The tumour suppressor OPCML promotes AXL inactivation by the phosphatase PTPRG in ovarian cancer
2018; Springer Nature; Volume: 19; Issue: 8 Linguagem: Inglês
10.15252/embr.201745670
ISSN1469-3178
AutoresJane Antony, Elisa Zanini, Zoe Kelly, Tuan Zea Tan, Evdoxia Karali, Mohammad N. Alomary, Youngrock Jung, Katherine Nixon, Paula Cunnea, Christina Fotopoulou, Andrew D. Paterson, Sushmita Roy‐Nawathe, Gordon B. Mills, Ruby Yun‐Ju Huang, Jean Paul Thiery, Hani Gabra, Chiara Recchi,
Tópico(s)Neuroendocrine Tumor Research Advances
ResumoArticle15 June 2018free access Transparent process The tumour suppressor OPCML promotes AXL inactivation by the phosphatase PTPRG in ovarian cancer Jane Antony orcid.org/0000-0002-2424-5470 Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, Singapore Search for more papers by this author Elisa Zanini orcid.org/0000-0002-5659-1626 Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Zoe Kelly Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Tuan Zea Tan orcid.org/0000-0001-6624-1593 Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore Search for more papers by this author Evdoxia Karali Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Mohammad Alomary Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Youngrock Jung Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Katherine Nixon Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Paula Cunnea Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Christina Fotopoulou Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Andrew Paterson Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Sushmita Roy-Nawathe Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Gordon B Mills Division of Basic Science Research, Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Ruby Yun-Ju Huang Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore Department of Obstetrics and Gynecology, National University Health System, Singapore, Singapore Search for more papers by this author Jean Paul Thiery orcid.org/0000-0003-0478-5020 Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore, Singapore Department of Biochemistry, National University of Singapore, Singapore, Singapore Search for more papers by this author Hani Gabra Corresponding Author [email protected] orcid.org/0000-0002-3322-4399 Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Early Clinical Development, IMED Biotech Unit, AstraZeneca, Cambridge, UK Search for more papers by this author Chiara Recchi Corresponding Author [email protected] orcid.org/0000-0003-1605-0945 Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Jane Antony orcid.org/0000-0002-2424-5470 Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, Singapore Search for more papers by this author Elisa Zanini orcid.org/0000-0002-5659-1626 Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Zoe Kelly Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Tuan Zea Tan orcid.org/0000-0001-6624-1593 Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore Search for more papers by this author Evdoxia Karali Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Mohammad Alomary Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Youngrock Jung Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Katherine Nixon Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Paula Cunnea Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Christina Fotopoulou Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Andrew Paterson Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Sushmita Roy-Nawathe Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Gordon B Mills Division of Basic Science Research, Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Ruby Yun-Ju Huang Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore Department of Obstetrics and Gynecology, National University Health System, Singapore, Singapore Search for more papers by this author Jean Paul Thiery orcid.org/0000-0003-0478-5020 Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore, Singapore Department of Biochemistry, National University of Singapore, Singapore, Singapore Search for more papers by this author Hani Gabra Corresponding Author [email protected] orcid.org/0000-0002-3322-4399 Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Early Clinical Development, IMED Biotech Unit, AstraZeneca, Cambridge, UK Search for more papers by this author Chiara Recchi Corresponding