0.4286 10 tFGFR1 SKOV-3 A2780 6000 of FR1 ) 5 pFGFR1 P = 0.4286 be r 5 pFG(Y766) 5 (Y766 pFGFR1(Y766)

++ ++ deposits tumor of Number 5 pFGFR1

N um (Y766) Number of tumor deposits tumor of Number Vehicle lin 4000 deposits tumor of Number 0 0 0 bu M) – + – + +– +– rOPCML (10 µM) β-Tubulin µ 0 – + – + rOPCM+– L (10 µM) +– rOPCML (10 µM) β-Tu β-Tubulin A2780+– SKOV-3 +– rOPCML (10 µM) β-Tubulin A2780 SKOV-3

per mouse per A2780 SKOV-3 rOPCML(2 2000 E Control r-OPCML Control A2780r-OPCML SKOV-3 HER2 Tumor burden Tumor burden A2780 RTKs T RTKs P = 0.4286 (mg) weight tumor Mean P = 0.4286 SKOV-3 A2780 SKOV-3 6000 A2780 6000 P = 0.4286 0 P = 0.4286 ++ ++ ++ ++ +– +– rOPCML (10 µM) pHER2 (Y1248) Vehicle VehicleEGFR 4000 4000 A2780 SKOV-3 T M) – + – + – + – + µ µM) Ascites per mouse per rOPCML(2 rOPCML(2 2000 mouse per 2000 SKOV-3 HER2 HER2 A2780 A2780 T pEGFR T(Y1173) 2500 P = 0.0218 Mean tumor weight (mg) weight tumor Mean FGFR1 (mg) weight tumor Mean T Figure 7. rOPCML abrogates an identical spectrum of RTKs and their downstream signaling in SKOV3 and A2780 in vitro and after the intraperitoneal 2000 administration of rOPCML in vivo. a, rOPCML specifically abrogates total/phospho-HER2, total/phospho-FGFR1 and phospho-, but not total EGFR 0 0 SKOV-3 (*A2780 does not express EGFR, although VEGFR1 was expressed and not altered). rOPCML abrogates downstream substrates pAkt (S-473) and pErk 1 +– +– rOPCML (10 µM) +– +– rOPCML (10 µM) pHER2 (Y1248) 1500 and 2 in both SKOV-3 and A2780. b, quantitation of mean tumor weight per mouse (n = 4). c, rOPCML significantly abrogates ascites both intraperitoneal pHER2 (Y1248) P = 0.0286 ++ EGFR EGFR A2780 SKOV-3 A2780tumor models SKOV-3 (n = 4) and (d) significantly reduces mean number of tumor deposits in the A2780 intraperitoneal model. e, comparison is shown between T T pFGFR1 (Y766) intraperitoneal tumors collected from BSA and rOPCML-IP-treated mice Vehicleand demonstrates the tumor suppressor effect of rOPCML in vivo. f, Western 1000 M) – + VEGFR1 blot analysis of recovered intraperitoneal tumor lysate demonstrating the impactµ of rOPCML protein treatment on cell signaling: rOPCML specifically * T Ascites abrogatesAscites total/phospho-HER2 (pHER2), total/phospho-FGFR1 and phospho- but not total EGFR.

AKT ( m L) ascites Peritoneal 500 SKOV-3 SKOV-3 T rOPCML (2 pEGFR (Y1173) pEGFR (Y1173) 2500 P = 0.0218 2500 P = 0.0218 tHER2 FEBRUARY 2012 CANCER DISCOVERY | 167 FGFR1 FGFR1 T 0 T 2000 2000 +– +– rOPCML (10 µM) Downloaded from cancerdiscovery.aacrjournals.orgSKOV-3 onpHER2 SeptemberSKOV-3 24, 2021. © 2012 American Association for pAKT (S473) A2780 SKOV-3 Cancer Research. 1500 1500 (Y1248) P = 0.0286 P = 0.0286 ++ ++ ERK 1/2 T pFGFR1 (Y766) pFGFR1 (Y766) Number of depositsVehicle – + Vehicle 1000 1000 µM) tEGFRM) – + VEGFR1 VEGFR1 µ T T * * 20

pERK 1/2 ( m L) ascites Peritoneal AKT AKT ( m L) ascites Peritoneal T 500 500 T P = 0.0142 rOPCML (2 rOPCMLpEGFR (2 tHER2 tHER2(Y1173) β-Tubulin 0 150 +– +– rOPCML (10 µM) +– +– rOPCML (10 µM) pHER2 pHER2 pAKT (S473) pAKT (S473) A2780 SKOV-3 A2780 SKOV-3 10 (Y1248) tFGFR1(Y1248)

