LPA Activates Wnt/β-catenin Signaling in Ovarian Cancer Lysophosphatidic Acid Initiates Epithelial To Mesenchymal Transition and Induces Beta-Catenin- Mediated Transcription In Epithelial Ovarian Carcinoma

During tumor progression, epithelial ovarian cancer (EOC) cells undergo epithelialto-mesenchymal transition (EMT), which influences metastatic success. Mutationdependent activation of Wnt/β-catenin signaling has been implicated in gain of mesenchymal phenotype and loss of differentiation in several solid tumors; however, similar mutations are rare in most EOC histotypes. Nevertheless, evidence for activated Wnt/β-catenin signaling in EOC has been reported and immunohistochemical analysis of human EOC tumors demonstrates nuclear staining in all histotypes. The current study addresses the hypothesis that the bioactive lipid lysophosphatidic acid (LPA), prevalent in the EOC microenvironment, functions to regulate EMT in EOC. Our results demonstrate that LPA induces loss of junctional β-catenin, stimulates clustering of β1 integrins, and enhances the conformationally active population of surface β1 integrins. Further, LPA treatment initiates nuclear translocation of β-catenin and transcriptional activation of Wnt/β-catenin target genes resulting in gain of mesenchymal marker expression. Together these data suggest that LPA initiates EMT in ovarian tumors through β1-integrin-dependent activation of Wnt/βcatenin signaling, providing a novel mechanism for mutation-independent activation of this pathway in EOC progression. Ovarian cancer is a heterogeneous group of cancers subdivided into four types based on cellular phenotype: germ cell tumors, stromal tumors, primary peritoneal tumors and epithelial tumors (1,2). Epithelial tumors represent 90% of all occurrences of ovarian cancer (epithelial ovarian cancer or EOC); of these high-grade serous EOC is the most commonly occurring (75% 1 http://www.jbc.org/cgi/doi/10.1074/jbc.M115.641092 The latest version is at JBC Papers in Press. Published on July 14, 2015 as Manuscript M115.641092 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. at U niersity of N tre D am e L ibaries on July 7, 2015 hp://w w w .jb.org/ D ow nladed from LPA Activates Wnt/β-catenin Signaling in Ovarian Cancer of cases, 90% of deaths; (2,3)). The lack of specific targeted therapies and acquisition of resistance to current standard therapies (combination platinumand taxol-based compounds), coupled with diagnosis at advanced stage, makes ovarian cancer the deadliest gynecological malignancy and fifth leading cause of cancer-related deaths among American women (4-6). Improved understanding the pathobiology and mechanisms underlying metastasis of EOC will aid the development of more efficacious treatments for women with this disease. The etiology of EOC remains unclear; however two major hypotheses predict that epithelial ovarian cancers arise from genetic aberrations in either the ovarian surface epithelium or the fallopian tube epithelium (3,7-11). Fallopian tube epithelial cells are derived from intermediate mesoderm and display classic epithelial markers such as epithelial cadherin (E-cadherin). Although normal ovarian surface epithelium (OSE) characteristically exhibits epithelial morphology, the mesodermally-derived tissue expresses mesenchymal markers (e.g. vimentin, Neural (N)cadherin), and OSE cells in culture often deposit extracellular matrix components characteristic of mesenchyme (collagen types I and III) (12,13). Typically, gain of mesenchymal phenotype (epithelial-to-mesenchymal transition, or EMT) is a hallmark tumor dissemination and progression in many carcinomas (14,15); however, ovarian carcinomas exhibit an early gain of epithelial phenotype, or mesenchymal-to-epithelial transition (MET). This gain of an epithelial gene profile aids disseminating cells in multicellular aggregate (spheroid) formation and anoikis resistance (16). Later in progression cells revert to a mesenchymal phenotype (EMT) and it is proposed that this phenotypic plasticity contributes to decreased response to conventional therapeutics (17-20). Further, recent work has demonstrated a role for mesenchymal phenotype in mesothelial cell invasion at metastatic sites in the peritoneal cavity (21). Understanding the mechanism(s) regulating this phenotypic plasticity may enable reversal of acquired resistance to chemotherapeutic agents. Acquisition of mesenchymal phenotype via EMT can be initiated by extracellular matrix context, disruption of adhesion, and soluble signaling factors (22,23). Of these factors, lysophosphatidic acid (LPA) has emerged as a potentially important mediator of EMT in ovarian carcinoma due in part to its high concentration in the ovarian cancer microenvironment (up to 80μM) (24,25). LPA modulates mitogenic activity, motility, escape from anoikis and survival effects through a class of heterotrimeric G-proteincoupled receptors, LPA1 – LPA5 (26,27). One mechanism