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Identification of Retinoid-Modulated Proteins in Squamous Carcinoma Cells Using High-Throughput Immunoblotting

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Identification of Retinoid-Modulated Proteins in Squamous Carcinoma Cells Using High-Throughput Immunoblotting [CANCER RESEARCH 64, 2439–2448, April 1, 2004] Identification of Retinoid-Modulated Proteins in Squamous Carcinoma Cells Using High-Throughput Immunoblotting Hyun-Jung Kim and Reuben Lotan Department of Thoracic/Head and Neck Medical Oncology, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas ABSTRACT in various cancer cell lines, including some that are resistant to ATRA. The apoptosis induced by CD437 in several cancer cells has Retinoids have shown clinical efficacy in cancer chemoprevention and been found to be independent of retinoid receptors. However, one therapy presumably by modulating the growth, differentiation, and apo- study suggested a role for RARs in mediating the effect of CD437 in ptosis of normal, premalignant, and malignant cells. To better understand the mechanisms by which retinoids exert their effects, we used a high- ovarian cancer cells (3). The receptor-independent mechanisms of throughput Western blotting method (Becton-Dickinson PowerBlot) to apoptosis induction attributed to CD437 include an increase in c-Myc evaluate changes in the levels of cellular signaling proteins in head and levels, an increase in activator protein 1 (AP-1), an increase in cell neck squamous cell carcinoma cells treated with the cytostatic all-trans- surface death receptors, and mitochondrial permeability transition retinoic acid or with the proapoptotic retinoids 6-[3-(1-adamantyl)-4- (reviewed in Ref. 4). hydroxyphenyl]-2-naphthalene carboxylic acid or N-(4-hydroxyphenyl) N-(4-Hydroxyphenyl)retinamide (4HPR), a synthetic retinoid, retinamide. Treatments of the head and neck squamous cell carcinoma which was examined in several clinical chemoprevention studies, can cells with these retinoids for 24 h resulted in increased levels of 14, 22, and transactivate nuclear receptors. 4HPR also induces apoptosis in vari- 22 proteins and decreased levels of 5, 10, and 7 proteins, respectively. The ous cancer cell lines, including those resistant to ATRA. Currently, changes in the levels of the following proteins were confirmed by conven- tional western immunoblotting: all-trans-retinoic acid increased ELF3, the mechanism of apoptosis induction by 4HPR is not fully elucidated. topoisomerase II ␣, RB2/p130, RIG-G, and EMAPII and decreased It may induce apoptosis by a receptor-independent mechanism (5), MEF2D and cathepsin L. N-(4-Hydroxyphenyl)retinamide up-regulated although in some cells receptors may be involved (6). Recently, ELF3, c-Jun, Rb2/p130, JAK1, p67phox, Grb2, O6-methylguanine-DNA generation of reactive oxygen species (ROS) and modulation of methyltransferase, and Ercc-1. 6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2- mitochondrial permeability transition and respiration have been found naphthalene carboxylic acid increased Rb2/p130, c-Jun, Sp1, Sin, and to mediate apoptosis induced by 4HPR (7). Increased ceramide pro- tomosyn and decreased cathepsin L, Mre11, and topoisomerase II ␣. Some duction (8), increased GADD153, and activation of 12-lipoxygenase of these proteins were also modulated by these retinoids in other human (9) have been also implicated in receptor-independent mechanisms of cancer cell lines. A subset of the proteins were modulated similarly by the 4HPR-induced apoptosis. different retinoids, whereas changes in other proteins were unique for each retinoid. These results suggest that the mechanisms by which these Our group is interested in chemoprevention and therapy of head and retinoids modulate proteins are distinct but may overlap. Some of the neck cancers. We have found that retinoids are effective against head retinoid-modulated proteins identified in this study may be novel candi- and neck squamous cell carcinoma (HNSCC) cells in vitro and in vivo. dates for mediating different responses to retinoids. For example, ATRA suppresses the proliferation of HNSCC cells in monolayer culture, inhibits the formation of colonies in semisolid agar, and decreases the growth of HNSCC multicellular spheroids INTRODUCTION (10). In addition, ATRA suppresses the squamous differentiation of Retinoids, a group of natural and synthetic vitamin A analogues, HNSCC cells (11). Several synthetic retinoids also showed antipro- including all-trans-retinoic acid (ATRA), are known to play a major liferative, apoptotic, and differentiation modulatory effects on role in regulating growth, differentiation, and apoptosis of many cell HNSCC cells (12). At low concentration, CD437 suppressed the types both in vivo and in vitro. Most of the biological activities of expression of squamous differentiation markers and induced apoptosis ATRA and some synthetic retinoids are thought to be mediated by two in HNSCC cells through a retinoid receptor-dependent mechanism classes of nuclear retinoids receptors, retinoic acid receptors (RAR␣, (13). 