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PROTOCADHERIN 7 Acts Through SET and PP2A to Potentiate MAPK Signaling by EGFR and KRAS During Lung Tumorigenesis Xiaorong Zhou1,2, Barrett L

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PROTOCADHERIN 7 Acts Through SET and PP2A to Potentiate MAPK Signaling by EGFR and KRAS During Lung Tumorigenesis Xiaorong Zhou1,2, Barrett L Published OnlineFirst November 7, 2016; DOI: 10.1158/0008-5472.CAN-16-1267-T Cancer Therapeutics, Targets, and Chemical Biology Research PROTOCADHERIN 7 Acts through SET and PP2A to Potentiate MAPK Signaling by EGFR and KRAS during Lung Tumorigenesis Xiaorong Zhou1,2, Barrett L. Updegraff1, Yabin Guo1, Michael Peyton3, Luc Girard3,4, Jill E. Larsen5, Xian-Jin Xie6,7, Yunyun Zhou6, Tae Hyun Hwang6, Yang Xie6, Jaime Rodriguez-Canales8, Pamela Villalobos8, Carmen Behrens9, Ignacio I. Wistuba8, John D. Minna3,4,10, and Kathryn A. O'Donnell1,7 Abstract Non–small cell lung cancer (NSCLC) is the leading cause versely, PCDH7 depletion suppressed ERK activation, sensi- of cancer-associated deaths worldwide. Given the efficacy of tized cells to MEK inhibitors, and reduced tumor growth. membrane proteins as therapeutic targets in human malig- PCDH7 potentiated ERK signaling by facilitating interaction nancies, we examined cell-surface receptors that may act as of protein phosphatase PP2A with its potent inhibitor, the drivers of lung tumorigenesis. Here, we report that the PRO- SET oncoprotein. By establishing an oncogenic role for TOCADHERIN PCDH7 is overexpressed frequently in NSCLC PCDH7 in lung tumorigenesis, our results provide a rationale tumors where this event is associated with poor clinical to develop novel PCDH7 targeting therapies that act at the outcome. PCDH7 overexpression synergized with EGFR and cell surface of NSCLC cells to compromise their growth. KRAS to induce MAPK signaling and tumorigenesis. Con- Cancer Res; 77(1); 1–11. Ó2016 AACR. Introduction KRAS. Significant effort has focused on the development of kinase inhibitors directed against several proteins downstream of KRAS, Non–small cell lung cancer (NSCLC) is the most prevalent including the RAF–MAPK and PI3K–AKT signaling cascades (3). form of lung cancer and is the leading cause of cancer-associated These targeted therapies are capable of inducing disease remis- deaths worldwide (1). Patients have few therapeutic options and sion; however, the development of drug resistance is inevitable disease progression is inevitable, underscoring the critical need to (4). Similarly, patients that harbor EGFR mutations initially identify new actionable targets. KRAS and EGFR mutations are respond well to EGFR inhibitor therapy, but drug resistance among the most common alterations in NSCLC and have been eventually develops (5, 6). Recently, high-throughput sequencing identified in 30% and 15% of patients, respectively (1, 2). Cur- studies have identified hundreds of additional mutations in rently, there are no clinically approved drugs that target mutant NSCLC tumors (7, 8). In spite of this progress, there remains a significant challenge to distinguish the key alterations that pro- mote lung tumorigenesis, which may lead to the identification of 1Department of Molecular Biology, UT Southwestern Medical Center, Dallas, new therapeutic targets. Texas. 2Department of Immunology, Nantong University School of Medicine, Protocadherins (PCDH) are members of the cadherin super- Nantong, China. 3Hamon Center for Therapeutic Oncology Research, UT South- family that regulate cell adhesion. Their extracellular domains 4 western Medical Center, Dallas, Texas. Department of Pharmacology, UT contain cadherin-like repeats, but they differ significantly from Southwestern Medical Center, Dallas, Texas. 5Department of Genetics and classical cadherins with respect to their unique cytoplasmic Computational Biology, QIMR Berghofer Medical Research Institute, Brisbane, Queensland. 6Department of Clinical Science, UT Southwestern Medical Center, domains that lack the conserved motifs for binding b-catenin Dallas, Texas. 7Harold C. Simmons Comprehensive Cancer Center, UT South- and p120-catenin (9, 10). While some PCDHs exhibit cell–cell western Medical Center, Dallas, Texas. 8Department of Translational Molecular adhesion properties, a new paradigm has recently emerged sug- Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas. gesting that PCDHs also control signal transduction pathways (9). 9 Department of Thoracic Head and Neck Medical Oncology, The University of Moreover, there is a rapidly expanding body of literature dem- Texas MD Anderson Cancer Center, Houston, Texas. 