by which LPA modulates motility and facilitates the gain of a mesenchymal phenotype in EOC is disruption of E-cadherin-based cell-cell adhesions (28-31). Of particular interest is the fate of adherens junction-associated and/or cytoplasmic β-catenin following disruption of cellcell adhesions. In intact epithelial tissues, βcatenin displays junctional localization and is associated with the cytoplasmic domain of Ecadherin. Junction disruption can induce pathways that subsequently target β-catenin for proteosome-mediated degradation or for nuclear translocation and transcriptional regulation (32). As previous work has demonstrated the intersection of cytoplasmic and nuclear β-catenin pools (33), this study investigated the consequences of LPA-induced disruption of Ecadherin-mediated cell junctions on subcellular βcatenin localization and Tcf/Lef/β-catenin transcriptional activity. These results provide additional support for the role of ligandindependent β-catenin activity in serous epithelial ovarian carcinomas. EXPERIMENTAL METHODS Cell Culture. OVCA429 and OVCA433 cell lines were generously provided by Dr. Robert Bast Jr. (M.D. Anderson Cancer Center, Houston, TX) and were maintained in MEM (Gibco Invitrogen, Carlsbad, CA), 10% fetal bovine serum (Gibco Invitrogen), penicillin/streptomycin (Gibco Invitrogen), amphotericin B, (Cellgro by Mediatech), non-essential amino acids (Cellgro by Mediatech, Herndon, VA), and sodium pyruvate (Cellgro by Mediatech) at 37°C in 5% CO2. Multicellular aggregates were formed in 96 well plates coated with 50 microliters of 0.5% agarose in serum-free media by seeding 5,000 cells per well in serum-free media and incubating overnight at 37°C in 5% CO2. Aggregate formation was confirmed by light microscopic visualization. Lysophosphatidic Acid. LPA was purchased from Cayman Chemical (Ann Arbor, MI) and was prepared for use by dehydrating the 2 at U niersity of N tre D am e L ibaries on July 7, 2015 hp://w w w .jb.org/ D ow nladed from LPA Activates Wnt/β-catenin Signaling in Ovarian Cancer lyophilized protein under a tissue culture hood, on ice, overnight. LPA was resuspended in 1% BSA in PBS at a final concentration of 2 mM, allowed to dissolve on a rotator at 4°C overnight, then aliquoted. Aliquots in use were stored at -20°C, and at -80°C for long-term storage. Where indicated, cells were pre-treated for 15 minutes prior to addition of LPA with the LPA receptor inhibitor, Ki16425, or the Rho signaling inhibitor, Y27632, which were both purchased form Cayman Chemical (Ann Arbor, MI). Antibodies. Mouse monoclonal anti-active β-catenin (clone 8E7) recognizes β-catenin that is de-phosphorylated on Ser37 and/or Thr41 and was purchased from Upstate Biotechnology (Lake Placid, NY). Purified mouse anti-β-catenin monoclonal antibody (clone 14/β-catenin), mouse anti-E-cadherin (clone 36/E-cadherin), and antiactive β1 integrin (clone HUTS21) were purchased from BD Transduction Laboratories (San Jose, CA). Anti-β1 integrin (function-blocking; MAB1959), anti-β1 integrin (MAB2250) and a mouse monoclonal antibody recognizing FAK phosphorylated at tyrosine 397 (clone 18) were purchased from EMD Millipore (Billerica, MA). Anti-HDAC1 was purchased from Thermo Fisher Scientific (Rockford, IL). Anti-PCNA (clone PC10) was purchase from Dako (Glostrup, Denmark). Mouse anti-E-cadherin (clone HECD1) was purchased from Zymed (San Francisco, CA). Monoclonal mouse anti-vinculin (clone VIN-11-5) was purchased from Abcam (Cambridge, MA). Monoclonal mouse antivimentin (clone VIM-13.2), polyclonal mouse anti-IgG, peroxidase-conjugated anti-mouse IgG and peroxidase-conjugated anti-Rabbit IgG were purchased from Sigma-Aldrich (St. Louis, MO). Rhodamine phalloidin, Alexa Fluor 488 goat antimouse IgG (H+L) was purchased from Molecular Probes (Eugene, OR). Rat anti-Tcf was purchased from Kamiya Biomedical (Tukwila, WA). Polyclonal anti-mouse IgG was purchased from Chemicon (Temecula, CA). Rabbit polyclonal anti-FAK was purchased from Santa Cruz Biotechnology (Dallas, TX). Immunofluorescence. Cells were plated on 22-mm glass coverslips coated by passive adsorption with type I collagen (from rat tail, BD Biosciences, San Jose, CA) and placed in 6 well tissue culture plates and were treated with LPA at the concentrations and times described in the Figure Legends. Following treatment, cells were gently washed in phosphate buffered saline (PBS), fixed in paraformaldehyde (4%) and permeabilized with 0.3% Triton X-100 at room temperature, washed in PBS, and blocked in PBS/1% bovine serum albumin (BSA) or PBS/2.5% goat serum followed by the addition of primary antibody diluted in PBS/1% BSA or PBS/2.5% goat serum at 37°C. After two washes in PBS, coverslips were incubated with Alexa Fluor 488 or Alexa Fluor 594-conjugated goat anti-mouse or goat anti-rabbit IgG (1:500 dilution) in the dark at room temperature. Coverslips were washed in PBS twice and in distilled water once, fixed using