4HPR also induces apoptosis in specific HNSCC cells where RAR␤, and RAR␥) and retinoid X receptors (RXR␣, RXR␤, and ROS production level was high (14). However, in other cells, apo- RXR␥). These receptors form RXR-RAR heterodimers or RXR-RXR ptosis induction by 4HPR partially involved retinoid receptor pathway homodimers after binding to selective retinoids. These dimers then (6). bind consensus DNA sequence or response elements called RAREs Many of the effects of ATRA, CD437, and 4HPR, whether they are and RXREs and transactivate target genes, thereby altering the growth mediated by retinoid receptors or by other mechanisms, involve and differentiation of cells (1). Recently, certain synthetic retinoids changes in gene expression and posttranscriptional changes (e.g., have been demonstrated to exert their effects via receptor-independent protein stability; Refs. 15–19). To improve our understanding of the mechanisms. For example, the synthetic retinoid 6-[3-(1-adamantyl)- mechanisms by which retinoids affect HNSCC cells, we tried to 4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) that has identify proteins that are differentially modulated after treatment with been shown to transactivate RAR␥ receptors (2) can induce apoptosis different retinoids. For this purpose, we used the Becton-Dickinson PowerBlot Western Array Screening system. This is a high-through- Received 8/23/03; revised 12/3/03; accepted 12/18/03. put Western blotting method, which uses carefully formulated mix- Grant support: The Irving and Nadine Mansfield and Robert David Levitt Cancer tures of subsets of ϳ800 monoclonal antibodies to evaluate differ- Research Chair (to R. Lotan). The costs of publication of this article were defrayed in part by the payment of page ences in levels of cellular signaling proteins between total cell extracts charges. This article must therefore be hereby marked advertisement in accordance with from different cells or tissues. Treatments with ATRA, CD437, and 18 U.S.C. Section 1734 solely to indicate this fact. 4HPR resulted in changes in many proteins that included transcription Note: This report is part of the research performed toward obtaining a Ph.D. degree for Hyun-Jung Kim from the University of Texas Graduate School of Biomedical Sciences at factors, signaling mediators, DNA synthesis and repair proteins, and Houston. tumor suppressor. Requests for reprints: Reuben Lotan, Department of Thoracic/Head and Neck Medical Oncology-432, The University of Texas, M. D. Anderson Cancer Center, Houston, TX 77030. The retinoid-modulated proteins identified here include some that Phone: (713) 792-8467; Fax: (713) 745-5656; E-mail: [email protected]. have not been previously associated with retinoid response. Our 2439 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2004 American Association for Cancer Research. PROTEIN CHANGES IN RETINOID-TREATED SCC CELLS results provide additionally support to the contention that retinoids sample for this analysis.2 The blot was removed from the manifold, washed, modulate specific genes and proteins associated with cell growth and and hybridized with goat antimouse antibody conjugated with horseradish apoptosis. peroxidase. The blots were developed with chemiluminescence system (Su- perSignal West Pico, Pierce, Rockford, IL). Each of the samples was analyzed in triplicate blots. After exposure to X-ray films, the developed films were scanned, and the MATERIALS AND METHODS “trace” of each band was processed for densitometric analysis. Briefly, trace includes a measure of both band intensity and band area. Standard average is Retinoids. ATRA, CD437, and 4HPR were obtained from Dr. Werner the average of Trace for all bands on a blot or bands from the standards lane. Bollag (F. Hoffmann-La Roche, Basel, Switzerland), Dr. Brahm Shroot (Gal- The individual band intensity was expressed as a percentage of the correspond- derma RϩD, Sophia Antipolis, France), and Dr. Ronald Lubet (National ing trace or standards average and designated as “percent lane average” to Cancer Institute, Bethesda, MD), respectively. Retinoids were dissolved in normalize for different exposure and protein loading between gels. The percent DMSO at a concentration of 10 mM and stored under nitrogen gas at Ϫ80°C. lane average for treated bands was expressed as a percentage of the percent Stock solutions were diluted to the appropriate final concentration with growth lane average for the corresponding control bands. The “percent of control” was medium just before use. determined to express increases or decreases in protein expression. Changes Cell Culture and Treatment with Retinoids. Human HNSCC cell line are expressed as fold increase or decrease between control
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