Author [email protected] orcid.org/0000-0003-1605-0945 Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK Search for more papers by this author Author Information Jane Antony1,2,3,†, Elisa Zanini1, Zoe Kelly1, Tuan Zea Tan2, Evdoxia Karali1, Mohammad Alomary1, Youngrock Jung1, Katherine Nixon1, Paula Cunnea1, Christina Fotopoulou1, Andrew Paterson1, Sushmita Roy-Nawathe1, Gordon B Mills4, Ruby Yun-Ju Huang2,5, Jean Paul Thiery2,6,7, Hani Gabra *,1,8 and Chiara Recchi *,1 1Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, London, UK 2Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore 3NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, Singapore 4Division of Basic Science Research, Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 5Department of Obstetrics and Gynecology, National University Health System, Singapore, Singapore 6Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore, Singapore 7Department of Biochemistry, National University of Singapore, Singapore, Singapore 8Early Clinical Development, IMED Biotech Unit, AstraZeneca, Cambridge, UK †Present address: Institute for Stem Cell Biology and Regenerative Medicine, Stanford, CA, USA *Corresponding author. Tel: +44 20 7594 2792; E-mail: [email protected] *Corresponding author. Tel: +44 20 7594 1549; E-mail: [email protected] EMBO Rep (2018)19:e45670https://doi.org/10.15252/embr.201745670 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract In ovarian cancer, the prometastatic RTK AXL promotes motility, invasion and poor prognosis. Here, we show that reduced survival caused by AXL overexpression can be mitigated by the expression of the GPI-anchored tumour suppressor OPCML. Further, we demonstrate that AXL directly interacts with OPCML, preferentially so when AXL is activated by its ligand Gas6. As a consequence, AXL accumulates in cholesterol-rich lipid domains, where OPCML resides. Here, phospho-AXL is brought in proximity to the lipid domain-restricted phosphatase PTPRG, which de-phosphorylates the RTK/ligand complex. This prevents AXL-mediated transactivation of other RTKs (cMET and EGFR), thereby inhibiting sustained phospho-ERK signalling, induction of the EMT transcription factor Slug, cell migration and invasion. From a translational perspective, we show that OPCML enhances the effect of the phase II AXL inhibitor R428 in vitro and in vivo. We therefore identify a novel mechanism by which two spatially restricted tumour suppressors, OPCML and PTPRG, coordinate to repress AXL-dependent oncogenic signalling. Synopsis The GPI-anchored tumour suppressor OPCML interacts with the oncogenic RTK AXL inducing its redistribution into cholesterol-rich membrane domains. There, AXL is de-phosphorylated by the resident phosphatase PTPRG, inhibiting AXL-dependent signalling, motility and invasion. Binding of Gas6 to the oncogenic RTK AXL promotes its interaction with the tumour suppressor OPCML. The Gas6/AXL/OPCML complex accumulates in cholesterol-rich lipid domains, where the phosphatase PTPRG de-phosphorylates AXL. The coordinated action of OPCML and PTPRG blocks AXL signalling and inhibits Gas6-stimulated motility and invasion in ovarian cancer cells. Introduction Epithelial ovarian cancer (EOC) is one of the leading causes of cancer-related deaths in women across the world and the most lethal gynaecological malignancy 1. Loco-regional dissemination of the tumour is deadly, with only 10–15% of patients surviving beyond 10 years 2. Unfortunately, due to its relatively asymptomatic nature and early propensity to dissemination, EOC is typically diagnosed at late stage. Patients' tumours ultimately become resistant to existing treatments, and thus, there is an unmet need for alternative treatment strategies. We have recently identified the receptor tyrosine kinase (RTK) AXL as a pivotal node of oncogenic RTK cross-talk in ovarian cancer 3, 4. AXL is an important driver of epithelial-to-mesenchymal transition (EMT) and tumour progression, and has been implicated in promoting cell adhesion, survival, proliferation, motility and invasion 5. AXL is a member of the TAM subfamily of RTKs 6 with its only known ligand being growth arrest-specific-6 (Gas6). Binding of Gas6 to AXL results in homodimerisation of AXL in a 2:2 stoichiometry with Gas6 and subsequent activation of the AXL kinase domain 7. In ovarian cancer, AXL overexpression confers worse prognosis 3 and