gelvatol, and visualized using fluorescence microscopy (Nikon Microphot FXA or Leica Microsystems DM5500B). Semiquantitative analysis of surface-associated (junctional) β-catenin staining was determined by counting a minimum of 12 fields per treatment and scoring as positive the number of cells with two remaining fluorescent cell-cell borders (30). Immunohistochemistry. Immunohistochemical analysis was performed on a human tumor tissue microarray prepared at the Robert H. Lurie Comprehensive Cancer Center at Northwestern University with Institutional Review Board approval. Tumor specimens were cut 3 to 5 microns thick (1 mm in diameter) and deparaffinized. Antigen retrieval was accomplished by heat induction at 99°C in an antigen retrieval solution (10 mM Tris, 1mM EDTA, pH 9.0) for approximately 1 hour. Immunohistochemical staining with anti-β-catenin (BD Transduction Laboratories; 1:50) was performed according to standard procedures and as previously described (31). A total of 105 tissues of varying histotypes were evaluated (41 serous, 26 endometrioid, 3 MMMT, 6 mucinous, clear cell, 19 borderline, 1 untyped). A determination of nuclear β-catenin positivity or negativity was determined based on presence of β-catenin staining coinciding with hematoxylin-stained nuclei (34). Cell Fractionation. Cells were fractionated following a 2 h incubation with LPA (40 uM) or LiCl (control, 40 uM) as previously described (35). Briefly, cells were washed twice with cold PBS, and lysed with a cold hypotonic lysis buffer (10.0 mM NaCl, 20.0 mM HEPES pH 7.9, 1.0 mM EDTA, 2.0 mM MgCl2, 20.0 mM β3 at U niersity of N tre D am e L ibaries on July 7, 2015 hp://w w w .jb.org/ D ow nladed from LPA Activates Wnt/β-catenin Signaling in Ovarian Cancer glycerophosphate, 1.0 mM Na3VO4, 1.0 mM PMSF, 1.0 mM DTT, 200 mM sucrose, 0.5% NP40, and 10 μg/mL of each aprotinin, pepstatin A and leupeptin). Lysate was collected by scraping, passed through a 26 gauge syringe and centrifuged at 16,000 × g for 1 minute at 4°C after a 10 minute incubation on ice. The cytoplasmic fraction was collected (supernatant), and the pellet was washed twice with hypotonic lysis buffer before treatment with nuclear extraction buffer (420.0 mM NaCl, 20.0 mM HEPES pH 7.9, 1.0 mM EDTA, 2.0 mM MgCl2, 20.0 mM β-glycerophosphate, 1.0 mM Na3VO4, 1.0 mM PMSF, 1.0 mM DTT, 25% glycerol and 10 μg/mL of each aprotinin, pepstatin A and leupeptin). Following a 10-15 minute incubation and 5-minute centrifugation (16,000 × g at 4°C), the nuclear fraction was collected. Protein concentration was measured using a detergent-compatible protein assay kit (Bio-Rad, Hercules, CA). Western blot analysis was performed as described below. Control blots were probed for HDAC1 and β-actin to assess nuclear and cytoplasmic fractions, respectively. Immunoprecipitation. The β-catenin/Tcf co-immunoprecipitation protocol was adopted from chromatin immunoprecipitation protocols previously described (36,37). Cells were grown to 80% confluence, then serum-starved overnight. Appropriate samples were pre-treated with 40 μM Ki16425 (LPA receptor inhibitor, Cayman Chemical, Ann Arbor, MI) or DMSO control, followed by addition of 40 μM LPA for 2 hours. Non-treated cells, cells treated with Ki16425 alone, and cells treated with 40 μM LiCL served as controls. Cells were collected in a hypotonic lysis buffer (20 mM HEPES, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA), incubated on ice for 20 minutes, then centrifuged at 13,000 rpm for 10 minutes at 4°C. Nuclei isolated from the previous step were disrupted by resuspension in a ‘breaking’ buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl, 1% SDS, 2% Triton X-100), then passed through a 26 gauge syringe 8 times. The suspension was centrifuged at 13,000 rpm for 10 minutes at 4°C, diluted in 1 ml triton buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl, 0.1% Triton X-100) and cleared of nonspecific binding by incubation with 20 μl protein A/G beads (Sigma-Aldrich, St. Louis, MO) at 4°C overnight. After centrifugation and collection of supernatant, protein concentration was measured using a kit (Bio-Rad). Five hundred micrograms total protein were added to 1 ml triton buffer, 20 μl protein A/G beads and 5 μg anti β-catenin antibody, then incubated on a rotator overnight at 4°C. Beads were washed 5 times in triton buffer, and then resuspended in 2X sample buffer. Samples were boiled, then analyzed for Tcf expression by Western blot as described below. Antibody was removed from the PVDF membrane by washing in stripping buffer (50 mM Tris, pH 6.8, 1% SDS, 150 mM NaCl, 100 mM βmercaptoethanol, 0.02% sodium azide) then reprobed for β-catenin as an assay control. Experiments were repeated in triplicate. Western blots were quantified using Multigauge v.2 (FUJIFILM, Tokyo, Japan). Flow Cytometry Analysis. Cells were serum-starved overnight, followed by pretreatment of appropriate samples with the LPA receptor inhibitor, Ki16425 (Cayman Chemical, Ann Arbor, MI) for 30 minutes at 37°C, and LPA treatment for 2 hours at 37°C. Cells were trypsinized, resuspended in serum-free medium then incubated with anti-active β1 integrin (HUTS21 clone, BD Trans Labs, San Jose, CA) for 1 hour. Following three washings in PBS, cells were incubated, protected from light, with anti-mouse IgG-Alexa488 for 30 minutes at ambient temperature. Excess antibody was removed by washing, cells were resuspended in PBS, then analyzed with the CyAn ADP Analyzer (Beckman Coulter, Brea, CA). All assays were performed three times. β1 Integrin Crosslinking. In order to evaluate membrane localization of β1 integrins as individual heterodimers or as clusters, a crosslinking mechanism was utilized to capture integrin distribution following LPA treatment. Analysis of β1 integrin clustering was performed as described previously (49). Cells were serumstarved overnight, trypsinized and resuspended in fresh serum-free medium in 1.5 ml Eppendorf tubes. Suspensions were incubated with 40 μM LPA for 40 minutes at room temperature. Control cells were incubated with 1% BSA in PBS (LPA vehicle). In order to crosslink surface expressed β1 integrins, anti-β1 integrin (MAB1959) was added for a 40-minute incubation on ice. Cells were washed, resuspended in serum-free medium, then incubated with polyclonal anti-mouse IgG 4 at U niersity of N tre D am e L ibaries on July 7, 2015 hp://w w w .jb.org/ D ow nladed from LPA Activates Wnt/β-catenin Signaling in Ovarian Cancer (Chemicon) at 37°C for 30 minutes. Cell suspensions were affixed to glass 22-mm coverslips by cytology centrifugation using a Cytopro 7620 Centrifuge (Wescor, Logan, UT) with LO acceleration for 2000 rpm for 10 minutes. Clustering was analyzed by immunofluorescent staining (primary antibody: anti-β1 integrin (MAB2250); secondary antibody: anti-mouse IgG – Alexa488), as described below. Fluorescent data was quantified by counting the number of cells with punctate staining patterns indicating clusters, compared with the total number of cells in a given field (Nikon Microphot FXA, 40X magnification). A minimum of 10 high powered fields were analyzed per condition and the experiment was repeated in triplicate. Western Blotting. Samples to be analyzed by western blotting were loaded onto SDSpolyacrylamide gels, electrophoresed, and then transferred onto polyvinylidene fluoride (PVDF) microporous membranes (Millipore). After blocking non-specific binding to membranes in 3% BSA in TBST for 1 hour at room temperature, membranes were incubated with primary antibodies for 3 hours at room temperature or overnight at 4°C, and then with HRP-conjugated secondary antibodies. Immunoreactivity was determined by SuperSignal West Dura Extended Duration Substrate kit (Fisher Scientific). Western blots were quantified using Multigauge v.2 (FUJIFILM, Tokyo, Japan). PVDF membranes were stripped of antibody by washing in a stripping buffer (50 mM Tris, pH 6.8, 1% SDS, 150 mM NaCl, 100 mM β-mercaptoethanol, 0.02% sodium azide) then re-probed for appropriate assay control(s). Tcf Reporter Assay. TOPflash (TCF Reporter Plasmid) and FOPflash (TCF Mutant Reporter Plasmid) were generously provided by Dr. Hans Clevers (Hubrecht Laboratory and Utrecht University, Utrecht, the Netherlands). The Renilla luciferase vector, pRL-CMV, was purchased from Promega (Madison, WI). OVCA433 cells were plated at 40-50% confluence in 6 well plates and transiently cotransfected with a Renilla luciferase reporter construct (pRL-CMV) and either the firefly luciferase TOPflash TCF Reporter Plasmid or the FOPflash TCF mutant Reporter Plasmid using Fugene 6 Transfection Reagent according to the manufacturer’s instructions (Roche Diagnostics, Mannheim, Germany). Approximately 18 hours after transfection, cells were cultured in low calcium (0.1mM CaCl2), serum-containing MEM (S-MEM (Invitrogen) for 1 hour before the addition of LPA (40 uM) for 2, 4 or 30 h. Cells were then lysed in passive lysis buffer (Promega). Both Renilla and Firefly Luciferase readings were taken on a Veritas Microplate Luminometer (Turner Biosystems, Sunnyvale, CA) using the reagents and protocol provided in the Dual Luciferase Reporter Assay System (Promega). Firefly luciferase readings were first normalized to the reading for the corresponding Renilla luciferase reading to account for transfection efficiency. The adjusted TOPflash reading was then normalized to the corresponding adjusted FOPflash reading to account for background reading of the TOPflash construct. Primers. qPCR primers probing VIM, WNT5A and LRP6 were purchased from SA Biosciences (Frederick, MD). Primers used to probe GAPDH, PTGS2 and SNAI1 are as follows: GAPDH Forward: 5’-GAGTCAACGGATTTGGTCGT-3’; GAPDH Reverse: 5’TTGATTTTGGAGGGATCTCG-3’; PTGS2 Forward: 5’-GCCCAGCACTTCACGCATCAG3’; PTGS2 Reverse: 5’-AGACCAGGCACCAGACCAAAGAC-3’; SNAI1 Forward: 5’-TTCCAGCAGCCCTACGACCAG3’; SNAI1 Reverse: 5’-CGGACTCTTGGTGCTTGTGGA-3’. Quantitative