ERK 1/2 ERK 1/2 T T Number of deposits Number of deposits tEGFR 5 pFGFR1tEGFR(Y766) pERK 1/2 pERK 1/2 20 deposits tumor of Number 20 P = 0.0142 0 P = 0.0142 pEGFR pEGFR +– +– (Y1173)rOPCML (10 µM) β-Tubulin(Y1173) β-Tubulin β-Tubulin 15 15 A2780 SKOV-3

10 10 tFGFR1 tFGFR1

pFGFR1 5 5 (Y766) pFGFR1(Y766) Number of tumor deposits tumor of Number Number of tumor deposits tumor of Number

0 0 +– +– rOPCML (10 µM) +– β+– -TubulinrOPCML (10 µM) β-Tubulin A2780 SKOV-3 A2780 SKOV-3 research article McKie et al. cell-free pulldown experiments with RTKs demonstrated that discussed previously). The lack of identification of a single OPCML physically interacts with EPHA2, FGFR1, and HER2 frequently activated RTK candidate in ovarian cancer to via their extracellular domains but not with EGFR (the levels date leads us to speculate that this may be a plausible of which are unchanged by OPCML). The structural basis for mechanism. We further speculate that OPCML action may this specificity is currently under investigation. We further have even more relevance in other cancer types where a explored the mechanism of OPCML action by using HER2 more oncogenic “addicted” signaling state for these RTKs as a paradigm in the cancer SKOV-3 and the normal OSE-C2 exists (such as HER2 amplification in breast cancer), and cell model systems. this is currently under investigation. To demonstrate that OPCML mediates its function by These findings have immediate relevance for human interaction with the target RTK extracellular domain as a cancer. OPCML is an unusual example of an extracellular prerequisite for RTK downregulation, we transiently trans- tumor suppressor protein and therefore the possibility of fected full-length and truncated (extracellular domain-de- direct tumor suppressor protein therapy at the cell sur- leted) rat HER2/Neu constructs in the presence or absence face could be considered rather than gene therapy with all of OPCML. We demonstrated clear downregulation of the its attendant complexities. The construction of a protein intact 185-kD Neu receptor by greater than 75% in response consisting of domains 1–3 without the N-terminus signal to OPCML in contrast to the 95-kD extracellular domain- peptide or the C-terminus GPI anchor signal, and without less truncated neu that remained unaffected by OPCML ex- eukaryotic glycosylation, allowed us to test the hypothesis pression. In addition, we demonstrated that the extracellular that a recombinant OPCML-like protein (rOPCML) might domain-containing RTK’s negative regulation by OPCML have potential as a cancer therapeutic. In vitro, this recom- was functional and responsible for the observed tumor sup- binant protein therapeutic demonstrated dose-dependent pressor phenotype. OPCML-specific sequestration of HER2 growth inhibition in 6 of 7 epithelial ovarian cancers cell to the detergent-resistant membrane fraction (detergent- lines tested with no effect on a normal ovarian surface insoluble fraction, or cholesterol-rich “lipid-raft” domain) epithelial cell line (that expresses physiologic OPCML). was observed in OPCML-expressing SKOV-3 cells (BKS-2.1) The mechanism of this pharmacologic inhibition by as well as ­enriched colocalization of HER2 and OPCML. rOPCML was shown to be identical to that of the trans- Furthermore, IFM also demonstrated the redistribution of fected OPCML described in this study, with abrogation HER2 to lipid-resistant membrane domains consistent with of an identical repertoire of RTKs, similar downstream OPCML being localized and therefore internalized via a pERK and pAKT effects, evidence of both growth inhibi- “lipid-raft” non–clathrin-dependent endocytic pathway such tion and apoptosis, and no discernable effect on EGFR. as the clathrin-independent carriers/GPI-enriched early en- Furthermore, in 2 separate intraperitoneal models of ovar- dosomal compartment pathway (34). ian cancer in vivo, twice-weekly rOPCML intraperitoneal These data are supported by the findings from pulse-chase therapy inhibited peritoneal tumor growth and the forma- experiments that demonstrate accelerated loss/nonrecirculation tion of ascites and inhibited peritoneal dissemination in of biotinylated surface HER2 protein in OPCML-expressing the A2780 model. Future studies will define the potency, cells. We also demonstrated that OPCML binding mediated optimal dose, and schedule of rOPCML in these models. polyubiquitination of this specific spectrum of RTKs in both In summary, we have defined a novel mechanism of ac- OPCML-transfected cancer cells and normal ovarian surface tion for the frequently inactivated and prognostically impor- epithelial cells expressing physiologic levels of OPCML. MG- tant tumor suppressor OPCML as a systems-level repressor 132 studies in OPCML-expressing cancer and normal cells con- of a defined spectrum of receptor tyrosine kinases. We have firmed that these polyubiquitinated RTKs were then targeted also developed a recombinant OPCML-derived protein that for proteasomal degradation, thereby explaining how OPCML has potential as a treatment approach for ovarian and other negatively regulates its RTK binding partners. cancers. More generally, the discovery of the mechanism of Our hypothesis is therefore that by binding to specific action of OPCML may have serendipitously uncovered a RTKs via their extracellular domains, OPCML sequesters spectrum of RTKs that could be coinhibited with RTK in- these specific RTKs in detergent-resistant membrane do- hibitor combinations to avoid the problem of signaling re- mains and diverts those RTKs (in this case HER2) away dundancy and produce a more profound anticancer effect in from the canonical clathrin endocytic route, where rapid ovarian and other cancers. RTK recycling is prevalent, to a raft-dependent bulk endo- cytic pathway. This leads to OPCML-binding-specific poly- ubiquitination that results in degradation via a proteasomal Methods route, presumably via a specific E3 ubiquitin ligase. OPCML Antibodies can therefore abrogate the ligand-activated and steady-state phosphorylation of ERK 1 and 2 and AKT by regulating the Polyclonal goat and monoclonal mouse anti-OPCML antibodies availability of a spectrum of specific RTKs. were purchased from R&D Systems. Anti-HER2 antibodies were pur- chased from Calbiochem [anti-ErbB2 (Ab-4) and (3B5) mouse mono- We speculate that during carcinogenesis, the frequent clonal antibodies]. Anti-EGFR antibody (goat polyclonal antibody; loss of OPCML expression in epithelial ovarian cancers catalog number AF-231) was from R&D Systems. Antihemagglutinin (and many other types of cancer) by somatic methylation antibody was from Santa Cruz Biotechnology. Anti–EEA-1 and anti– and LOH (11) may therefore deregulate RTKs, confer- caveolin-1 were purchased from Abcam. Phospho-EGFR, HER2, ring a signaling-mediated selective growth advantage on FGFR1 phospho-FGFR1 (Y766), phospho-ERK total ERK, phospho- those cells (as seen in OSE-C2 OPCML knockdown cells AKT, total AKT, EPHA2, FGFR3, HER4, HER3, FGFR2, EphA10,