10Department of Internal onstrating that PCDH expression is dysregulated and/or mutated Medicine, UT Southwestern Medical Center, Dallas, Texas. in a wide variety of tumor types (11–13). Both tumor suppressive Note: Supplementary data for this article are available at Cancer Research and oncogenic roles of PCDHs have been reported. For example, Online (http://cancerres.aacrjournals.org/). PCDH8 is inactivated through either mutation or epigenetic Current address for Y. Zhou: Center of Biostatistics and Bioinformatics, Univer- silencing in a large fraction of breast carcinomas (14). PCDH10 sity of Mississippi Medical Center, Jackson, MS. was identified as a potential tumor suppressor in gastric, colo- Corresponding Author: Kathryn A. O'Donnell, UT Southwestern Medical Center, rectal, and pancreatic cancers (15). In addition, frequent silencing 6000 Harry Hines Boulevard, NA6.300A, Dallas, TX 75390-9148. Phone: 214- of PCDH17 has been reported in studies of esophageal and 648-5144; Fax: 214-648-5190; E-mail: [email protected] pancreatic carcinomas (16). In contrast, PCDH11Y is overex- doi: 10.1158/0008-5472.CAN-16-1267-T pressed in advanced prostate cancer primary tumors and metas- Ó2016 American Association for Cancer Research. tases, and ectopic expression of PCDH11Y in prostate cancer cell www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst November 7, 2016; DOI: 10.1158/0008-5472.CAN-16-1267-T Zhou et al. lines enhances tumorigenesis (17). Notably, high expression of transduction. PCDH7 depletion in H1944 or H441 was achieved PCDH7 has been linked to brain and bone metastasis of breast using Lenti-CRISPRv2 followed by selection with puromycin and lung cancer cells (11, 12, 18, 19). Collectively, these studies (1 mg/mL). SET knockout cell lines were generated using lenti- þ suggest that PCDHs may play important roles in tumorigenesis. CRISPR. Three days after infection, individual GFP cells were However, the roles of PCDHs in lung cancer and the mechanisms sorted into 96-well plates single-cell SET knockout cell lines were through which gain-of-function and loss-of-function of PCDHs established. drive tumorigenesis in vivo are poorly understood. Because these Cell lines were originally derived in the Minna laboratory with molecules are cell surface receptors and therefore are potentially the exception of PC9 cells that were obtained from the ATCC. All accessible to antibody-based therapeutic modalities, elucidation cell lines have been DNA fingerprinted using the PowerPlex 1.2 kit of the roles of PCDH family members in tumorigenesis, and the (Promega) and have been found to be mycoplasma free using the resulting consequences on signal transduction pathways, repre- e-Myco Mycoplasma PCR Detection kit V2.0 (Boca Scientific, sents a promising area of investigation in cancer biology. catalog #25235). Cell lines were obtained by the O'Donnell lab In this study, we describe the unexpected finding that a poorly in 2013, last tested for mycoplasma in April 2016, and authenti- characterized protocadherin, PCDH7, functions as an oncogene cated in the last year with the PowerPlex 1.2 kit (Promega). to promote transformation of human bronchial epithelial cells and lung tumorigenesis. This PCDH came to our attention Constructs because it is frequently overexpressed in lung cancer and high RNA was extracted from Hela cells using the RNeasy Mini Kit expression of PCDH7 in tumors strongly associates with poor (Qiagen) with an on-column DNase digestion. cDNA was gen- survival of NSCLC patients. Moreover, single-nucleotide poly- erated using the Superscript IV First-Strand Synthesis System PCDH7 fi morphisms located in PCDH7 have been associated with poor (Thermo Fisher). isoforms A-D were PCR ampli ed from overall survival in early-stage NSCLC patients (20). Here, we cDNA using PrimeSTAR HS DNA polymerase (Clontech) and demonstrate that overexpression of PCDH7 potently synergizes subsequently cloned into the gateway entry cloning vector pCR8/ fi PCDH7 with lung cancer drivers, including mutant KRAS and EGFR, GW/TOPO (Invitrogen) and con rmed by sequencing. inducing transformation of human bronchial epithelial cells isoforms were then subcloned into lentiviral vectors pLX304 (HBEC) and promoting tumorigenesis in vivo. These oncogenic (Addgene #25890) or CMV Hygro DEST (Addgene #17454) using effects are associated with the ability of PCDH7 to enhance MAP the LR Clonase II enzyme (Invitrogen). pENTR4-FLAG (Addgene kinase signaling by binding to protein phosphatase 2A (PP2A), #17423) and then FLAG-tagged SET was introduced into HBECs which directly dephosphorylates ERK/2, and the SET oncoprotein, using the pLX304 vector. Lentiviral packaging was performed in a potent inhibitor of PP2A (21, 22), thereby suppressing PP2A 293T cells to ectopically express PCDH7, SET, or EGFP in HBECs activity. Collectively, this work uncovers a new mechanism or PC9 cells. Depletion of PCDH7 was performed using Lenti- through which PCDH7 leads to hyperactivation of the MAPK CRISPR-V2 (Addgene #52961) to introduce Cas9 and sgRNA PCDH7 PCDH7 pathway, a critical driver of lung tumorigenesis, and suggests that directed against
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