Adaptive RSK‐EphA2‐GPRC5A signaling switch triggers chemotherapy resistance in ovarian cancer
2020; Springer Nature; Volume: 12; Issue: 4 Linguagem: Inglês
10.15252/emmm.201911177
ISSN1757-4684
AutoresLidia Moyano‐Galceran, Elina Pietilä, S. Pauliina Turunen, Sara Corvigno, Elisabet Hjerpe, Daria Bulanova, Ulrika Joneborg, Twana Alkasalias, Yuichiro Miki, Masakazu Yashiro, Anastasiya Chernenko, Joonas Jukonen, Madhurendra Singh, Hanna Dahlstrand, Joseph W. Carlson, Kaisa Lehti,
Tópico(s)PARP inhibition in cancer therapy
ResumoArticle2 March 2020Open Access Adaptive RSK-EphA2-GPRC5A signaling switch triggers chemotherapy resistance in ovarian cancer Lidia Moyano-Galceran Lidia Moyano-Galceran orcid.org/0000-0001-9219-6394 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Elina A Pietilä Elina A Pietilä orcid.org/0000-0003-0209-9369 Research Programs Unit, Individualized Drug Therapy, University of Helsinki and Helsinki University Hospital, Helsinki, Finland Search for more papers by this author S Pauliina Turunen S Pauliina Turunen Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Sara Corvigno Sara Corvigno Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Elisabet Hjerpe Elisabet Hjerpe Department of Obstetrics and Gynecology, Visby Hospital, Visby, Sweden Search for more papers by this author Daria Bulanova Daria Bulanova Institute for Molecular Medicine Finland, FIMM, University of Helsinki, Helsinki, Finland Search for more papers by this author Ulrika Joneborg Ulrika Joneborg Division of Pelvic Cancer, Department of Women's and Children's Health, Karolinska Institutet and University Hospital, Stockholm, Sweden Search for more papers by this author Twana Alkasalias Twana Alkasalias Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Research Centre, Salahaddin University-Erbil, Erbil, Iraq Search for more papers by this author Yuichiro Miki Yuichiro Miki Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Department of Gastroenterological Surgery, Osaka City University Graduate School of Medicine, Osaka, Japan Search for more papers by this author Masakazu Yashiro Masakazu Yashiro Department of Gastroenterological Surgery, Osaka City University Graduate School of Medicine, Osaka, Japan Search for more papers by this author Anastasiya Chernenko Anastasiya Chernenko Research Programs Unit, Individualized Drug Therapy, University of Helsinki and Helsinki University Hospital, Helsinki, Finland Search for more papers by this author Joonas Jukonen Joonas Jukonen Research Programs Unit, Individualized Drug Therapy, University of Helsinki and Helsinki University Hospital, Helsinki, Finland Search for more papers by this author Madhurendra Singh Madhurendra Singh Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Hanna Dahlstrand Hanna Dahlstrand Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Joseph W Carlson Joseph W Carlson Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Kaisa Lehti Corresponding Author Kaisa Lehti [email protected] orcid.org/0000-0001-9110-8719 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Research Programs Unit, Individualized Drug Therapy, University of Helsinki and Helsinki University Hospital, Helsinki, Finland Search for more papers by this author Lidia Moyano-Galceran Lidia Moyano-Galceran orcid.org/0000-0001-9219-6394 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Elina A Pietilä Elina A Pietilä orcid.org/0000-0003-0209-9369 Research Programs Unit, Individualized Drug Therapy, University of Helsinki and Helsinki University Hospital, Helsinki, Finland Search for more papers by this author S Pauliina Turunen S Pauliina Turunen Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Sara Corvigno Sara Corvigno Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Elisabet Hjerpe Elisabet Hjerpe Department of Obstetrics and Gynecology, Visby Hospital, Visby, Sweden Search for more papers by this author Daria Bulanova Daria Bulanova Institute for Molecular Medicine Finland, FIMM, University of Helsinki, Helsinki, Finland Search for