Author Manuscript Published OnlineFirst on May 28, 2014; DOI: 10.1158/0008-5472.CAN-13-3430 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 hMOB3 modulates MST1 apoptotic signaling and supports tumor growth in 2 glioblastoma multiforme 3 Fengyuan Tang1*, Lei Zhang1, Gongda Xue1, Debby Hynx1, Yuhua Wang1, Peter D. Cron1, 4 Christian Hundsrucker1,4, Alexander Hergovich3, Stephan Frank2, Brian A. Hemmings1, Debora 5 Schmitz-Rohmer1* 6 1 Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland ; 2 Division of 7 Neuropathology, Institute of Pathology, University of Basel, Switzerland; 3 Cancer Institute 8 University College London, London, UK; 4 Swiss Institute of Bioinformatics, Basel, Switzerland 9 Running Title: hMOB3 inhibits apoptotic MST1 cleavage and promotes tumor growth 10 Keywords: apoptosis, etoposide, proliferation, STK4, caspase cleavage 11 Financial Support: Gongda Xue and Debora Schmitz-Rohmer are supported by the Swiss 12 National Science Foundation SNF 31003A_130838 and 31003A_138287, respectively. Christian 13 Hundsrucker is supported by Swiss Initiative in Systems Biology (Systems Biology IT). 14 Alexander Hergovich is a Wellcome Trust Research Career Development fellow (grant 15 090090/Z/09/Z). The FMI is supported by the Norvartis Research Foundation. 16 *Corresponding authors. Mailing address: Friedrich Miescher Institute for Biomedical 17 Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland. E-mail: [email protected] and 18 [email protected] . Phone: +41-61-6974872 or +41-61-6974046; Fax: +41-61-6973976; 19 Conflict of interest: The authors disclose no potential conflicts of interest. 20 Word count: 5263 words 6 Figures +3 Supplemental Figures 1 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 28, 2014; DOI: 10.1158/0008-5472.CAN-13-3430 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 21 Abstract: 22 New therapeutic targets are needed that circumvent inherent therapeutic resistance of 23 glioblastoma multiforme (GBM). Here we report such a candidate target in the uncharacterized 24 adaptor protein hMOB3, which we show is upregulated in GBM. In a search for its biochemical 25 function, we found that hMOB3 specifically interacts with MST1 kinase in response to apoptotic 26 stimuli and cell-cell contact. Moreover, hMOB3 negatively regulated apoptotic signaling by 27 MST1 in GBM cells by inhibiting the MST1 cleavage-based activation process. Physical 28 interaction between hMOB3 and MST1 was essential for this process. In vivo investigations 29 established that hMOB3 sustains GBM cell growth at high cell density and promotes 30 tumorigenesis. Our results suggest hMOB3 as a candidate therapeutic target for the treatment of 31 malignant gliomas. 32 33 34 35 36 37 38 39 40 2 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 28, 2014; DOI: 10.1158/0008-5472.CAN-13-3430 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 41 Introduction 42 Glioblastoma multiforme (GBM) is the most common and aggressive primary human brain 43 tumor, with a median survival of approximately 14 months after diagnosis. Despite the benefits 44 of surgical resection and the use of adjuvant radiochemotherapies, patients almost invariably 45 succumb to recurrent widespread tumor growth (1, 2). Thus, defining the mechanism of 46 resistance of GBM cells and discovering further effective therapeutic targets are crucial medical 47 goals. 48 The Hippo pathway is an evolutionarily conserved tumor suppressive signal originally identified 49 in Drosophila as a tumor suppressive signal (3-9). Deregulation of Hippo signaling components, 50 such as MST and LATS/NDR kinases, MOB1 proteins, as well as the downstream effector YAP, 51 has been reported in numerous animal tumor models and human malignancies (10). 52 MST1 (Sterile 20-like kinase 1), the mammalian homolog of the Hippo kinase, plays a critical 53 role in regulating cellular apoptosis and proliferation (11-15). MST1 contains an N-terminal 54 kinase domain, followed by an auto-inhibitory domain and a C-terminal protein-protein 55 interaction domain called SARAH (Salvador-RASSF-Hippo) (16). In response to apoptotic 56 stimuli, MST1 is activated by dimerization-mediated trans-phosphorylation and caspase- 57 mediated cleavage (17-20). Cleaved MST1 translocates from the cytoplasm into the nucleus and 58 induces chromatin condensation by phosphorylation different targets (21-25). Although Akt and 59 JNK have been reported to phosphorylate MST1 and modulate its cleavage (26-29), the 60 regulation of apoptotic MST1 signaling has not been completely defined. 