168 | CANCER DISCOVERY FEBRUARY 2012 www.aacrjournals.org

Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2012 American Association for Cancer Research. OPCML Downregulates a Spectrum of RTKs research article

VEGFR1, and VEGFR 3 b-tubulin were all purchased from AbCam. siRNA Knockdown Horseradish peroxidase-conjugated secondary antibodies were from Endogenous OPCML was knocked down in OSE-C2 cells by Dako. Alexa Fluor 488 goat antirabbit IgG and Alexa Fluor 555 goat transient transfection of a specific pool of 3 siRNAs (StealthKD- antimouse were from Molecular Probes. Invitrogen) with Lipofectamine RNAiMAX reagent following proto- col guidelines. Cell Lines PEO1, PEA1, and PEA2 were recently authenticated and tested Stable shRNA Cell Line Generation in OSE-C2 Cells by Dr. Katherine Hale Stemke in the laboratory of Dr. Gordon Mills Stable expression of shRNAs against physiologic OPCML was (Cancer Center Support grant for Characterized Cell Line core at achieved in OSE-C2 cells via the use of the MISSION cloned shRNAs University of Texas MD Anderson Cancer Center, National Cancer in pLKO.1-puro, supplied as glycerol stocks from Sigma-Aldrich. Five Institute grant number CA16672). These 3 cell lines were originally such cloned shRNAs were transfected into OSE-C2 and selected on 3 developed by Dr. Simon Langdon in 1992 and obtained by us in that µg/mL puromycin for 3 to 4 weeks. laboratory. For PEO1, PEA1, and PEA2, we used Applied Biosystem’s Identifyler kit, which tests 16 STR loci, and then we compared the MTT Proliferation Assay output to public databases. Only 8 loci are published to avoid iden- Cell proliferation assays were performed in quadruplicate with tification of individuals (Supplementary Table S2). The testing was the MTT assay. Cells were plated out in 96-well plates at a density of undertaken in March 2011. 2,000 cells/well and cultured in low-serum medium (0.25% fetal calf OVISE and OSE-C2 were received from their originating labo- serum) or low-serum medium supplemented with 50 ng/mL EGF. ratories but not tested. OVISE was received from Dr. Junzo Kigawa Cells were incubated MTT for 2 hours at 37°C and the purple foma- (Tottori University, Japan) in November 2004. OSE-C2 was re- zan product was solubilized in 100 μL of dimethyl sulfoxide resus- ceived from Dr. Richard Edmondson (Newcastle University, UK) in pended and read on a plate reader at 540 nm. 2008. We have not undertaken authentication of the remaining cell lines, all of which were received from the American Type Culture Coimmunoprecipitation and Pull-Down Assays Collection. Cell layers were washed in PBS and incubated for 30 minutes in lysis buffer (1% Triton X-100; 10 mM Tris, pH 8.0; 150 mM NaCl; 2.5

Cell Culture mM MgCl2; 5 mM EGTA; 1mM Na3VO4; 50 mM sodium fluoride; The SKOV-3-derived OPCML-expressing lines (SKOBS-3.5, and protein inhibitor cocktail; Roche). Cell lysates were then cleared BKS2.1, and empty-vector SKOBS-V1.2) have been described previ- by centrifugation at 13,000 rpm (16,000 3 g) for 20 minutes at 4°C, ously (11). Stimulation time courses were undertaken with 50 ng/ and aliquots containing equal amounts of protein were incubated mL human recombinant EGF (Promega) or 10 ng/mL acidic and with the appropriate antibody before addition of secondary antibody basic FGFs (FGF1/2; R&D Systems) following serum-starvation over- conjugated to Sepharose resin. Beads were then washed 3 times with night. The immortalized normal ovarian surface epithelium (OSE) lysis buffer and eluted by heating for 5 minutes