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Protein Kinase C Inhibitor AEB071 Targets Ocular Melanoma Harboring GNAQ Mutations Via Effects on the PKC/Erk1/2 and PKC/NF-Kb Pathways

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Protein Kinase C Inhibitor AEB071 Targets Ocular Melanoma Harboring GNAQ Mutations Via Effects on the PKC/Erk1/2 and PKC/NF-Kb Pathways Published OnlineFirst May 31, 2012; DOI: 10.1158/1535-7163.MCT-12-0121 Molecular Cancer Therapeutic Discovery Therapeutics Protein Kinase C Inhibitor AEB071 Targets Ocular Melanoma Harboring GNAQ Mutations via Effects on the PKC/Erk1/2 and PKC/NF-kB Pathways Xinqi Wu1,2, Jingjing Li1,2, Meijun Zhu3, Jonathan A. Fletcher3, and F. Stephen Hodi1,2 Abstract Somatic GNAQ mutations at codon 209 have been identified in approximately 50% of uveal melanomas and have been reported to be oncogenic through activating PLCb/PKC/Erk1/2 pathways. We hypoth- esized that protein kinase C (PKC) may provide new opportunities for therapeutic targeting of uveal melanoma carrying GNAQ mutations. To test this hypothesis, uveal melanoma cells harboring wild-type or mutant GNAQ were treated with the PKC inhibitor AEB071 (sotrastaurin) or infected with lentivirus- expressing short hairpin RNAs (shRNA) targeting PKC isoforms. Notably, AEB071 at low micromolar concentrations significantly inhibited the growth of uveal melanoma cells harboring GNAQ mutations through induction of G1 arrest and apoptosis. However, AEB071 had little effect on uveal melanoma cells carrying wild-type GNAQ. AEB071-mediated cell inhibition in the GNAQ-mutated uveal melanoma was accompanied by inhibition of extracellular signal–regulated kinase (Erk)1/2 phosphorylation, NF-kB, decreased expression of cyclin D1, survivin, Bcl-xL, and XIAP, and increased expression of cyclin- dependent kinase inhibitor p27Kip1. AEB071 suppressed the expression of PKC a, b, d, e,andq in GNAQ-mutated uveal melanoma cells. Our findings from shRNA-mediated knockdown studies revealed that these PKC isoforms are functionally important for uveal melanoma cells harboring GNAQ mutations. Furthermore, inhibitors of Erk1/2 and NF-kB pathways reduced viability of uveal melanoma cells. Together, our findings show that AEB071 exerts antitumor action on uveal melanoma cells carrying GNAQ mutations via targeting PKC/Erk1/2 and PKC/NF-kB pathways. Targeted PKC inhibition with drugs such as AEB071 offers novel therapeutic potential for uveal melanoma harboring GNAQ mutations. Mol Cancer Ther; 11(9); 1905–14. Ó2012 AACR. Introduction identified in approximately 50% of uveal melanoma and GNAQ Uveal melanoma is the most common primary intraoc- 83% blue nevi (3–5). The gene encodes for the a ular malignant tumor in adults, with an incidence of 7 subunit of q class of heterotrimeric GTP-binding proteins cases per million annually (1). Approximately half of (G proteins) that are composed of 3 subunits (Ga,Gb, patients with uveal melanoma develop metastases to the and Gg) and transduce signals from 7-transmembrane liver within 15 years of initial diagnosis. With no effective G-protein–coupled receptors (GPCR) to intracellular cas- treatment modality available the median survival time of cades (6). Activation of GPCRs results in exchange of GDP patients with uveal melanoma with metastasis is less than for GTP on the Ga subunit, resulting in the dissociation of a bg a bg 6 months (2). the GTP bound form of G from G . Both G and G The etiology of uveal melanoma has not been fully can then activate downstream cellular signaling path- understood. Mutations in the GNAQ gene have been ways. The signal is terminated when GTP is hydrolyzed to GDP by the intrinsic GTPase activity of the Ga subunit. The majority of GNAQ mutations occur at codon 209 Authors' Affiliations: 1Department of Medical Oncology, 2Melanoma within the GTPase catalytic domain, resulting in loss of Disease Center, Dana-Farber Cancer Institute and Harvard Medical School; and 3Department of Pathology, Brigham and Women's Hospital and the intrinsic GTPase activity and constitutively activation Harvard Medical School, Boston, Massachusetts of GNAQ. Expression of mutated GNAQ results in mela- Note: Supplementary data for this article are available at Molecular Cancer nocyte transformation and increased extracellular signal– Therapeutics Online (http://mct.aacrjournals.org/). regulated kinase (Erk)1/2 phosphorylation, indicating GNAQ Corresponding Author: F. Stephen Hodi, Dana-Farber Cancer Institute, that mutant behaves as a dominant acting onco- 450 Brookline Avenue, Boston, MA 02215. Phone: 617-632-5053; Fax: gene (4, 5). Currently, there are no available therapies 617-582-7992; E-mail: [email protected] targeting GNAQ. doi: 10.1158/1535-7163.MCT-12-0121 The protein kinase C (PKC) family is a widely expressed Ó2012 American Association for Cancer Research. group of serine/threonine kinases comprising multiple www.aacrjournals.org 1905 Downloaded from mct.aacrjournals.org on September 29, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst May 31, 2012; DOI: 10.1158/1535-7163.MCT-12-0121 Wu et al. isoforms that can be divided into 3 structurally and Cell-cycle analysis functionally distinct subgroups (7, 8). These are the Cells were collected by trypsinization and fixed in cold conventional PKCs (PKCa,PKCb,andPKCg), which ethanol. After incubation with RNase A and PI, cell-cycle are activated by diacylglycerol (DAG) and phospholip- distribution was determined by flow cytometric analysis þ id and are Ca2 dependent; the novel PKCs (PKCd, (fluorescence-activated cell sorting, FACS). PKCe,PKCq,andPKCh), which are also activated þ by DAG and phospholipids, but are not Ca2 depen- Analysis of apoptosis dent; and the atypical PKCs, which do not require DAG Apoptotic cells were detected by Annexin V-fluorescein þ or Ca2 for activation. PKCs regulate key biologic isothiocyanate staining and FACS as described pre- processes including cell proliferation, apoptosis, differ- viously (17). Compensation for AEB071 autofluorescence entiation, angiogenesis, tumor development, and was conducted. chemoresistance (7, 9–15). PKCs are involved in GNAQ-mediated activation of the MAPK/Erk1/2 path- Knockdown of PKC isotypes by shRNA ways (6, 16). It has been known that GNAQ transduces The constructs (pLKO.1-puro) containing shRNA tar- signals from GPCRs to phospholipase Cb (PLCb;ref.6). get sequences for PKCa,PKCd,orGFPwereprovided PLCb enzymes catalyze the hydrolysis of phosphatidy- by Dana-Farber Cancer Institute shRNA Core Facility. linositol biphosphate to release inositol trisphosphate Lentivirus expressing PKC shRNA was produced as and DAG that function as second messengers propa- describe previously (17). Cells were infected with virus gating and amplifying the Ga-mediated signal through for 3 days and cell viability was determined using MTS activation of PKCs. Active PKCs further activate Erk1/2 assay. through the RAF/MAPK/Erk1/2 pathway (16). Using short hairpin RNA (shRNA)-mediated downregulation Immunoblotting of PKC isoforms b, e,andq we have recently shown that Preparation of whole-cell lysates and immunoblotting these isoforms are functionally important for GNAQ- have been described previously (19). Antibodies against mutated uveal melanoma cells (17). The oncogenic PKC isoforms were: PKCa (Cell Signaling #2056), PKCd properties of mutant GNAQ and the important (Cell Signaling #2058), PKCq (BD Biosciences #610090); PKC roles in GNAQ-mediated Erk1/2 activation and PKCq Thr538 (Cell Signaling #9377), PKCq/d Ser643/676 GNAQ-mutated uveal melanoma cells (4, 16) suggested (Cell Signaling #9376), and PKCe (BD Biosciences that PKC may provide new opportunities for therapeu- #610085). Antibodies against Akt, phospho-Akt, Erk1/2, tic intervention of uveal melanoma carrying GNAQ phospho-Erk1/2, cyclin D1, Bcl-xL, XIAP, survivin, mutations. To test this hypothesis, uveal melanoma cells HADC1, and glyceraldehyde-3-phosphate dehydroge- carryingwild-typeGNAQorGNAQmutatedatcodon nase (GAPDH) were purchased from Cell Signaling Tech- 209 were treated with the PKC inhibitor AEB071 (sotras- nology. Antibodies against PKCbII, p27Kip1, and RelA taurin), a PKC inhibitor that has potent activity against (p65) were purchased from Santa Cruz Biotechnology. classical and novel PKC isotypes (18). AEB071 selec- Actin antibody was purchased from Sigma-Aldrich. Pro- tively inhibited the growth of uveal melanoma cells tein signal intensity was measured using NIH ImageJ harboring GNAQ mutations by targeting PKC/Erk1/2 software and normalized to that of actin. and PKC/NF-kB pathways. Nuclear extract preparation and electrophoresis mobility shift assay Materials and Methods Nuclear extraction and NF-kB electrophoresis mobility Cell lines shift assay (EMSA) kits purchased from Signosis were The sources and GNAQ mutational status of uveal used to isolate nuclear extracts from uveal melanoma cells melanoma cell lines C918, Ocm1, Ocm3, Mel285, Mel202, and conduct EMSA. The instructions provided by the 92.1, and Omm1.3 have been described previously (19). manufacturer were followed. Uveal melanoma cells were cultured in RPMI-1640 con- taining 10% FBS, 50 mg/mL penicillin, and 100 mg/mL Measurement of interleukin-6 in cell culture medium streptomycin at 37 C and 5% CO2. These cell lines were Cells were treated with dimethyl sulfoxide (DMSO) or 5 recently authenticated by short tandem repeat PCR anal- mmol/L AEB071 for 72 hours in 6-well plates. Medium ysis at Biosynthesis Inc. Human epidermal melanocytes was collected and centrifuged to remove cells. Interleukin were purchased from Lifeline Cell Technology and grown (IL)-6 in the supernatant was determined using the in the medium provided by the company. Human IL-6 Quantikine ELISA Kit purchased from R&D Systems. Viability assay Cells were seeded in 96-well plates at 2 Â 103 cells per Statistical analysis well and incubated over night
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