Real-Time PCR. Total RNA was extracted from treated and control cells using TRizol reagent (Life Technologies, San Diego, CA) according to manufacturer’s instructions. cDNA was synthesized from 1 μg RNA using the RT First Strand cDNA Synthesis Kit (SA Biosciences, Frederick, MD). Amplification was performed during using iCycler (Bio-Rad, Hercules, CA) for 40 cycles, with each cycle consisting of 15 seconds denaturation at 95.0°C followed by 1 minute of annealing at 60.0°C. SYBR Green Master Mix and primer sets for RAC1, VIM, LRP6, and WNT5A were purchased from SA Biosciences (Frederick, MD). GAPDH was used as an internal control in each reaction. Statistics. P-values were determined using the T-test function (two sample, unequal variance, one tailed distribution) using Excel (Microsoft Corporation, Redmond, WA). 5 at U niersity of N tre D am e L ibaries on July 7, 2015 hp://w w w .jb.org/ D ow nladed from LPA Activates Wnt/β-catenin Signaling in Ovarian Cancer RESULTS LPA Initiates Epithelial-to-Mesenchymal Transition (EMT) and Loss of Junctionally Localized β-catenin in Ovarian Carcinoma. Previous work by our laboratory and others has demonstrated a potential link between LPA and EMT via LPA-induced activation of RhoA, an initial target in the EMT transduction pathway (38), and via regulation of cell-cell adhesion in EOC (28-31,39,40). Ovarian cancers metastasize as both single cells and multi-cellular aggregates and it has been proposed that E-cadherin stabilizes aggregate formation and contributes to chemotherapy resistance (12,16,21). To assess the potential mechanistic link between LPA and EMT, multi-cellular aggregates generated from EOC cell lines were treated with LPA and evaluated for changes in mesenchymal (vimentin) and epithelial (E-cadherin) marker expression. E-cadherin cell surface expression was downregulated in multicellular aggregates (Fig. 1A-D, green) while vimentin expression increased in response to LPA treatment (Fig. 1A-D, red). Further confirmation of gain of mesenchymal phenotype was demonstrated by evaluation of F-actin stress fiber arrangement and vinculin expression. Reorganization of F-actin stress fibers (Fig. 1E-H, red) and punctate vinculin staining (Fig. 1E-H, green) were observed, indicating gain of mesenchymal phenotype in response to LPA treatment. We have previously demonstrated that LPA disrupts E-cadherin junctional integrity in ovarian cancer monolayer cultures (30). In order to validate LPA-induced adherens junction disruption in multi-cellular aggregate cultures and to evaluate whether adherens junction disruption alters the subcellular localization of β-catenin, multi-cellular aggregates of OVCA 433 cells were treated with LPA (40 μM LPA, 2 h) and evaluated for E-cadherin and β-catenin expression by immunofluorescence microscopy. Junctionally localized β-catenin is significantly decreased following LPA treatment compared with control (Fig. 2C,D,K) and corresponds temporally to loss of surface-associated E-cadherin (Fig. 2A,B). Furthermore, β-catenin perinuclear and nuclear accumulation can be observed in response to LPA (Fig. 2D). To determine whether LPA-mediated loss of β-catenin junctional localization is a LPA receptor-dependent event, cells were either treated with LPA (40 μM) alone, pre-treated with the pharmacologic LPA receptor inhibitor Ki16425 (40 μM, 30 min), or pre-treated with inhibitor and then treated with LPA. LPA treatment led to loss of β-catenin junctional localization with nuclear/perinuclear accumulation of β-catenin (Fig. 2G,K) compared with untreated control (Fig. 2E), vehicle control (Fig. 2F), and Ki16425 alone (Fig. 2H). This loss of junctional localization was rescued by pre-treatment with Ki16425 prior to LPA treatment (Fig. 2I,L). In positive controls, loss of junctional β-catenin was also observed following treatment with lithium chloride (LiCl, GSK3-β inhibitor, Fig. 2J,L). Similar results were obtained with OVCA 429 cells (data not shown). Lysophosphatidic Acid Induces β1 Integrin Activation and Clustering. We have demonstrated that ligand-induced β1 integrin clustering activates pathways leading to enhanced nuclear translocation of β-catenin and activation of β-catenin target genes in ovarian cancer (24). Furthermore, LPA – LPA receptor interaction has been previously shown to trans-activate a number of surface-expressed signaling receptors (27). In order to assess the potential for cross-talk between LPA signaling and integrin activation, OVCA433 cells were treated with either 30 μM or 70 μM LPA for 1 hour at 37°C, and then evaluated for β1 integrin activation by flow cytometry analysis using a conformation-specific antibody that detects activated β1 integrin (HUTS21). Both 30 and 70 μM LPA induced β1 integrin activation, and this activation was partially blocked by pretreatment with an LPA receptor inhibitor, Ki16425 (Fig. 3A,B). Disseminating ovarian tumor cells are exposed to LPA in