in 50 μL of SDS line OSE-C2 (32) was held in culture at the permissive temperature sample buffer. Pull-down assays were performed with recombinant GST-OPCML fusion proteins bound to magnetic glutathione beads of 33°C in 95% air and5% CO2. We peformed transient transfections with the use of the Effectene lipofectamine reagent (QIAGEN), fol- (Promega). Cell lysates that were prepared as for immunoprecipita- lowing the manufacturer’s guidelines. tion and proteins that were produced with the use of the in vitro TNT Rabbit Reticulocyte Lysate Expression System (Promega) or expressed in bacteria were analyzed for interactions. Plasmid Constructs The OPCML cDNA expression plasmids in pcDNA3.1zeo pre- Expression of Recombinant OPCML and FGFR viously described (11) were used for transient transfections. The Extracellular Domain cDNA encoding all 3 OPCML Ig domains was generated by PCR Recombinant proteins were produced in the BL21 bacterial cell and introduced into the bacterial GST-fusion expression vector line (Promega). Protein expression was induced by the addition of pGEX-6P-1 (GE Healthcare) and pHisTRx (kindly provided by Dr 1 mM final concentration IPTG at 37°C for 4 hours. Cells were then Edward McKenzie, University of Manchester). The hemaggluti- harvested by centrifugation at 5000 rpm for 20 minutes at 4°C. Cell nin-tagged Ubiquitin vector (pRK5-HA-Ubiquitin-WT) was ob- pellets were resuspended in PBS pH 7.4 and prepared for lysis with tained from Dr. Luke Gaughan (Newcastle University), and the the use of lysozyme from the egg white of a chicken (1 mg/mL; EGFR and HER2 cDNA in pcDNA-3.1zeo was provided by Prof. Sigma-Aldrich), 10 units/mL Dnase I (Sigma-Aldrich), and 1% Triton Bill Gullick (University of Kent). Rat Neu full-length cDNA (185- X-100. After 1 hour of incubation at room temperature, the lysate kD pNeu) and deletion mutant form with no extracelluar domain was subject to freeze-thaw to enable complete lysis. (95-kD ∆5) were cloned in pSecTagB2 as previously described (33). A full-length FGFR1 cDNA clone and extracellular domain con- Solubilization and Refolding of Inclusion Bodies structs were provided by Prof. Graeme Guy and Prof. Kyung Hyun Inclusion bodies were solubilized in denaturation buffer (8 M Kim, respectively. Urea; 20 mM Tris-HCl, pH 8.0; 150 mM NaCl; and 10 mM DTT) to a final concentration of 5 mg/mL. Refolding of proteins was under- Immunofluorescence Microscopy taken by extensive dialysis against cold PBS in 10-kDa MWCO dialy- Cells grown on glass slides were fixed in 4% paraformaldehyde and sis tubing. Protein concentrations were monitored throughout the permeabilized for 20 minutes with PBS containing 0.2% saponin be- experiment with protein assay reagent (Bio-Rad Laboratories) with fore blocking in PBS containing 10% goat serum, 2% albumin and 2% the use of bovine serum albumen as a standard. fetal calf serum for 1 hour. Slides were incubated with appropriate combinations of mAb OPCML, mAb HER2, and pAb EGFR primary Basic Detergent-Insoluble Fraction Fractionation antibodies for 1 hour at room temperature, followed by incubation Cells were harvested by treatment with EDTA and centrifugated at for 1 hour with animal antimouse Alexa-555 (OPCML) and animal 3000 rpm for 5 minutes, and then washed with ­ice-cold PBS and fol- antirabbit Alexa 488 (HER2) before they were mounted and imaged lowed by a second centrifugation step. Cells were then osmotically lysed on a Zeiss LSM 510 confocal microscope. by the use of a low-salt buffer (20 mM Tris-HCl, pH 7.0, containing

