GPCR Expression in Cancer Cells and Tumors Identifies New
Total Page：16
File Type：pdf, Size：1020Kb
fphar-09-00431 May 17, 2018 Time: 16:38 # 1 ORIGINAL RESEARCH published: 22 May 2018 doi: 10.3389/fphar.2018.00431 GPCRomics: GPCR Expression in Cancer Cells and Tumors Identifies New, Potential Biomarkers and Therapeutic Targets Paul A. Insel1,2*†, Krishna Sriram1†, Shu Z. Wiley1, Andrea Wilderman1, Trishna Katakia1, Thalia McCann1, Hiroshi Yokouchi1, Lingzhi Zhang1, Ross Corriden1, Dongling Liu1, Michael E. Feigin3, Randall P. French4,5, Andrew M. Lowy4,5 and Fiona Murray1,2,6† 1 Department of Pharmacology, University of California, San Diego, San Diego, CA, United States, 2 Department of Medicine, University of California, San Diego, San Diego, CA, United States, 3 Department of Pharmacology and Therapeutics, Roswell Edited by: Park Comprehensive Cancer Center, Buffalo, NY, United States, 4 Department of Surgery, University of California, San Diego, Ramaswamy Krishnan, San Diego, CA, United States, 5 Moores Cancer Center, University of California, San Diego, San Diego, CA, United States, Harvard Medical School, 6 School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Aberdeen, United Kingdom United States Reviewed by: G protein-coupled receptors (GPCRs), the largest family of targets for approved drugs, Kevin D. G. Pfleger, Harry Perkins Institute of Medical are rarely targeted for cancer treatment, except for certain endocrine and hormone- Research, Australia responsive tumors. Limited knowledge regarding GPCR expression in cancer cells likely Deepak A. Deshpande, has contributed to this lack of use of GPCR-targeted drugs as cancer therapeutics. Thomas Jefferson University, United States We thus undertook GPCRomic studies to define the expression of endoGPCRs *Correspondence: (which respond to endogenous molecules such as hormones, neurotransmitters and Paul A. Insel metabolites) in multiple types of cancer cells. Using TaqMan qPCR arrays to quantify [email protected] the mRNA expression of ∼340 such GPCRs, we found that human chronic lymphocytic †These authors have contributed equally to this work. leukemia (CLL) cells/stromal cells associated with CLL, breast cancer cell lines, colon cancer cell lines, pancreatic ductal adenocarcinoma (PDAC) cells, cancer associated Specialty section: fibroblasts (CAFs), and PDAC tumors express 50 to >100 GPCRs, including many This article was submitted to Translational Pharmacology, orphan GPCRs (which lack known physiologic agonists). Limited prior data exist a section of the journal regarding the expression or function of most of the highly expressed GPCRs in these Frontiers in Pharmacology cancer cells and tumors. Independent results from public cancer gene expression Received: 06 February 2018 Accepted: 12 April 2018 databases confirm the expression of such GPCRs. We propose that highly expressed Published: 22 May 2018 GPCRs in cancer cells (for example, GPRC5A in PDAC and colon cancer cells and Citation: GPR68 in PDAC CAFs) may contribute to the malignant phenotype, serve as biomarkers Insel PA, Sriram K, Wiley SZ, and/or may be novel therapeutic targets for the treatment of cancer. Wilderman A, Katakia T, McCann T, Yokouchi H, Zhang L, Corriden R, Keywords: breast cancer, cancer microenvironment, chronic lymphocytic leukemia, colon cancer, GPCR array, Liu D, Feigin ME, French RP, orphan receptors, pancreatic cancer Lowy AM and Murray F (2018) GPCRomics: GPCR Expression Abbreviations: BMNK cell, Bone Marrow Natural Killer cell; CLL/B-CLL, Chronic Lymphocytic Leukemia/B-cell Chronic in Cancer Cells and Tumors Identifies Lymphocytic Leukemia; Ct/1Ct, Cycle threshold/Difference in Cycle threshold; EBI, European Bioinformatics Institute; New, Potential Biomarkers FPKMs, Fragments Per Kilobase of transcript per Million mapped reads; GPCR, G protein-coupled receptor; GtoPdb, Guide and Therapeutic Targets. to Pharmacology database; IUPHAR, International Union of Basic and Clinical Pharmacology; PDAC, Pancreatic Ductal Front. Pharmacol. 9:431. Adenocarcinoma; PDEC, Pancreatic Ductal Epithelial Cell; PSCs, Pancreatic stellate cells; RNA-seq, High throughput RNA doi: 10.3389/fphar.2018.00431 sequencing; TCGA, The Cancer Genome Atlas; TMM, trimmed mean of M values. Frontiers in Pharmacology| www.frontiersin.org 1 May 2018| Volume 9| Article 431 fphar-09-00431 May 17, 2018 Time: 16:38 # 2 Insel et al. GPCRomics in Cancer INTRODUCTION could serve as novel biomarkers and therapeutic targets in various types of cancer. Knowledge regarding the biology of tumors and malignant cells has greatly expanded in recent years. Several hallmarks of cancer have been identified: proliferative signaling, replicative MATERIALS AND METHODS immortality, evasion of growth suppressors, resistance to cell death, induction of angiogenesis, and the