Published OnlineFirst August 4, 2020; DOI: 10.1158/1541-7786.MCR-20-0075 MOLECULAR CANCER RESEARCH | SIGNAL TRANSDUCTION AND FUNCTIONAL IMAGING Phosphorylation of PLCg1 by EphA2 Receptor Tyrosine Kinase Promotes Tumor Growth in Lung Cancer Wenqiang Song1,2, Laura C. Kim3, Wei Han4, Yuan Hou5, Deanna N. Edwards1, Shan Wang1, Timothy S. Blackwell2,4,6, Feixiong Cheng5,7,8, Dana M. Brantley-Sieders1,9, and Jin Chen1,2,3,6,9 ABSTRACT ◥ EphA2 receptor tyrosine kinase (RTK) is often expressed at EphA2 decreased phosphorylation of PLCg1andlossofPLCg1 high levels in cancer and has been shown to regulate tumor inhibited tumor cell growth in vitro.KnockoutofPLCg1by growth and metastasis across multiple tumor types, including CRISPR-mediated genome editing also impaired tumor growth non–small cell lung cancer. A number of signaling pathways in a KrasG12D-p53-Lkb1 murine lung tumor model. Collectively, downstream of EphA2 RTK have been identified; however, these data show that the EphA2-PLCg1 signaling axis promotes mechanisms of EphA2 proximal downstream signals are less tumor growth of lung cancer and provides rationale for disrup- well characterized. In this study, we used a yeast-two-hybrid tion of this signaling axis as a potential therapeutic option. screen to identify phospholipase C gamma 1 (PLCg1) as a novel EphA2 interactor. EphA2 interacts with PLCg1andthekinase Implications: The EphA2-PLCG1 signaling axis promotes tumor activity of EphA2 was required for phosphorylation of PLCg1. In growth of non–small cell lung cancer and can potentially be targeted human lung cancer cells, genetic or pharmacologic inhibition of as a therapeutic option. Introduction inhibitor–resistant tumor cells (5). Loss of EphA2 reduced viability of erlotinib-resistant tumor cells harboring EGFRT790M muta- Receptor tyrosine kinases (RTK) regulate signal transduction path- tions in vitro and inhibited tumor growth in an inducible ways that control cell proliferation, survival, and motility. Dysregula- þ EGFRL858R T790M-mutant lung cancer model in vivo (5). Several tion of RTKs by mutations, amplifications, or overexpression can lead EphA2 inhibitors including an antibody, a peptide, and a small- to oncogenic transformation and malignant progression (1). A num- molecule inhibitor have been developed (6). An EphA2-targeting ber of RTKs have been identified as potential drivers of non–small cell DOPC-encapsulated siRNA is currently in phase I clinical trials for lung cancer (NSCLC), one of which is EphA2 (2). The EphA2 RTK advanced or recurrent solid tumors (NCT01591356). However, belongs to the EPH family, the largest family of RTKs, and is despite the interest in EphA2 as a therapeutic target, molecular commonly overexpressed in NSCLC and associated with poor clinical mechanisms mediating EphA2 function, particularly its proximal outcomes (3). Targeted disruption of EphA2 impairs tumor growth in downstream signals, are not well characterized. KRAS-mutant mouse models and in human NSCLC xenografts (4). Phospholipase C gamma (PLCg) is a lipase activated by receptors in Furthermore, EphA2 is overexpressed in EGFR tyrosine kinase the cellular membrane, including RTKs and adhesion receptors. Once activated, PLCg hydrolyzes phosphatidylinositol 4,5-bisphosphate to form diacylglycerol and inositol 1,4,5-trisphosphate, the latter pro- þ 1Division of Rheumatology and Immunology, Department of Medicine, moting the transient release of intracellular Ca2 , another important 2 Vanderbilt University Medical Center, Nashville, Tennessee. Veterans Affairs signaling molecule. PLCg is ubiquitously expressed and exists in two Medical Center, Tennessee Valley Healthcare System, Nashville, Tennessee. isoforms, PLCG1 and PLCG2, each with distinct functions in a variety 3Program in Cancer Biology, Vanderbilt University, Nashville, Tennessee. 4Division of Allergy, Pulmonary and Critical Care Medicine, Department of of cell types and disease states (7, 8). PLCG1 plays a role in vascu- Medicine, Vanderbilt University Medical Center, Nashville, Tennessee. 