FEBRUARY 2012 CANCER DISCOVERY | 169

Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2012 American Association for Cancer Research. research article McKie et al. complete protease inhibitor cocktail; Roche); then, we passed the cell receiving twice-weekly 1-mL injections of 10 µM rOPCML in PBS. suspension through a 22-gauge needle (an aliquot was retained for The experiment was terminated after 3 weeks of treatment because of analysis; whole-cell lysate). After that, we performed a second centrifu- disease progression in control animals. gation at 45,000 rpm, 4°C for 1 hour. The membrane pellet was resus- pended (total membrane fraction; aliquot retained) in ice-cold PBS plus Immunohistochemical Staining inhibitors and centrifuged again 45,000 rpm, 4°C for 30 minutes. The Sections (2.5 microns thick) were cut from formalin-fixed, par- membrane pellet was resuspended in ice-cold PBS, 1% Triton-X100, plus affin-embedded tissue. The tissue sections were dewaxed and rehy- inhibitors, and allowed to incubate for 30 minutes at 4°C. The detergent- drated by immersing in 3 changes of xylene, 3 changes of 74 OP, treated membrane fraction was then centrifuged at 45,000 rpm at 4°C and 1 70% alcohol for 2 minutes each change, then in tap water. for 1 hour, the supernatant was removed and retained for analysis (de- Endogenous peroxidase blocking was done by immersing the slides tergent-soluble membranes), and the remaining pellet was solubilized in in hydrogen peroxide block (H2O2)–0.6% hydrogen peroxide in tap 1% SDS (detergent-resistant membrane fraction). water for 15 minutes. The sections were then stained, developed, and counterstained on the 16000 Biogenex machine set according to Hemagglutinin Ubiquitination Assay manufacturer protocol and using super sensitive polymer HRP IHC SKOBS-V1.2 and BKS-2.1 were transiently transfected with a hem- detection kit (Biogenex). agglutinin-tagged ubiquitin vector. At 24 hours after transfection, cells were serum starved for an additional 24 hours before being treated Statistical Analyses with the MG-132 for 1 hour at 37°C. Cells were then treated with EGF Data are expressed as mean ± SEM. Differences were analyzed (50 ng/mL) for 1 hour at 37°C (+EGF) or left untreated (–serum). A by the Fishers exact or Student t test. P  0.05 was considered rabbit anti-HER2 monoclonal antibody was used for immunoprecipi- significant. tation. Ubiquitinated proteins were detected by immunoblotting with an antihemagglutinin antibody. Samples were run in parallel to probe Disclosure of Potential Conflicts of Interest for HER2 with a mouse anti-HER2 monoclonal antibody. No potential conflicts of interest were disclosed. Biotin Pulse-Chase Labeling of Cell-Surface Proteins Acknowledgments Cell-surface proteins were labeled via the Pierce cell-surface pro- tein isolation kit where cell-impermeable, cleavable biotinylation We wish to acknowledge the support of the NIHR Biomedical reagent (Sulfo-NHS-SS-Biotin) labels exposed primary amines of Research Centre, the Cancer Research UK Clinical Centre, and the proteins on the surface of cells. After a 30-minute “pulse” incubation Experimental Cancer Medicine Centre at Imperial College London. with biotinylation reagent at 4°C, the cells are returned to growth We thank Prof. K. H. Kim, from the University of South Korea, for media and incubated for required periods at 37°C in 5% CO2. Cells His-tagged FGFR1 ECD; Dr. Luke Gaughan, of Newcastle University, are then harvested, lysed, and the labeled surface proteins are