activation of Primary Cells and Cell Lines invasion and metastasis (Hanahan and Weinberg, 2011). A key Table 1 summarizes the cells used in the current studies and recent focus for basic and clinical investigators has been the the source from which they were obtained. Patient cells were identification of “driver mutations,” including ones that are isolated after informed consent, under an Institutional Review shared among anatomically distinct types of cancer and predicted Board-approved protocol, and within the guidelines of the Health to be responsive to molecularly targeted therapeutics. In parallel Insurance Portability and Accountability Act. All cells were has been the growth of personalized (precision) medicine grown under standard tissue culture conditions and studied when approaches guided by genetic analyses that seek to identify such growing at log phase and at low passage number. driver mutations (Schwaederle et al., 2015; Borad and LoRusso, 2017; Chae et al., 2017; Hyman et al., 2017). In addition, increased Isolation of mRNA and Preparation of understanding of the immune suppression that contributes to cDNA tumor growth and metastasis and development of therapeutics RNA was isolated using a Qiagen RNeasy MiniKit, with on- directed at this immune suppression have yielded improved column DNase-1 digestion (Qiagen, Valencia, CA, United States). clinical outcomes for a variety of cancers (Cogdill et al., 2017; cDNA was synthesized using a Superscript III First Strand Gotwals et al., 2017; Lim and June, 2017). Synthesis Kit (Invitrogen, Carlsbad, CA, United States) as per the In spite of such progress, new therapies are needed for manufacturer’s instructions, using random hexamer priming. most cancers. In this regard, GPCRs, the largest family of signaling receptors in humans and the largest family of protein Analysis of GPCR Expression targets for approved drugs (Sriram and Insel, 2018), have We used TaqMan GPCR arrays (Thermo Fisher Scientific) to rarely been exploited as therapeutic targets in cancer with identify and quantify GPCR expression (Snead and Insel, 2012). the exception of certain endocrine cancers (e.g., pituitary, adrenal, testes, ovarian) and hormone-responsive tumors (e.g., breast and prostate cancer). Even though GPCRs are TABLE 1 | Human cells and cell lines used for assessment of GPCR expression. not thought to be functionally mutated and commonly expressed (i.e., they are not “genetic drivers”) in cancers, Type of cancer Cells or cell lines Source of cells or cell used lines GPCRs and post-GPCR signaling mechanisms play an important role in regulating cellular functions integral to B-cell chronic Patient-derived UCSD Moores the hallmarks of cancer (e.g., growth/proliferation, metabolism, lymphocytic primary cells Normal Cancer Ctr death/apoptosis, ion and nutrient transport, and migration; leukemia (CLL) human B cells Dorsam and Gutkind, 2007; Hanahan and Weinberg, 2011; Bone marrow NK cells immortalized UCSD Moores stromal natural killer with telomerase Cancer Ctr O’Hayre et al., 2014). GPCRs are not only expressed by (NK) cells that reverse transcriptase cancer cells themselves, but also by cell-types in the tumor support CLL cell microenvironment, including stromal (fibroblast), vascular, growth immune, and inflammatory cells. Numerous effects of GPCRs, Colon cancer T-84, Caco-2 cell ATCC including their regulation of apoptotic cell death, are mediated by lines the second messenger cAMP (Insel et al., 2012, 2014). Moreover, Triple-negative BT-20, HS-578, ATCC as plasma membrane proteins, GPCRs should be targetable as breast cancer MDA-MB-157, MDA-MB-436 cell are numerous other types of cell surface proteins in various lines cancers. Pancreatic ductal 34E/79E UCSD Moores Cancer The lack of consideration of GPCRs as therapeutic targets adenocarcinoma patient-derived cells Ctr. ATCC may relate, at least in part, to the limited information (PDAC) cells and regarding their expression by cancer cells and cells of the PDAC tumors Human tumor microenvironment. We therefore have used an unbiased control pancreatic ductal epithelial cells (GPCRomic) approach to identify and quantify GPCR expression AsPC-1, MiaPaCa-2 in cancer cells in order to identify GPCRs that may contribute to cell lines the malignant phenotype and might be therapeutic targets. Here, Pancreatic ductal 5 primary Lowy lab, UCSD Moores we present findings that utilize GPCR-specific PCR-based arrays, adenocarcinoma patient-derived CAFs Cancer Ctr ScienCell RNA-seq, and mining of databases to define GPCR expression Human PSCs: Research Laboratories Corresponding (#3830; ScienCell of primary cancer cells, cancer cell lines, cells in tumor tissue, “normal” precursor Research Laboratories, and the tumor microenvironment. The findings reveal previously cells Carlsbad, CA, unrecognized