more papers by this author Ulrika Joneborg Ulrika Joneborg Division of Pelvic Cancer, Department of Women's and Children's Health, Karolinska Institutet and University Hospital, Stockholm, Sweden Search for more papers by this author Twana Alkasalias Twana Alkasalias Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Research Centre, Salahaddin University-Erbil, Erbil, Iraq Search for more papers by this author Yuichiro Miki Yuichiro Miki Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Department of Gastroenterological Surgery, Osaka City University Graduate School of Medicine, Osaka, Japan Search for more papers by this author Masakazu Yashiro Masakazu Yashiro Department of Gastroenterological Surgery, Osaka City University Graduate School of Medicine, Osaka, Japan Search for more papers by this author Anastasiya Chernenko Anastasiya Chernenko Research Programs Unit, Individualized Drug Therapy, University of Helsinki and Helsinki University Hospital, Helsinki, Finland Search for more papers by this author Joonas Jukonen Joonas Jukonen Research Programs Unit, Individualized Drug Therapy, University of Helsinki and Helsinki University Hospital, Helsinki, Finland Search for more papers by this author Madhurendra Singh Madhurendra Singh Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Hanna Dahlstrand Hanna Dahlstrand Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Joseph W Carlson Joseph W Carlson Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Kaisa Lehti Corresponding Author Kaisa Lehti [email protected] orcid.org/0000-0001-9110-8719 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Research Programs Unit, Individualized Drug Therapy, University of Helsinki and Helsinki University Hospital, Helsinki, Finland Search for more papers by this author Author Information Lidia Moyano-Galceran1, Elina A Pietilä2,‡, S Pauliina Turunen1,‡, Sara Corvigno3,4, Elisabet Hjerpe5, Daria Bulanova6, Ulrika Joneborg7, Twana Alkasalias1,8, Yuichiro Miki1,9, Masakazu Yashiro9, Anastasiya Chernenko2, Joonas Jukonen2, Madhurendra Singh1, Hanna Dahlstrand3,4, Joseph W Carlson3 and Kaisa Lehti *,1,2 1Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden 2Research Programs Unit, Individualized Drug Therapy, University of Helsinki and Helsinki University Hospital, Helsinki, Finland 3Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden 4Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden 5Department of Obstetrics and Gynecology, Visby Hospital, Visby, Sweden 6Institute for Molecular Medicine Finland, FIMM, University of Helsinki, Helsinki, Finland 7Division of Pelvic Cancer, Department of Women's and Children's Health, Karolinska Institutet and University Hospital, Stockholm, Sweden 8Research Centre, Salahaddin University-Erbil, Erbil, Iraq 9Department of Gastroenterological Surgery, Osaka City University Graduate School of Medicine, Osaka, Japan ‡These authors contributed equally to this work *Corresponding author. Tel: +46 8 524 852 54; E-mail: [email protected] EMBO Mol Med (2020)12:e11177https://doi.org/10.15252/emmm.201911177 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 Metastatic cancers commonly activate adaptive chemotherapy resistance, attributed to both microenvironment-dependent phenotypic plasticity and genetic characteristics of cancer cells. However, the contribution of chemotherapy itself to the non-genetic resistance mechanisms was long neglected. Using high-grade serous ovarian cancer (HGSC) patient material and cell lines, we describe here an unexpectedly robust cisplatin and carboplatin chemotherapy-induced ERK1/2-RSK1/2-EphA2-GPRC5A signaling switch associated with cancer cell intrinsic and acquired chemoresistance. Mechanistically, pharmacological inhibition or knockdown of RSK1/2 prevented oncogenic EphA2-S897 phosphorylation and EphA2-GPRC5A co-regulation, thereby facilitating a signaling shift to the canonical tumor-suppressive tyrosine phosphorylation and consequent downregulation of EphA2. In combination with platinum, RSK inhibitors