61 MOB1 (Mps One Binder 1) proteins were first characterized in yeast, where they are essential 62 components of mitotic exit and septation initiation networks (30, 31). Drosophila mob1/mats 3 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 28, 2014; DOI: 10.1158/0008-5472.CAN-13-3430 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 63 functions as a tumor suppressor by regulating the activation of the Warts kinase (32, 33). The 64 mammalian genome encodes 6 MOB proteins through 6 different genes, namely MOB1A/B, 65 MOB2 and MOB3A/B/C (34, 35). Mammalian Mob1A and Mob1B are essential for embryonic 66 development and prevent tumorigenesis in a broad range of tissues via a mechanism similar to 67 that reported in flies (36, 37). The function of human MOB1 has been characterized as a co- 68 activator of the MST-NDR/LATS kinase cascade (38, 39). Human MOB2 has been reported to 69 restrict NDR kinase signaling (34). Although hMOB3 shares higher amino acid sequence 70 identity (50%) with hMOB1 than hMOB2 (37%), it neither interacts with nor activates 71 NDR/LATS kinases (34, 35). Its biochemical functions remain unknown. Therefore, the 72 molecular roles of hMOB3 in the context of the mammalian Hippo pathway merit further 73 investigation. 74 In the present study we have found that the previously uncharacterized hMOB3 is overexpressed 75 in GBM. Biochemically, hMOB3 directly interacts with MST1 kinase in response to apoptotic 76 stimuli and at high cell density. Functionally, hMOB3 negatively regulates MST1 cleavage 77 during etoposide-induced apoptosis and attenuates the apoptotic response. Moreover, hMOB3 is 78 required to sustain tumor cell proliferation and growth in vitro and in vivo. Taken together, our 79 study reveals that hMOB3 restricts the crosstalk between MST1 and caspases during apoptosis 80 and supports tumorigenesis in GBM suggesting hMOB3 as a potential target for GBM therapy. 81 82 Materials and Methods 83 Patients. Tissue samples of primary GBM and adjacent non-neoplastic brain were processed in 84 accordance with the guidelines of the Ethical Committee of the University Hospital of Basel. 4 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 28, 2014; DOI: 10.1158/0008-5472.CAN-13-3430 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 85 Tumors were diagnosed and graded according to the World Health Organization (WHO) 86 Classification of Tumors of the Nervous System (40). 87 Cell culture, transfection, and stimulation. HEK293 cell line was obtained from American 88 Type Culture Collection (ATCC). Glioma cell lines were described previously (41, 42). All the 89 cell lines in this study were confirmed with absence of mycoplasma contamination 90 (MycoAlertTM, Lonza) and regularly authenticated by growth and morphological observations. 91 HEK293 and glioma cell lines were maintained in Dulbecco's modified Eagle's medium 92 supplemented with 10% fetal calf serum. Transfection of HEK293 and GBM cells were carried 93 out using jetPEI (PolyPlus Transfections, Dietikon, Switzerland) and Lipofectamine 2000 94 (Invitrogen, CA, USA) according to the manufacturer's instructions, respectively. Apoptosis was 95 induced as indicated in the figure legends. Okadaic acid was purchased from Alexis 96 Biochemicals (Enzo Life Sciences, Lausen, Switzerland). Cyclohexylamine (CHX), actinomycin 97 D and etoposide were obtained from Sigma (St Louis, MO, USA). 98 Annexin V assay. Annexin V staining was performed according to the manufacturer's 99 instructions (BD Bioscience) and analyzed by FACSCalibur. The results were from three 100 independent experiments and presented as mean ± standard deviation. Statistical analysis is 101 performed in Excel with two tailed-paired-student t test. 102 Tumor Implantation: Aythymic Nude–Foxn1nu mice (Harlan, France) were maintained in 103 Specific and Opportunistic Pathogen Free (SOPF) facility with food and water ad libitum. 104 U87MG cells (8x105 in 200μl DMEM:Matrigel(1:1 ratio)) were implanted into left flanks. 105 Tumor diameters were regularly measured via caliper and tumor volumes calculated as follows: 106 Volume = d2 D π/6, where d is shorter tumor diameter and D is longer tumor diameter. All 5 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2014 American Association