af- for HA-tagged ubiquitin expression plasmid; and Prof. Bill Gullick, finity-purified using Thermo Scientific NeutrAvidin Agarose resin, of the University of Kent, for EGFR and HER2 cDNA in pcDNA- followed by elution in 2× SDS lysis buffer. Before spun-column puri- 3.1zeo. The immortalized normal OSE line, OSE-C2, was a kind gift fication of the biotinylated protein, 25% of the cell lysate was kept as from Dr. Richard Edmondson of Newcastle University. We would a total cellular input sample and added to SDS-gel loading buffer for also like to thank Mr. Feras Al-Jayoosi for technical assistance with subsequent Western analysis. some of these experiments. We are extremely grateful to Dr. Fiona Simpson and Prof. Henning Walczak for critical review of this work. Annexin V/Propidium Iodide Fluorescence-Activated Cell Sorting Apoptosis Assay Grant Support A total of 105 cells were seeded into 6-well plates, with 1 mL of Supported by the Ovarian Cancer Action Research Centre Core RPMI (10% fetal calf serum, 2 mM L-glutamine, and 50 units/mL Grant (principal investigator H. Gabra) and Cancer Research UK penicillin/streptomycin) for 24 hours. Then, 24 hours later they Discovery Award C8220/A14254 (principal investigator H. Gabra). were either treated with BSA (3 mg/mL) or OPCML (3 mg/mL) for 6 hours. Cells were analyzed by flow cytometry after harvesting by Received October 3, 2011; revised November 7, 2011; accepted gentle cell scraping and dual staining with annexin V and PI (FITC November 15, 2011; published online February 13, 2012. Annexin V Apoptosis Detection Kit II; BD Pharmingen) as per manu- facturer’s protocol. Flow cytometric analysis was performed using a FACSCalibur and CellQuest software (Becton Dickinson) and FlowJo References (Tree Star) data analysis. 1. Bray F, Loos AH, Tognazzo S, Vecchia CL. Ovarian cancer in Europe: cross-sectional trends in incidence and mortality in 28 countries, Caspase-Glo Apoptosis Assay 1953–2000. Int J Cancer 2005;113:977–90. Casapse-3/7 apoptosis assays were performed in quadruplet by 2. Hall J, Paul J, Brown R. Critical evaluation of p53 as a prognostic using a commercially available kit following manufacturer’s instruc- marker in ovarian cancer. Exp Rev Mol Med 2004;6:1–20. tions (Caspase-Glo 3/7 assay, Promega) according to the aforemen- 3. Radice P. Mutations of BRCA genes in hereditary breast and ovarian tioned scheme. cancer. J Exp Clin Cancer Res 2002;21:9–12. 4. Meng Q, Xia C, Fang J, Rojanasakul Y, Jiang B. Role of PI3K and AKT spe- In Vivo Intraperitoneal Model cific isoforms in ovarian cancer cell migration, invasion and proliferation through the p70S6K1 pathway. Cellular Signalling 2006;18:2262–71. Two groups of 10 animals were intraperitoneally administered ei- 5. Maihle NJ, Baron AT, Barrette BA, Boardman CH, Christensen TA, 7 ther 1 × 10 cells of the A2780 or SKOV-3 cell line suspending in Cora EM, EGF/ErbB receptor family in ovarian cancer. Cancer Treat 0.5 mL of sterile PBS. Tumors were allowed to develop for 10 days, Res 2002;107:247–58. and 2 animals from each cohort were euthanized to analyze tumor 6. Le Page C, Ouellet V, Madore J, Hudson TJ, Tonin PN, Provencher take. The remaining animals were segregated into 2 groups: control DM, From gene profiling to diagnostic markers: IL-8 and FGF2 animals (4 animals per cell line) receiving twice-weekly 1-mL injec- complement CA125 serum-based markers in ovarian cancer. Int J tions of 10 µM BSA in PBS and trial animals (4 animals per cell line) Cancer 2006;118:1750–8.