it is primarily expressed during advanced-stage disease and at higher levels in peritoneal deposits and metastases compared to the primary tumour 8. Silencing AXL in ovarian cancer cells abrogates peritoneal dissemination of the tumour, and additionally, inhibiting the Gas6/AXL pathway hinders further progression of established metastatic disease in vivo 9. We have recently shown that the adversely prognostic mesenchymal subtype in ovarian cancer 10 presents sustained AXL signalling, which induces motility 3, 11. However, the mechanisms regulating AXL signalling remain unknown 12. Given AXL is known to extensively transactivate other RTKs 3, 13, identifying tumour suppressors that can repress these networks of oncogenic interactions would provide a promising platform for developing novel therapeutics. Opioid-binding protein/cell adhesion molecule-like (OPCML) is a glycosylphosphatidylinositol (GPI)-anchored tumour suppressor that is silenced in over 83% of ovarian cancer patients by loss of heterozygosity (LOH) and epigenetic mechanisms 14 and correlates with poor patient progression-free survival in ovarian and breast cancers 15. Hypermethylation of OPCML is also common in many other cancers such as lung, brain, breast, cervical, and gastrointestinal cancers and lymphomas, suggesting a conserved tumour suppressor function across various tissues and functional significance in their derived cancers 16. We previously demonstrated that OPCML inhibits proliferation in vitro and abrogates tumorigenicity in vivo 14 by negatively regulating a repertoire of RTKs, such as EPHA2, FGFR1, FGFR3, HER2 and HER4 15. Hence, we sought to understand whether re-establishing this tumour suppressor would also repress other oncogenic drivers such as AXL in ovarian cancer. Results AXL overexpression-mediated impaired survival is mitigated by OPCML expression First, we analysed the impact of AXL expression within the ICGC dataset of 154 patients 17. AXL overexpression (above median) conferred a significantly worse overall survival in this dataset (HR = 1.997, P = 0.0170; Fig EV1A). In order to delineate the advantage of OPCML expression, we analysed the OPCML promoter methylation status in these patients. In the subset where there was gene promoter methylation of OPCML, AXL overexpression again demonstrated a significantly worse overall survival (HR = 1.929, P = 0.0411; Fig EV1B). However, in the cohort without OPCML promoter methylation, there was no such difference (P = 0.1505, non-significant; Fig EV1C). Furthermore, when considering the expression levels of AXL and OPCML using the CSIOVDB dataset for 1,868 EOC patients 18, a similar pattern was observed. AXL overexpression conferred significantly worse overall survival (HR = 1.335, P = 0.0013; Fig EV1D), and this was accentuated for patients with low expression of OPCML (HR = 1.431, P = 0.0015; Fig EV1E). However, in patients with high levels of OPCML, there was no significant difference between overall survival for AXL-low and AXL-high expression states (HR = 1.322, P = 0.0651; Fig EV1F). Click here to expand this figure. Figure EV1. OPCML improves the survival of AXL-overexpressing patients in silico A–C. Kaplan–Meier curves showing overall patient survival of AXL-high (above median, red) and AXL-low (below median, blue) patient groups from ICGC dataset: (A) all patients, (B) OPCML-methylated state, (C) OPCML-unmethylated state. OPCML promoter methylation was defined using the cg15885337 and cg18710784 probe sets. D–F. Kaplan–Meier curves showing overall patient survival of AXL-high (above median, red) and AXL-low (below median, blue) patient groups from CSIOVDB dataset: (D) all patients, (E) OPCML-low expression state (below median), (F) OPCML-high expression state (above median). G–I. Kaplan–Meier curves showing progression-free patient survival of AXL-high (above median, red) and AXL-low (below median, blue) patient groups from TCGA dataset: (G) all patients, (H) OPCML-low expression state (below median), (I) OPCML-high expression state (above median). Data information: HR, hazard ratio. Y-axis = month. Download figure Download PowerPoint In terms of progression-free survival from the TCGA, AXL overexpression (highest quartile) tended to confer worse prognosis (HR = 1.284, P = 0.1030, not significant; Fig EV1G), and this was significantly accentuated in patients with low OPCML expression (HR = 1.56, P = 0.0419; Fig EV1H). In patients with high levels of OPCML, the negative impact of AXL overexpression was reduced (HR = 1.12, P = 0.5960; Fig EV1I). These findings suggest that the AXL overexpression-associated worsened prognosis could be mitigated by OPCML expression, underscoring the clinical and prognostic importance of OPCML. This suggested that the tumour suppressor OPCML could modulate AXL signalling and so we explored this hypothesis. OPCML interacts with AXL To assess whether OPCML could interact with and subsequently abrogate the oncogenic properties of AXL, we transduced AXL-expressing, OPCML-null (through somatic methylation) SKOV3 and PEA1 ovarian cancer cell lines with OPCML or the control (Empty) lentivirus to generate SKOV3-OPCML, SKOV3-Empty, PEA1-OPCML and PEA1-Empty cell lines, which were used in subsequent experiments (Fig 1A). In a mammalian 2-hybrid assay, a positive signal was detected in SKOV3-OPCML cells that express both AXL and OPCML (Fig 1B), suggesting for the first time an interaction between OPCML and AXL. A GST pull-down assay was also carried out using SKOV3-Empty cells, and AXL was detected in the eluate together with GST-OPCML (Fig 1C). The interaction between endogenous AXL and OPCML was also observed by co-immunoprecipitation in SKOV3-OPCML cells, where OPCML co-precipitated with AXL (Fig 1D). Figure 1. OPCML interacts with AXL A. Western blotting of AXL and OPCML protein levels in AXL-expressing, OPCML-null SKOV3 and PEA1 cell lines transduced with OPCML "O" or control Empty vector "E". B. Mammalian 2-hybrid assay between OPCML and AXL. C. Western blotting of the OPCML-GST pull down. Input: 1/20 of SKOV3-Empty whole-cell lysate. D. Western blotting of the anti-AXL immunoprecipitation. Input: 1/100 of SKOV3-OPCML whole-cell lysate. IP, immunoprecipitated protein; IB, immunoblotted protein. E. Immunostaining of OPCML (red) and AXL (green) in SKOV3-OPCML and PEA1-OPCML cells. Scale bar = 10 μm. F. Pearson's correlation R for AXL-OPCML co-localisation in (E). G. FRET efficiency for AXL-OPCML interaction in SKOV3-Empty "E" and SKOV3-OPCML "O", using AXL-AXL FRET as a positive control. Cells were labelled with anti-Axl rabbit (conjugated to donor probe) and anti-OPCML mouse (conjugated to acceptor probe), or anti-Axl mouse (conjugated to acceptor probe) and anti-Axl rabbit (conjugated to donor probe). H. PLA of AXL-OPCML (red) in SKOV3 and PEA1 cells transduced with empty vector "E" or OPCML "O". Scale bar = 50 μm. I, J. Primary ovarian tumour cells were characterised for AXL and OPCML levels by (I) Western blotting and (J) immunostaining. PAX8 was used to identify ovarian cancer cells. K. Quantitation of AXL-OPCML PLA in primary cells. PLA = PLA secondary antibodies only. FM = primary antibodies anti-AXL and anti-OPCML plus PLA secondary antibodies. This assay was performed in full medium. L. Western blotting of membrane fractionation of SKOV3-Empty cells: total cell lysate "T", liquid-disordered soluble fraction "S", detergent-resistant membrane fraction "R". Caveolin-1 is used as a marker of the R fraction, and Calnexin is used as a marker for the S fraction, E, SKOV3-Empty; O, SKOV3-OPCML. M. Densitometry of AXL band intensities in (J) in S and R, normalised to AXL intensity in input. Data information: Data are representative of at least three experiments with graphs depicting means ± SEM; (E) was performed once due to the limited numbers of primary cells; *P < 0.05, ***P < 0.01 and ****P < 0.0001 by Student's t-tests. Download figure Download PowerPoint The localisation of the proteins at the plasma membrane was analysed by immunofluorescence microscopy, and this revealed co-localisation between AXL and OPCML in both SKOV3-OPCML and PEA1-OPCML cells (Pearson's correlation R = 0.804 and 0.754, respectively, Fig 1E and F). This association was confirmed using Förster resonance energy transfer (FRET; Fig 1G), as well as in situ proximity ligation assay (PLA) Duolink (Fig 1H), both of which demonstrated a clear proximity between AXL and OPCML. The AXL-OPCML interaction was further confirmed in primary ovarian tumour cells expressing both AXL and OPCML (Fig 1I and J) using PLA (Fig 1K). Since GPI-anchored proteins like OPCML are located in cholesterol-enriched lipid domains, which can be isolated as "liquid-ordered" detergent-resistant membrane (DRM) fractions, we also investigated the effect of the OPCML-AXL