effectively sensitized even the most platinum-resistant EphA2high, GPRC5Ahigh cells to the therapy-induced apoptosis. In HGSC patient tumors, this orphan receptor GPRC5A was expressed exclusively in cancer cells and associated with chemotherapy resistance and poor survival. Our results reveal a kinase signaling pathway uniquely activated by platinum to elicit adaptive resistance. They further identify GPRC5A as a marker for abysmal HGSC outcome and putative vulnerability of the chemo-resistant cells to RSK1/2-EphA2-pS897 pathway inhibition. Synopsis Platinum chemotherapy induces RSK1/2-EphA2-GPRC5A oncogenic signaling switch associated to intrinsic and acquired resistance. This study identifies GPRC5A as a marker for poor therapy response and vulnerability of the resistant cells to RSK1/2-EphA2-pS897 pathway inhibition in ovarian cancer. The oncogenic switch is mediated by platinum-activated ERK1/2-RSK1/2 pathway coupled with co-localization of EphA2 and the EphA2-interacting orphan receptor GPRC5A. RSK inhibition impairs, through EphA2-pY588 activation and consequent EphA2 downregulation, the platinum-induced resistance-associated GPRC5A-EphA2-pS897 co-regulation, thus sensitizing HGSC cell to platinum. GPRC5A expression is associated with HGSC cell sensitivity to RSKi-platinum combination treatment ex vivo. In human HGSC tumors, high GPRC5A levels are correlated with poor treatment response, as well as adverse overall and progression-free patient survival. Combined EphA2-GPRC5A expression predict shorter progression-free survival. The paper explained Problem Surgical removal of tumor masses and platinum chemotherapy are the standard treatment for HCSC, which is often widely spread at the time of diagnosis. Albeit initially effective in reducing tumor burden, the cycles of platinum treatment induce changes in the surgically inoperable treatment-escaping micro-metastases. These increasingly resistant, residual cells give rise to incurable, recurrent disease. This study assessed the long-neglected aspect of chemotherapy—the potentially oncogenic rewiring of cancer cell signaling induced by the platinum treatment per se as means to confer and sustain resistance. Results Using OC cell lines and patient-derived cultures, we have identified a platinum-induced, adaptive resistance mechanism involving EphA2 and RSK1/2 kinases and GPRC5A receptor. Inhibition of the oncogenic RSK-EphA2-pS897 signaling restored the tumor-suppressive EphA2-pY588 and specially sensitized HGSC cells with high GPRC5A expression to platinum ex vivo. Histological analysis of GPRC5A in a TMA with primary and metastatic HGSC specimens revealed its potential as a predictive marker for patient survival and treatment response. Impact The herein identified mechanism on how platinum chemotherapy induces an oncogenic RSK1/2-EphA2-GPRC5A signaling switch to sustain residual resistant cells reveals a targetable vulnerability to tackle them for complete tumor eradication. Importantly, this platinum-induced signaling axis entails also a potential prognostic marker for predicting survival and platinum treatment response: GPRC5A marker expression in HGSC could be used to stratify the unresponsive patients to combinatorial treatments targeting the oncogenic RSK-EphA2-pS897 axis. Introduction Despite advances in anti-cancer treatments, majority of patients with disseminated metastases eventually recur with an increasingly therapy-resistant disease (Dagogo-Jack & Shaw, 2018). Both intrinsic and acquired drug resistance mechanisms contribute to tumor heterogeneity and evolution of genetically resistant cancer clones (McGranahan & Swanton, 2017; Dagogo-Jack & Shaw, 2018). Extensive evidence also indicates that tumor microenvironment (TME)-dependent phenotypic plasticity contributes to the therapy resistance and recurrent growth (Fischer et al, 2015; Zheng et al, 2015; Senthebane et al, 2017). Although chemotherapy-induced changes in both the cancer cells and the TME have been linked to tumor aggressiveness (Norouzi et al, 2018; Redfern