170 | CANCER DISCOVERY FEBRUARY 2012 www.aacrjournals.org

Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2012 American Association for Cancer Research. OPCML Downregulates a Spectrum of RTKs research article

7. Birrer M, Johnson M, Hao K, Wong KK, Park DC, Bell A, Whole 20. Levitt P. A monclonal antibody to limbic system neurons. Science genome oligonucleotide-based array comparitive genomic hybridi- 1984;223:229–301. sation analysis identified fibroblast growth factor 1 as a prognostic 21. Chen J, Lui W, Vos M, Clark GJ, Takahashi M, Schoumans J, The marker for advanced-stage serous adenocarcinomas. J Clin Oncol t(1;3) breakpoint-spanning genes LSAMP and NORE1 are involved 2007;25:2281–7. in clear cell renal cell carcinomas. Cancer Cell 2003;4:405–13. 8. Lafky J, Wilken J, Baron A, Maihle N. Clinical implications of the 22. Struyk A, Canoll P, Wolfgang M, Rosen C, D’Eustachio P, Salzer ErbB/epidermal growth factor (EGF) receptor family and its ligands J. Cloning of neurtrimin defines a new subfamily of differentially in ovarian cancer. Biochim Biophys Acta 2008;1785:232–65. ­expressed neural cell adhesion molecules. J Neurosci 1995;15. 9. Trinh XB, Tjalma WA, Vermeulen PB, Van den Eynden G, Van der 23. Fanatsu N, Miyata S, Kumanogoh H. Characterisation of a novel Auwera I, Van Laere SJ, The VEGF pathway and the AKT/mTOR/ rat brain GPI-anchored protein (Kilon),a member of the IgLON cell p70S6K1 signalling pathway in human epithelial ovarian cancer. Br J adhesion molecule family. J Biol Chem 1999;274:8224–30. Cancer 2009;100:971–8. 24. Lodge A, Howard M, McNamee C, Moss D. Co-localisation, 10. Gómez-Raposo C, Mendiola M, Barriuso J, Casado E, Hardisson D, Redondo ­heterophilic interactions and regulated expression of IgLON ­family A. Angiogenesis in ovarian cancer. Clin Transl Oncol 2009;11:564–71. proteins in the chick nervous system. Brain Res Mol Brain Res 11. Sellar GC, Watt KP, Rabiasz GJ, Stronach EA, Li L, Miller EP, OPCML 2000;82. at 11q25 is epigenetically inactivated and has tumor-suppressor 25. Simons K, Ikonen E. Functional rafts in cell membrane. Nature function in epithelial ovarian cancer. Nat Genet 2003;34:337–43. 1997;387:569–72. 12. Czekierdowski A, Czekierdowska S, Szymanski M, Wielgos M, 26. Simons K, Toomre D. Lipid rafts and signal transduction. Nat Rev Kaminski P, Kotarski J. Opioid-binding protein/cell adhesion mole- Mol Cell Biol 2000;1:31–9. cule-like (OPCML) gene and promoter methylation status in women 27. Chatterjee S, Mayor S. The GPI-anchor and protein sorting. Cell Mol with ovarian cancer. Neuroendocrinol Lett 2006;27:609–13. Life Sci 2001;58:1969–87. 13. Zhang J, Ye F, Chen H, Ye D, Lu W, Xie X. Deletion of OPCML 28. van Zantena T, Cambib A, Koopmanc M, Joostenb B, Figdorb C, gene and promoter methylation in ovarian epithelial carcinoma. Garcia-Parajo M. Hotspots of GPI-anchored proteins and integrin Zhangguoe Yi Xue Ke Xue Yuan Xue Bao 2006;28:173–7. nanoclusters function as nucleation sites for cell adhesion. Proc Natl 14. Chen H, Ye F, Zhang J, Lu W, Cheng Q, Xie X. Loss of OPCML ex- Acad Sci U S A 2009;106:18557–62. pression and the correlation with CpG island methylation and LOH 29. Hancock J. Lipid rafts: contentious only from simplistic standpoints. in ovarian serous carcinoma. Eur J Gynaecol Oncol 2007;28:464–7. Nat Rev Mol Cell Biol 2006;7:456–62. 15. Reed JE, Dunn JR, du Plessis DG, Shaw EJ, Reeves P, Gee AL, 30. Cancer Genome Atlas Research Network. Integrated genomic Expression of cellular adhesion molecule ‘OPCML’ is down-­regulated analyses­ of ovarian carcinoma. Nature 2011;474:609–15. in gliomas and other brain tumours. Neuropathol Appl Neurobiol 31. Langdon SP, Lawrie SS, Hay FG, Hawkes MM, McDonald A, 2007;33:77–85. Hayward IP, Characterization and properties of nine human ovarian 16. Tsou JA, Galler JS, Siegmund KD, Laird PW, Turla S, Cozen W, adenocarcinoma cell lines. Cancer Res 1988;48:6166–72. Identification of a panel of sensitive and specific DNA methylation 32. Davies BR, Steele IA, Edmondson RJ, Zwolinski SA, Saretzki G, von markers for lung adenocarcinoma. Mol Cancer 2007;6:70. Zglinicki T, Immortalisation of human ovarian surface epithelium 17. Duarte-Pereira S, Paiva F, Costa VL, Ramalho-Carvalho J, with telomerase and temperature-sensitive SV40 large T antigen. Exp Sawa-Bor­ dalo J, Rodrigues A, Prognostic values of opioid ­binding Cell Res 2003;288:390–402. protein/cell adhesion molecule-like promoter methylation in 33. Kumagai T, Davis J, Horie T, O’Rourke D, Greene M. The role of dis- bladder­ carcinoma. Eur J Cancer 2011;47:1106–14. tinct p185-neu extracellular subdomains for dimerization with the 18. Sriiaksa R, Zeller C, El-Bahrawy MA, Dai W, Daduang J, Jearanaikoon epidermal growth factor (EGF) receptor and EGF-mediated signal- P, CpG-island methylation study of liver fluke-related cholangiocar- ing. Proc Natl Acad Sci U S A 2001;98:5526–31. cinoma. Br J Cancer 2011;104:1313–8. 34. Howes MT, Kirkham M, Riches J, Cortese K, Walser PJ, Simpson 19. Cui Y, Ying Y, van Hasselt A, Ng KM, Yu J, Zhang Q, OPCML is a F, Clathrin-independent carriers form a high capacity endocytic broad tumour suppressor for multiple carcinomas and lymphomas sorting system at the leading edge of migrating cells. J Cell Biol with frequent epigenetic inactivation. PLoS One 2008;3:e29902–11. 2010;190:675–91.