ascites as anchorageindependent cells and multicellular aggregates. In order to evaluate LPA-induced β1 integrin clustering in anchorage-independent cells, OVCA433 cell suspensions were treated with 20 μM or 40 μM LPA for 1 hour at room temperature. Surface-expressed β1 integrin was crosslinked using a non-activating anti-β1 integrin antibody and cells were processed for immunostaining for β1 integrin (41). Representative high-powered fields are shown in Fig. 3C,D. β1 integrin clustering was potentiated by LPA treatment in a dose-dependent manner, with a 1.5-fold increase of the number of β1 integrin clusters (green) following 20 μM LPA treatment and a 2-fold 6 at U niersity of N tre D am e L ibaries on July 7, 2015 hp://w w w .jb.org/ D ow nladed from LPA Activates Wnt/β-catenin Signaling in Ovarian Cancer increase of clusters following 40 μM LPA (Fig. 3C,D). To further examine the effect of LPA on β1 integrins and junctional integrity, cells were treated with 30 uM LPA for various time points prior to processing for dual label immunofluorescence microscopy to examine LPAinduced changes in localization of β1 integrin and E-cadherin. Following LPA treatment, a timedependent aggregation of β1 integrin was observed in both OVCA429 and OVCA433 cells (Fig. 4A,B). Higher magnification examination of sites of β1 integrin clustering show recruitment of E-cadherin into clustered integrin complexes (Fig. 4C,D). Lysophosphatidic Acid Potentiates Nuclear Accumulation of β-catenin and Transcriptional Activation. Loss of junctional Ecadherin can target β-catenin to the nucleus or for degradation (33). Although the exact mechanism of β-catenin nuclear transport is unknown, cytoplasmic and perinuclear accumulation of βcatenin is coupled to increased nuclear activation of β-catenin target genes. In order to evaluate the effect of LPA on nuclear β-catenin accumulation, OVCA429 and OVCA433 cells were treated with LPA, nuclear proteins were isolated by subcellular fractionation as described in the Experimental Methods section, and fractions were evaluated by Western blot. In control experiments cells were treated with LiCl (positive control treatment) or left untreated. Results show that LPA treatment potentiates a ~50% increase in nuclear β-catenin localization in both OVCA429 and OVCA433 cells (Fig. 5A,B). Mechanisms for activation of Wnt signaling in human EOC tumors exhibit histotype dependence, with endometrioid EOC exhibiting mutations in β-catenin resulting in constitutively active Wnt signaling while other histotypes do not harbor a significant level of activating mutations in this pathway (42). Immunohistochemical evaluation of a human ovarian tumor tissue microarray demonstrates nuclear localization of β-catenin in each of the four EOC subtypes (Fig. 5C-F, Table 1). Levels of nuclear β-catenin in endometrioid tumor samples were consistent with literature values (84.6%). In small cohorts of mucinous and clear cell samples, 66.6% and 62.5% were positive for nuclear β-catenin expression, respectively while 50.0% of serous tumors exhibited positive nuclear staining. Eight metastatic lesions (7 serous, 1 clear cell) were also analyzed for nuclear β-catenin with 5 of the 7 serous tumors exhibiting positive staining (71.4%), in addition to positive immunoreactivity in the clear cell metastasis. These data support the hypothesis that factors other than mutational activation may regulate Wnt/β-catenin signaling in human EOC. The β-catenin protein sequence does not contain a nuclear localization signal domain, and the mechanism of nuclear transport is unclear (4345). Nevertheless, β-catenin activates transcription by displacing the transcriptional repressor Groucho in Tcf/Lef binding sitecontaining gene promoters, and subsequently binding the co-activators Tcf/Lef (and potentially other co-factors such as CBP) to initiate transcription (46-48). To assess the potential for LPA regulation of protein:protein interactions between β-catenin and Tcf, nuclear proteins were isolated as described and subjected to immunoprecipitation using anti-β-catenin antibodies. Immunoprecipitates were then probed for βcatenin or Tcf. Results show that Tcf/β-catenin interaction was enhanced in cells treated with LPA relative to untreated or DMSO treated controls (Fig. 6A and inset). Pre-treatment with Ki16425 reduced this protein:protein interaction to baseline levels, suggesting that LPA-induced Tcf/β-catenin interaction is LPA receptor-dependent. In order to measure activation of the β-catenin/Tcf/Lef transcriptional complex, cells were transfected with a Renilla luciferase reporter construct and either a TOP (Tcf) reporter construct or a FOP (control) reporter construct then treated with LPA for 2, 8 or 24 hours. β-catenin/Tcf/Lef transcriptional activity is increased after 2-hour LPA treatment, and sustained at 8 and 30 hours in (Fig. 6B,C). Consequentially, LPA-induced βcatenin/Tcf/Lef transcriptional activity led to