interaction on the membrane distribution of AXL. Upon fractionating the plasma membrane into DRM and the "liquid-disordered" detergent-soluble membrane (DSM) fraction, we observed that AXL was present in both compartments of the membrane in control cells lacking OPCML (Fig 1L). However, in cells expressing OPCML there was a shift in AXL localisation from the DSM to the DRM compartment where OPCML resides (Fig 1L and M), demonstrating that OPCML relocates AXL into cholesterol-enriched domains. To our knowledge, this is the first time that the interaction between OPCML and AXL and the subsequent AXL redistribution have been evidenced. Gas6 stimulation promotes OPCML-AXL interaction, thereby altering AXL localisation and activation To further understand the nature of the OPCML-AXL association, we decided to investigate how the binding of AXL to its ligand Gas6 would affect the functional interaction between AXL and OPCML. Given that the previous experiments were carried out in complete medium, which contains many growth factors, we used serum-free conditions to exclusively evaluate the effects of Gas6 addition. In serum-free non-stimulated conditions, we demonstrated that there was minimal co-localisation between AXL and OPCML in SKOV3-OPCML as observed by immunofluorescence staining (Pearson's correlation R = 0.22, Fig 2A and B). However, addition of Gas6 strongly induced co-localisation within 30 min, and at 3 and 12 h, suggesting a sustained interaction between AXL and OPCML in the presence of the ligand (Pearson's correlation R = 0.68, 0.73, 0.83, respectively, Fig 2A and B). Interestingly, when we quantified this interaction using PLA, we found negligible association between the two proteins in non-stimulating conditions, but very strong and temporally progressive interaction upon addition of Gas6 (Fig 2C and D). This Gas6-induced AXL-OPCML interaction was also observed in primary ovarian tumour cells (Fig 2E), though the effect of serum starvation was limited probably due to the secretion of autocrine growth factors. Furthermore, we could observe an increase in the amount of OPCML immunoprecipitated with AXL upon Gas6 treatment, suggesting again that the addition of Gas6 increases the affinity between AXL and OPCML (Fig 2F). We additionally determined that this interaction also occurred between OPCML and the phosphorylated form of AXL (pAXL), as demonstrated by co-immunoprecipitation of OPCML by anti-pAXL (Fig 2G). A GST pull-down assay was also carried out, where pAXL was detected in the eluate together with GST-OPCML, again enhanced by Gas6 (Fig 2H). Thus, OPCML also binds to the active form of AXL upon Gas6 stimulation. Figure 2. Gas6 stimulation promotes OPCML-AXL interaction, thereby altering AXL localisation and activation A–D. SKOV3-OPCML cells stimulated with Gas6 over a 12-h time course and (A) stained with DAPI (cyan), anti-OPCML (green) and anti-AXL (red) antibodies (scale bar = 10 μm), and (B) Pearson's correlation R was calculated; (C, D) OPCML-AXL interaction (red) was visualised by PLA (scale bar = 50 μm) and quantified, n = 3. E. Quantitation OPCML-AXL PLA interaction in primary ovarian tumour cells, n = 1. The six data points represent the quantifications from six separate images (from the same experiment). F. Co-immunoprecipitation with anti-AXL antibody in cells stimulated with Gas6 as indicated. Input: 1/50 of whole-cell lysate from SKOV3-OPCML cells. IP, immunoprecipitated protein; IB, immunoblotted protein. G. Co-immunoprecipitation with anti-pAXL antibody in SKOV3-OPCML cells stimulated with Gas6. H. OPCML-GST pull-down assay in SKOV3-Empty cells stimulated with Gas6. I. Membrane fractionation in SKOV3-Empty "E" and SKOV3-OPCML "O" cells into the detergent-resistant "R" membrane fraction and the detergent-soluble "S" fraction upon Gas6 stimulation. J. Ratio of AXL band intensity in "R" to total AXL "T" (T = S + R) from panel (I). K–M. (K) FRAP recovery curves. Black boxes represent median value, coloured boxes represent 1st–3rd quartile, and whiskers represent minimum to maximum values, (L) diffusion co-efficient and (M) percentage of AXL localised in insoluble lipid domain or soluble plasma membrane fraction in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with Gas6. N. FRET efficiency in SKOV3-OPCML "O" cells stimulated with Gas6, and labelled with anti-Axl (donor probe) and anti-OPCML (acceptor