et al, 2018), the effects of chemotherapy itself on the non-genetic, adaptive signaling mechanisms activated in the treatment-escaping cancer cells remain elusive. To dynamically communicate within the TME, tumor cells utilize cell surface receptors (Friedl & Alexander, 2011). The erythropoietin-producing hepatocellular receptor A2 (EphA2) is a widely expressed member of the largest receptor tyrosine kinase (RTK) family, the Eph receptors. EphA2 signals in a context-dependent and dual manner: (i) via ephrinA ligand-induced auto-phosphorylation at the cytoplasmic tyrosine residues, which can occur in connection with epithelial cell adhesion, and generally inhibits oncogenic signaling; or (ii) by ligand-independent signaling, whereby EphA2 is phosphorylated at cytoplasmic S897 residue, driving downstream pro-tumorigenic signaling upon crosstalk with other RTKs and signaling molecules (Gucciardo et al, 2014; Riedl & Pasquale, 2015; Kania & Klein, 2016; Zhou & Sakurai, 2017). Reportedly, the kinases Akt, PKA, and p90 ribosomal S6 kinases (RSK/p90-RSK/S6KA) can mediate tumor-promoting EphA2-S897 phosphorylation (Miao et al, 2009; Zhou et al, 2015; Barquilla et al, 2016). Among the RSK family, RSK1 and RSK2 support tumor growth and survival, whereas RSK3 and RSK4 are frequently downregulated in aggressive cancers (Casalvieri et al, 2017). In the context-dependent regulation, EphA2-pS897 signaling has been linked to over-activation of EphA2, Src-kinase activation, and EphA2 cleavage by matrix metalloproteinase MMP14/MT1-MMP (Sugiyama et al, 2013; Koshikawa et al, 2015; Hamaoka et al, 2018). Through such oncogenic signaling crosstalk, EphA2 can alter cell–cell contacts and extracellular matrix (ECM) adhesion or degradation to promote anchorage-independence, invasion in collagen-rich TME, drug resistance, and stem-like properties (Thaker et al, 2004; Lu et al, 2008; Sugiyama et al, 2013; Zhou & Sakurai, 2017; Giorgio et al, 2018). Ovarian cancer (OC) is the most lethal gynecologic malignancy (Siegel et al, 2018). High-grade serous ovarian cancer (HGSC) accounts for approximately 70% of diagnosed cases, majority of which are in metastatic stages (Seidman et al, 2004; Kobel et al, 2010; Torre et al, 2018). Metastatic HGSC is associated with aggressive dissemination in the abdominal cavity, which occurs via detachment of OC cells from the primary tumor to the peritoneal fluid, followed by accumulation of the metastatic cells and multicellular aggregates in ascites (Kenny et al, 2007; Hjerpe et al, 2018). Upon exposure to specific cues, OC cells adhere and grow as solid metastatic lesions in peritoneal organs, including the fatty omentum as the preferred site for invasion and induction of collagen-rich desmoplastic TME (Kenny et al, 2007, 2011; Luo et al, 2016). The relatively effective first-line therapy for HGSC patients is debulking surgery coupled to platinum-based chemotherapy (Marchetti et al, 2010). Despite initial treatment response, most HGSCs recur, often as a repeatedly chemo-sensitive disease (Pfisterer & Ledermann, 2006; Armbruster et al, 2018). This indicates that besides genetic changes and selection, more plastic resistance mechanisms are activated upon the aggressive disease progression (Friedl & Alexander, 2011). Targeting these mechanisms could provide effective combinatorial treatments urgently needed to eliminate also the chemotherapy-escaping OC (micro)metastases from sustaining aggressive tumor evolution. Frequently overexpressed in OC, EphA2 associates with high tumor grade, advanced stage, and poor clinical outcome (Thaker et al, 2004). It has been recognized as a putative target to block HGSC progression, although currently developed molecular-targeted therapies lack proof for specificity and efficacy (Landen et al, 2005b; Petty et al, 2018). In adhesion-dependent signaling, EphA2 cooperates with integrins, the transmembrane receptors that link the ECM to cell cytoskeleton (Hamidi & Ivaska, 2018). Moreover, the G-protein coupled receptor Class C, Group 