upregulation of five known β-catenin/Tcf/Lef target genes: VIM (Vimentin), WNT5A, LRP6, PTGS2 (Cox-2) and SNAI1 (Snail) (Fig. 7A). To evaluate a potential functional link between LPAinduced β1 integrin clustering and transcriptional activation of Wnt/β-catenin targets, cells were treated with LPA and activation of focal adhesion kinase (FAK) was evaluated by western blotting for phospho-Tyr397 (Fig. 7B, inset). LPA 7 at U niersity of N tre D am e L ibaries on July 7, 2015 hp://w w w .jb.org/ D ow nladed from LPA Activates Wnt/β-catenin Signaling in Ovarian Cancer treatment enhanced FAK phosphorylation (Fig. 7Bb, inset) and this effect was abrogated by the addition of antibodies that prevent β1 integrin clustering as well as by the LPA receptor inhibitor Ki16427 (Fig. 7Bc,d, inset). As recent studies have demonstrated that LPA can modulate integrin function through Rho/ROCK activation, we also examined the effect of the ROCK inhibitor Y27632 (Fig. 7Be, inset), also resulting in inhibition of FAK phosphorylation. To address the resulting effects on transcription, we examined expression of vimentin, as this β-catenin target gene was most significantly upregulated following LPA treatment (Fig. 7A). LPA-induced vimentin expression was blocked by inhibition of β1 integrin clustering (Fig. 7C), supporting the hypothesis that LPA-integrin crosstalk and subsequent integrin clustering regulate Wnt/βcatenin signaling and gene expression. Similar results were obtained following inhibition of LPA receptor or Rho/ROCK activity (Fig. 7C). DISCUSSION Lysophosphatidic acid (LPA) regulates a multitude of ovarian tumor cell responses including proliferation, migration and invasion (19,24,28). LPA is expressed as high as 80 μM in the ascites fluid and serum of patients with ovarian cancer (24,25,49-51), underlying the importance of understanding its pathophysiological role in ovarian cancer. Previous studies in cancer cells have shown that LPA induces β-catenin nuclear translocation. For example, in colon cancer cells, LPA activates β-catenin via modulation of glycogen synthase kinase 3β, resulting in enhanced transcription of a β-catenin/TCF reporter gene (52,53). Similar results were obtained in A431 epidermoid carcinoma cells (33,54), resulting in loss of junctional E-cadherin and gain of vimentin expression. Data in the current study define a role for LPA in activation of β-cateninregulated transcription and induction of an EMT program in ovarian cancer cells and multi-cellular aggregates and provide novel mechanistic insight on the role of LPA-induced clustering of β1 integrins in modulation of this signaling pathway. We have recently shown that LPA modulates loss of epithelial cohesion as a functional result of disrupting cell-cell junctions, through protease-dependent cleavage and altered cadherin trafficking in ovarian carcinoma (30). The fate of β-catenin in response to LPA-mediated adherens junction dissolution, however, had not been previously reported. Data presented here are consistent with our previous work and demonstrate loss of cell surface-associated β-catenin following LPA treatment. Interestingly, immunofluorescence microscopy results demonstrate that perinuclear accumulation of β-catenin is observed following LPA treatment, corresponding with data that first identified an intersection between the cell:cell adhesion-related and transcription-related cytoplasmic pools of β-catenin (33). The theory that cytoplasmic β-catenin pools intersect nuclear β-catenin pools is further supported by data demonstrating increased nuclear localization, Tcf/Lef reporter activity and β-catenin target gene transcription in response to LPA treatment. While the precise mechanism by which LPA treatment modulates β-catenin translocation is unknown, the current data support the hypothesis that LPA-integrin crosstalk results in β1 integrin activation. Many studies have suggested convergent signaling between growth factors and integrin-mediated adhesion processes [reviewed in (55-57)]. Several G-protein coupled agonists, including lysophosphatidic acid (LPA), have been shown to induce phosphorylation of the integrin effector p125FAK (58). Additionally, LPA-induced migration in fibroblasts is dependent on β1 integrin expression (59) and LPA-LPAR2 interaction activates TGF-β signaling in an integrin-mediated manner (60). Interestingly, LPA treatment also leads to increased ovarian cancer cell adhesion to collagen, as well as enhanced β1 integrin protein expression (29). We have previously reported that β1 integrin aggregation alters β-catenin dynamics via disruption of adherens junctions and inhibition of glycogen synthase kinase 3β activity, leading to enhanced transcriptionally active β-catenin (23). This is consistent with data in the current report showing co-localization of E-cadherin at sites of clustered β1 integrins. Furthermore, 3-dimensional collagen culture induces β1 integrin aggregation and downregulates the expression of dickkopf-1, an inhibitor of canonical Wnt signaling (61,62). These data support a mechanism whereby LPAinduced β1 integrin aggregation activates β catenin signaling. 