FEBRUARY 2012 CANCER DISCOVERY | 171

Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2012 American Association for Cancer Research. The OPCML Tumor Suppressor Functions as a Cell Surface Repressor−Adaptor, Negatively Regulating Receptor Tyrosine Kinases in Epithelial Ovarian Cancer

Arthur B. McKie, Sebastian Vaughan, Elisa Zanini, et al.

Cancer Discovery 2012;2:156-171.

Updated version Access the most recent version of this article at: http://cancerdiscovery.aacrjournals.org/content/2/2/156

Supplementary Access the most recent supplemental material at: Material http://cancerdiscovery.aacrjournals.org/content/suppl/2011/12/28/2159-8290.CD-11-0256.DC 1 http://cancerdiscovery.aacrjournals.org/content/suppl/2012/02/07/2159-8290.CD-11-0256.DC 2

Cited articles This article cites 34 articles, 7 of which you can access for free at: http://cancerdiscovery.aacrjournals.org/content/2/2/156.full#ref-list-1

Citing articles This article has been cited by 8 HighWire-hosted articles. Access the articles at: http://cancerdiscovery.aacrjournals.org/content/2/2/156.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerdiscovery.aacrjournals.org/content/2/2/156. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.

Downloaded from cancerdiscovery.aacrjournals.org on September 24, 2021. © 2012 American Association for Cancer Research.