5Genomic logenesis and erythrogenesis as well as T-cell development and Medicine Institute, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio. activity (9). Importantly, loss of PLCG1 is embryonic lethal in 6Department of Cell and Developmental Biology, Vanderbilt University, mice (10). PLCG2, meanwhile, is critical for B-cell development and 7 Nashville, Tennessee. Department of Molecular Medicine, Cleveland Clinic maturation (8, 11). Both PLCg isoforms are enriched and mutated in Lerner College of Medicine, Case Western Reserve University, Cleveland, Ohio. many cancers (8). Elevated PLCg1 has been shown to drive metastasis 8Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, Ohio. 9Vanderbilt-Ingram Cancer Center, Vanderbilt and progression of breast cancer (12, 13), and its phosphorylation University Medical Center, Nashville, Tennessee. status is prognostic for metastatic risk (14). PLCg has also been implicated in resistance to cancer treatment. In glioblastoma, Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). PLCg/HIF1a mediated FGFR1-induced radioresistance (15) while in head and neck and esophageal squamous cell carcinoma, the W. Song and L.C. Kim contributed equally to this article. AXL-EGFR-PLCg1 axis mediated resistance to PI3K inhibition (16). Corresponding Authors: Jin Chen, Vanderbilt University Medical Center, An acquired PLCG2 mutation also caused resistance to ibrutinib in T-3207E, Medical Center North, Vanderbilt University School of Medicine, 1161 chronic lymphocytic leukemia (17). While important roles for PLCg 21st Avenue South, Nashville, TN 37232. Phone: 615-343-3819; Fax: 615-343- fi g 8648; E-mail: [email protected]; and Dana M. Brantley-Sieders, have been identi ed in several cancer types, PLC role in lung cancer [email protected] has yet to be elucidated. In this report, we show that PLCg is a novel target of the EphA2 RTK Mol Cancer Res 2020;XX:XX–XX in lung cancer. We show that EphA2 interacts with and directly doi: 10.1158/1541-7786.MCR-20-0075 phosphorylates PLCg for activation. In addition, knockdown of Ó2020 American Association for Cancer Research. PLCG1 significantly reduces the growth of KRAS-mutant lung cancer AACRJournals.org | OF1 Downloaded from mcr.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst August 4, 2020; DOI: 10.1158/1541-7786.MCR-20-0075 Song et al. cells in vitro and inhibits lung tumor growth in an orthotopic Kras- interactome are given in our recent studies (18–20). We then mapped p53-Lkb1–mutant mouse model in vivo. Collectively, these studies the EPHA2, PLCG1, and PLCG2 into the PPIs network to construct identify the EphA2-PLCg1 axis as a potential therapeutic target for EPHA2-PLCG1/PLCG2 subnetwork. Next, we performed Kyoto KRAS-mutant lung cancer. Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to identify the functional pathway related with PLCG1 and PLCG2. Materials and Methods Cell growth assays Cell lines, plasmids, and reagents MTT assay was used to evaluate the short-term proliferation of cells. 293FT, COS-7, and mouse Kras, p53, and Lkb1 (KPL) lines were A total of 2 Â 103 cells were plated into 96-well plates with six replicates cultured in DMEM supplemented with penicillin/streptomycin in growth media. MTT reagent was added and the plates were read and10% FBS. Human lung cancer cell lines (A549, H23, H358, using plate reader (Synergy HT, BioTek) on days 1 to 6. Cell viability H2030, H2009, and HCC44) and BEAS2B cells were cultured in was normalized to day 1. Colony formation assays were used to RPMI1640 supplemented with penicillin/streptomycin and 10% FBS. evaluate the long-term proliferation of cells. A total of 400 cells were All cell lines were purchased from ATCC, except the murine KPL line plated into 12-well plates with 3 to 4 replicates in growth media. For which was generated in our lab as shown in Fig. 5. Cell lines were used drug treatment experiments, drugs were added the day after cell between passages 1 and 50 after thaw and authenticated using attachment with an initial plating of 2 Â 104 cells. Cell colonies were short tandem repeat profiling at ATCC, most recently in June 2019. visualized by crystal violet staining after 2 weeks for human cells or Mycoplasma was routinely tested approximately every 6 months to 1 week for mouse KPL cells. exclude possible contamination, most recently in November 2019, using the PlasmoTest Kit from InvivoGen. Y2H screen For transient knockdown, siRNAs were purchased from Dharma- Y2H screening was carried out by Hybrigenics Services (hybrigenics- con (smart pool siEphA2: catalog no. L-003116-00-0005; nontargeting services.com). The cytoplasmic EphA2 tail (AA 559-976) was cloned as pool: catalog no. D-001810-10-05; individual siPLCG1 #1–3: catalog a N-LexA-EPHA2-C fusion to be the bait against a lung cancer cDNA no. J-003559-05, 07, 08). For stable short hairpin (shRNA) knock- library (mix of A549, H1703, and H460) and 79 positive clones were down, lentiviral vector pLKO.1 was used (EphA2 shRNA #1 CGGA- selected on DO-3–selective medium plates. A confidence score CAGACATATGGGATATT; EphA2 shRNA#2 GCGTATCTT- (predicted biological score, PBS) was assigned to each interaction, then CATTGAGCTCAA; PLCG1 shRNA #1 ATGACAAAGCAATGT- scores were stratified into categories based on the degree of confidence. GACTGG; PLCG1 shRNA