probe). O. Western blotting of the "S" and "R" membrane fractions in SKOV3-Empty "E" and OPCML "O" cells stimulated with Gas6 for 3 h, for Gas6, AXL and pAXL. Data information: Data are representative of at least three experiments with graphs depicting means ± SEM, (E) was performed once due to the limited numbers of primary cells; *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 by Student's t-tests. Download figure Download PowerPoint When we analysed the effect of Gas6 addition on AXL distribution by membrane fractionation in SKOV3-Empty, the total AXL protein was distributed between the DSM and DRM and this distribution remained constant over time (Fig 2I). Surprisingly, the pool of activated pAXL was detected almost exclusively in the DSM (Fig 2I). Interestingly, in SKOV3-OPCML the increased interaction between OPCML and AXL caused by the addition of Gas6 induced a progressive shift of AXL from the DSM into the DRM, where the GPI-anchored protein resides (Fig 2I and J). As a consequence, since in the DRM AXL is mostly not phosphorylated, the total levels of phosphorylated and activated AXL decreased in the presence of OPCML (Fig 2I). As it is known that the association with lipid domain affects protein diffusion, we performed fluorescence recovery after photobleaching (FRAP) experiments on SKOV3-Empty and OPCML cells stimulated with Gas6 (Fig 2K). AXL recovery was less in SKOV3-OPCML cells, particularly upon Gas6 stimulation, compared to SKOV3-Empty cells (Fig 2K). The diffusion co-efficient dropped from 0.51 to 0.36 in SKOV3-OPCML cells upon Gas6 stimulation, while the reduction in SKOV3-Empty decreased from 0.66 to 0.57 (Fig 2L); this could be due to the increased association of AXL with the immobile lipid fraction upon Gas6 stimulation in SKOV3-OPCML (Fig 2M). To solidify our findings, FRET experiments were also performed to quantitate the level of AXL-OPCML interaction upon addition of Gas6 and a clear increase was observed at 30 min and 3 h (Fig 2N). Given that OPCML continued to retain dephosphorylated AXL in the DRM, we analysed Gas6-stimulated cells for the presence of the ligand in the membrane fractions (Fig 2O). In the cells lacking OPCML, there were elevated AXL, pAXL and Gas6 in the DSM (Fig 2O). However in the presence of OPCML, Gas6 is sequestered in the DRM along with AXL, which is dephosphorylated, implying that, even though the RTK is still bound to its ligand, it has been deactivated (Fig 2O). This suggests that Gas6 serves to trigger as well as retain an enhanced OPCML-AXL interaction on the extracellular side of the plasma membrane, regardless of the intracellular phosphorylation status of the AXL kinase domain. OPCML inhibits sustained Gas6/AXL signalling Observing the changes in membrane distribution and activation status of AXL upon binding to OPCML, we sought to understand how OPCML would therefore affect the Gas6/AXL signalling cascade. In order to ascertain that Gas6-induced effects were exclusively through the AXL signalling node, AXL-depleted SKOV3 cells were stimulated with Gas6. Upon AXL depletion, Gas6 addition did not induce phosphorylation of AXL, or of AXL downstream effector molecules such as ERK (Fig EV2A), or motility (Fig EV2B and C), confirming that Gas6 activates the ERK pathway and motility exclusively through AXL in these tumour cells. Click here to expand this figure. Figure EV2. OPCML expression negates Gas6-mediated ERK signalling and motility through AXL axis A. Western blotting of SKOV3 transfected with non-targeting siRNA or three independent siRNA for AXL and stimulated with Gas6 over time. B, C. Displacement and speed of SKOV3 transfected with non-targeting siRNA or three independent siRNA for AXL and stimulated with Gas6 over time. D, E. Immunostaining of SKOV3-Empty "E" and SKOV3-OPCML "O" cells in response to Gas6 stimulation. Scale bar = 50 μm. Data information: Data in are representative of at least three experiments with graphs depicting means ± SEM: *P < 0.05 by Student's t-tests. Download figure Download PowerPoint SKOV3-Empty and PEA1-Empty cell lines were stimulated with Gas6 over a 24-h time course. Gas6 induced the phosphorylation of AXL leading to sustained activation of ERK for up to 24 h (Fig 3A and B) and the induction of the EMT transcription factor Slug, a key factor in cell motility and invasiveness 19, 20 (F
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