8 at U niersity of N tre D am e L ibaries on July 7, 2015 hp://w w w .jb.org/ D ow nladed from LPA Activates Wnt/β-catenin Signaling in Ovarian Cancer Further support for this mechanism is provided by the observation that five prometastatic β-catenin target genes are upregulated following LPA treatment: VIM (vimentin), WNT5A, LRP6, PTGS2 (cox-2), and SNAI1 (snail1). Expression of these genes was also upregulated following multivalent β1 integrin engagement (23) and inhibition of LPA-induced β1 integrin aggregation blocked vimentin transcription. Cox-2 contributes to tumorigenesis by inhibiting apoptosis, increasing growth factor expression to promote angiogenesis, and by enhancing matrix metalloproteinase (MMP) expression to stimulate invasion (63). Cox-2 protein is expressed in ovarian carcinoma, and functions as a downstream effector of LPAmediated ovarian tumor cell migration and invasion (64). Genes commonly associated with EMT including vimentin, snail, LRP6, and Wnt5a were also induced by LPA treatment. Ovarian cancers typically display both epithelial and mesenchymal characteristics and the mesenchymal marker vimentin is widely expressed in human tumor specimens (16). Recent molecular profiling studies support these findings, showing upregulation of EMT and EMT-associated invasion programs in primary ovarian tumors and their matched metastases (65,66). Snail is a key inducer of EMT and functions as a negative regulator of E-cadherin transcription. Several studies have demonstrated that nuclear localization of Snail correlates with tumor progression, with enhanced Snail immunoreactivity in metastatic lesions (67,68). Snail and other mesenchymal genes were found to be upregulated a panel of ovarian cancer cells competent for mesothelial clearance, a surrogate assay that models initial events in intra-peritoneal metastasis (21). Furthermore, patients with both primary and metastatic tumors positive for Snail expression showed a significant decrease in overall survival (69). LRP6 functions as a Wnt co-receptor that recruits Axin and Dishevelled to the plasma membrane, thereby disrupting the degradation of β-catenin and facilitating β-catenin nuclear translocation (70). LRP6 is expressed by ovarian carcinoma cell lines (current data and unpublished observations) and tissues (71). Expression of Wnt5a,a ligand for the Frizzled receptor, is high in ovarian cancer tumors and ascites fluid and is linked to poor overall survival (33,69,70). Emerging data suggest that activation of canonical vs non-canonical Wnt signaling by Wnt5a is dependent on the receptor/co-receptor context and further studies in ovarian cancer cells are warranted (72-75). In summary, the current data demonstrate that lysophosphatidic acid, expressed in high concentration in ascites fluid of women with ovarian cancer, potentiates β-catenin-dependent Wnt signaling via a mechanism that involves activation and aggregation of β1 integrin. Currently, a number of therapeutic agents that target Wnt signaling, both loss of function and gain of function alterations, are being investigated in the treatment of cancer, bone disease, cardiac and vascular disease and arthritis (76,77). Therapeutics under development are designed to target each level of Wnt signaling (extracellular, cytoplasmic, nuclear and pathway crosstalk), and include small molecule and biotherapeutic agents (78-80). Although not yet extensively evaluated in ovarian cancer, recent pre-clinical studies support a role for further evaluation of Wnt/β-catenin signaling as a potential therapeutic target (71,81). Understanding the mechanisms by which microenvironmental factors such as lysophosphatidic acid may contribute to ovarian cancer progression and metastasis via modulation of this pathway may thereby contribute to identification of alternative treatment options for women with ovarian cancer. ACKNOWLEDGEMENTS: This work was supported by Research Grants RO1 CA109545 (M. S. S.) and RO1 CA086984 (M. S. S.) from the National Institutes of Health/National Cancer Institute, a Research Supplement to Promote Diversity CA086984-11S1 (R. J. B.) and the Leo and Ann Albert Charitable Trust (M.S.S). CONFLICT OF INTEREST: The authors declare that they have no conflicts of interest with the contents of this article. AUTHOR CONTRIBUTIONS: RJB and MSS designed the study and wrote the paper. SDW performed the experiments shown in Figure 1. YL performed and analyzed the experiments shown in Figure 4. RJB performed and analyzed the experiments shown in Figures 9 at U niersity of N tre D am e L ibaries on July 7, 2015 hp://w w w .jb.org/ D ow nladed from LPA Activates Wnt/β-catenin Signaling in Ovarian Cancer 2,3,5-7. All authors reviewed the results and approved the final version of the manuscript.