5, Member A (GPRC5A) has been identified as an interactor of EphA2 and β1-integrin (Bulanova et al, 2017). While tumor-suppressive and oncogenic functions have been reported for this orphan receptor, possible GPRC5A functions in OC remain unknown (Zhou & Rigoutsos, 2014). Intrigued by our unexpected observation of platinum-induced EphA2 upregulation in ex vivo 3D collagen cultures of HGSC patient cells, we used relevant cell models and clinical tumor material to understand the EphA2-GPRC5A pathway and its clinical implications in OC. Our results uncover a robust platinum-induced switch in EphA2 signaling duality via RSK activation, which pharmacological reversal allowed elimination of the otherwise resistant GPRC5A overexpressing cells. Results Cisplatin treatment leads to EphA2 upregulation in patient-derived HGSC cells ex vivo For investigating HGSC signaling and TME-dependent resistance to platinum chemotherapy, we established ex vivo cultures from the ascites of treatment-naïve patients with metastatic disease (Table 1). The fresh patient cells were plated to ascites-like culture growing spontaneously as suspension cells and spheres, or embedded in 3D collagen, which typifies the collagen-rich desmoplastic microenvironment around solid HGSC metastatic lesions (Kenny et al, 2007). By immunofluorescence, these cells were 60–90% positive for the nuclear HGSC marker PAX8 (Fig EV1A; Laury et al, 2010). Ex vivo cell responses to cisplatin were variable with part of the patient cultures showing treatment resistance particularly when embedded in collagen (Fig EV1B). In such culture, cisplatin affected the cell viability by increased apoptosis (Fig EV1C–E, cleaved caspase-3). Table 1. Patient information Patient OriginA Stage Residual tumor size BRCA status Platinum–taxane regimen Response Follow-up OCKI_p01 HGS-O IVB 0 mm Mut Yes CR NED OCKI_p02 HGS-O IIIC 0 mm WT Yes CR PD OCKI_p03 HGS-O IVB > 2 cm Mut Yes PR PD OCKI_p04 HGS-FP IIB 0 mm WT Yes CR NED OCKI_p06 HGS-FP IIIC 0 mm WT Yes CR PD OCKI_p10 HGS-FP IIIC 0 mm WT Yes CR NED OCKI_p11 HGS-O IVB > 2 cm WT Yes PR PD OCKI_p13 HGS-FP IIIC 0 mm WT Yes CR NED OCKI_p20 HGS-FP IIIC 0 mm Mut Yes CR NED OCKI_p22 HGS-FP IIIC < 5 mm WT Yes CR NED OCKI_p25 HGS-FP IIIB < 1 cm WT Yes PD PD OCKI_p27 HGS-FP IIIC 0 mm NA NA NA NA OCKI_p28 CCC IIIC > 2 cm NA Yes NA NA HGSC originA: O, ovary; FP, fallopian tube. Abbreviations: CCC, clear cell carcinoma; WT, wild type; Mut, mutant; CR, complete response; PR, partial response; PD, progressive disease; NED, no evidence of disease; NA, no available data. Click here to expand this figure. Figure EV1. Patient-derived HGSC cells show variable cisplatin resistance in 3D collagen A. Confocal micrographs show PAX8 (green) in primary patient-derived HGSC cells after 7-days culture in 3D collagen. Cells were 62–87% positive for the nuclear HGSC marker. Scale bars: 20 μm. B, C. Viability of patient-derived HGSC cells cultured for 7 days in ascites-like suspension or 3D collagen (B, C; viability of freshly isolated primary cells could only be assessed once with N = 3 experimental replicates) as well as in an adherent culture (C; N = 3) after 0–50 μM cisplatin treatment for 72 h. Cells derived from patient OCKI_p01, OCKI_p03, OCKI_p06, and OCKI_p13 showed treatment resistance particularly when embedded in 3D collagen. D, E. Representative confocal micrographs show cytokeratin 7 (CK7, green), phalloidin (F-actin, red), and cleaved caspase-3 (clCasp3, orange) in 3D OCKI_p13 HGSC culture treated without or with 20 µM cisplatin for 72 h, 4 days after 3D embedding. Corresponding quantification for clCasp3 (E). Scale bars: 50 μm. N = 4. Data information: In (B, C and E), data are presented as mean (SD). *P < 0.05. Exact P-values are provided in Appendix Table S10, Student's t-test. Download figure Download PowerPoint The cells grew in 3D collagen as colonies positive for cytokeratin 7 (CK7; epithelial HGSC marker; Lengyel, 2010), with or without surrounding residual CK7−, vimentin+ mesenchymal cells (Fig 1A; see OCKI_p01 and OCKI_p02, respectively). The CK7+ cell morphology ranged from compact sphere-forming cells, prominent in cultures OCKI_p01 and OCKI_p02, to round cells in looser grape-like colonies in relatively resistant cultures OCKI_p03 and OCKI_p06 (Fig 1A). Considering the rounded collagen invasive phenotype of OCKI_p03 and OCKI_p06 cells, resembling the reported EphA2-dependent breast cancer cell phenotypes (Sugiyama et al, 2013), and EphA2 association with OC clinical outcome (Thaker et al, 2004), we analyzed EphA2 in these ex vivo cultures by immunofluorescence. Significantly, cisplatin treatment led to over twofold increased EphA2 intensity in the treatment-escaping OCKI_p01, OCKI_p03, and OCKI_p06, while OCKI_p02 cells were positive for EphA2 also prior treatment (Fig 1B and C; OCKI_p01: 3.8 ± 0.2, OCKI_p03: 3.5 ± 0.2, and OCKI_p06: 2.0 ± 0.1-fold increase, P ≤ 0.008). Figure 1. Cisplatin treatment leads to EphA2 upregulation in ex vivo HGSC cultures A. Confocal micrographs show cytokeratin 7 (CK7, green) and vimentin (red) in patient-derived HGSC cells cultured in 3D collagen for 7 days. Scale bars: 50 μm. B. Confocal micrographs of EphA2 (red) in HGSC cells cultured in 3D collagen for 7 days and treated for 72 h with 10 μM cisplatin (5 μM for OCKI_p01). The intensity of EphA2 is comparable only between mock and treatment conditions for each patient. Scale bars: 20 μm. C. Chart illustrates EphA2 fold change after treatment. Mock is set to one. N = 3. Data information: In (C), data are presented as mean fold change (SD). **P < 0.01. Exact P-values are provided in Appendix Table S10, Student's t-test. Download figure Download PowerPoint Platinum induces an oncogenic feedback response via EphA2 tyrosine–serine phosphorylation switch in OC cell lines and patient-derived cells The context-dependent EphA2 signaling can occur via ligand-induced tyrosine auto-phosphorylation, generally considered tumor-suppressive, or via oncogenic, ligand-independent phosphorylation of the S897 residue (Gucciardo et al, 2014; Zhou & Sakurai, 2017). To examine whether platinum chemotherapy affects this EphA2 signaling duality, OVCAR3, OVCAR4, and OVCAR8 cells were first treated with up to 20 μM cisplatin for 72 h (see Appendix Fig S1A and B for cell characterization). In all these human OC cell lines, EphA2 was constitutively expressed and increased after platinum treatment (Fig 2A). The ligand-independent EphA2-pS897 was likewise enhanced. The tumor-suppressive EphA2-pY588 was increased in OVCAR3 and to a less extent in OVCAR4, but was low in OVCAR8 with and without cisplatin (Fig 2A–C). Notably, the pS897/pY588 ratio increased in all three cell models compared to untreated controls (Fig 2B; OVCAR3: 5 μM cisplatin 2.7 ± 0.6, OVCAR4: 10 μM cisplatin 4.2 ± 0.5, and OVCAR8: 20 μM cisplatin 3.5 ± 0.1-fold increase, P ≤ 0.047). Moreover, OVCAR3 and OVCAR4 with low pS897/pY588 ratio prior treatment were sensitive to cisplatin, whereas platinum-resistant OVCAR8 had constitutive oncogenic EphA2-pS897 dominance (Fig 2C and D; cell viability at 20 μM cisplatin: OVCAR3 24.3 ± 10.7% and OVCAR4 20.9 ± 6.8% vs. OVCAR8 84.7 ± 3.3%, P < 0.001). Figure 2. OC cell lines and patient-derived cells undergo an EphA2 phosphorylation switch upon platinum treatment A, B. EphA2 (total and phosphorylated at S897 or Y588) in OVCAR3, OVCAR4, and OVCAR8 after treatment with 0–20 μM cisplatin for 72 h was assessed by immunoblotting (A) and quantified for pS897/pY588 ratio (B). N = 4. C. EphA2 (total and phosphorylated) in corresponding untreated cells. The same β-actin detection for these samples is shown in Appendix Fig S1B. D. Cytotoxicity assay results after cell treatment with 0–20 μM cisplatin for 72 h. N = 6. E. Quantitative assessment of EphA2 (total and phosphorylated) and pS897/pY588 ratio in early passage patient-derived HGSC cultures treated with 0–20 μM cisplatin for 72 h (see immunoblots in Appendix Fig S2A). N = 6 patients, pooled. F. Corresponding EphA2 pS897/pY588 ratios for individual patient cells. G, H. EphA2 (total and phosphorylated) in OVCAR4 and OVCAR8 after treatment with 0–80 μM carboplatin for 72 h (G) along with pS897/pY588 quantification (H). N = 3. Data information: In (B, D–E, and H), data are presented as mean (SD). *P < 0.05, **P < 0.01, ***P < 0.001. Exact P-values are provided in Appendix Table S10, Student's t-test. Source data are available online for this figure. Source Data for Figure 2 [emmm201911177-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint Patient-derived HGSC cultures likewise expressed EphA2, which was increased after cisplatin treatment (Fig 2E, Appendix Fig S2A, see Appendix Fig S2B and C for PAX8 positivity and mutant TP53 pattern of nutlin unresponsiveness). Coincidentally, oncogenic EphA2-pS897 increased in all six patient cells, whereas EphA2 auto-phosphorylation showed an opposite pattern with EphA2-pY588 in the untreated cells declining progressively after treatment with increasing concentrations of cisplatin (Fig 2E, Appendix Fig S2A). Thus, the pS897/pY588 ratio was significantly increased in these patient-derived cells (Fig 2E and F; 5 μM cisplatin 3.0 ± 1.3, 10 μM cisplatin 4.5 ± 1.7, 20 μM cisplatin 6.1 ± 1.3-fold increase, P ≤ 0.02). The current suggested platinum chemotherapy for OC patients is carboplatin, a non-inferior but less toxic platinum-derivate that also causes less unspecific apoptosis in vitro (du Bois et al, 2003; Goodisman et al, 2006). To validate the specific effect of platinum on EphA2, OVCAR4 and OVCAR8 were treated with up to 80 μM carboplatin (higher concentrations than cisplatin due to lower chemical reactivity; Alberts & Dorr, 1998). After treatment, EphA2 (total and pS897) was enhanced and the tumor-suppressive EphA2-pY588 decreased in OVCAR4 (Fig 2G). As a result, carboplatin significantly increased the pS897/pY588 ratio (Fig 2G and H; 3.0 ± 0.2-fold at 80 μM carboplatin, P = 0.014), whereas the platinum-resistant OVCAR8 had high pS897/pY588 prior and after treatment (Fig 2G and H; 2.1 ± 0.1-fold higher in untreated OVCAR8 than corresponding OVCAR4, P = 0.048). Altogether, these results reveal a previously unappreciated induction of a robust oncogenic EphA2 phosphorylation switch by platinum chemotherapy in HGSC cells. Platinum triggers an oncogenic EphA2-S897 phosphorylation in vivo To define the effect of platinum in EphA2 signaling in vivo, OVCAR4 cells were lentivirally transduced to express Renilla luciferase and injected intraperitoneally in severe combined immunodeficient (SCID) female mice. All mice developed tumors in the abdominal cavity (Figs 3A and EV2A). These tumors grew as widely disseminated foci in the omentum and other peritoneal organs, coincident with the accumulation of ascites, thus mimicking HGSC dissemination in patients (Fig EV2A; Kenny et al, 2011). Carboplatin effectively reduced the tumor burden and eliminated the ascites (Fig 3B and C; P ≤ 0.01). In the solid omental and peritoneal tumors, carboplatin had negligible effects on proliferation (Ki67), whereas apoptosis (TUNEL and clCasp3) was increased (Figs 3H and I, and EV2B–D; P ≤ 0.042). However, residual tumor foci remained, modeling the resistant metastatic lesions with potential for aggressive disease progression in patients (Fig 3D; Pfisterer & Ledermann, 2006; Armbruster et al, 2018). By immunofluorescence, total EphA2 and the oncogenic EphA2-pS897 were enhanced in the residual carboplatin-treated tumors as compared to untreated controls (Fig 3E–H; EphA2 1.4 ± 0.4, EphA2-pS897 1.6 ± 0.1-fold increase; P ≤ 0.009). In the carboplatin-treated tumors, EphA2-pS897 and clCasp3 localized to different tumor cells and areas (Fig 3H). These results suggest that the treatment-escaping HGSC cells activated oncogenic EphA2 signaling in response to platinum chemotherapy also in vivo. Figure 3. Platinum treatment promotes ligand-independent oncogenic EphA2 phosphorylation in vivo A. Bioluminescence images visualize mock- and carboplatin-treated OVCAR4 xenograft tumors (day 53; af
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