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An Integrative Study Published OnlineFirst January 12, 2010; DOI: 10.1158/1535-7163.MCT-09-0321 Molecular Spotlight on Molecular Profiling Cancer Therapeutics Multifactorial Regulation of E-Cadherin Expression: An Integrative Study William C. Reinhold1, Mark A. Reimers1,2, Philip Lorenzi1,3, Jennifer Ho1, Uma T. Shankavaram1,4, Micah S. Ziegler1, Kimberly J. Bussey1,5, Satoshi Nishizuka1,6, Ogechi Ikediobi1,7, Yves G. Pommier1, and John N. Weinstein1,3 Abstract E-cadherin (E-cad) is an adhesion molecule associated with tumor invasion and metastasis. Its down- regulation is associated with poor prognosis for many epithelial tumor types. We have profiled E-cad in the NCI-60 cancer cell lines at the DNA, RNA, and protein levels using six different microarray platforms plus bisulfite sequencing. Here we consider the effects on E-cad expression of eight potential regulatory factors: E-cad promoter DNA methylation, the transcript levels of six transcriptional repressors (SNAI1, SNAI2, TCF3, TCF8, TWIST1, and ZFHX1B), and E-cad DNA copy number. Combined bioinformatic and pharmacological analyses indicate the following ranking of influence on E-cad expression: (1) E-cad pro- moter methylation appears predominant, is strongly correlated with E-cad expression, and shows a 20% to 30% threshold above which E-cad expression is silenced; (2) TCF8 expression levels correlate with (−0.62) and predict (P < 0.00001) E-cad expression; (3) SNAI2 and ZFHX1B expression levels correlate positively with each other (+0.83) and also correlate with (−0.32 and −0.30, respectively) and predict (P =0.03and 0.01, respectively) E-cad expression; (4) TWIST1 correlates with (−0.34) but does not predict E-cad expres- sion; and (5) SNAI1 expression, TCF3 expression, and E-cad DNA copy number do not correlate with or predict E-cad expression. Predictions of E-cad regulation based on the above factors were tested and ver- ified by demethylation studies using 5-aza-2′-deoxycytidine treatment; siRNA knock-down of TCF8, SNAI2, or ZFHX1B expression; and combined treatment with 5-aza-2′-deoxycytidine and TCF8 siRNA. Finally, levels of cellular E-cad expression are associated with levels of cell-cell adhesion and response to drug treatment. Mol Cancer Ther; 9(1); 1–16. ©2010 AACR. Introduction tions (2). Down-regulation of E-cad has been described in multiple carcinoma types during tumor progression E-cadherin (E-cad) is a transmembrane glycoprotein (3–6). Its down-regulation, a sign of poor prognosis for that functions to maintain stable cell-cell contacts in epi- multiple types of epithelial carcinomas (7–9), is asso- thelial cell types (1). It forms Ca+2-dependent homodi- ciated with increases in both invasion (3, 10, 11) and me- mers that bind to their counterparts in adjacent cells, tastasis (8, 12). In melanocytes, E-cad down-regulation resulting in the formation of intercellular adherens junc- and a concurrent up-regulation of N-cadherin lead to altered cell-cell relationships; whereas normal melano- cytes interact primarily with keratinocytes, melanoma cells interact more strongly with melanocytes and fibro- Authors' Affiliations: 1Laboratory of Molecular Pharmacology, Center for blasts (13, 14). Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland; 2Virginia Commonwealth University, Richmond, Multiple single factors have been reported to regulate Virginia; 3Department of Bioinformatics and Computational Biology and E-cad expression in one or another cancer type (3–6, 15–23). Department of Systems Biology, M. D. Anderson Cancer Center, Houston, Texas; 4Radiation Oncology Branch, National Cancer However, those factors have not been studied together Institute, National Institutes of Health, Bethesda, Maryland; 5Clinical in combination as a system and across the spectrum of Translational Research Division, Translational Genomics Research cancers. Accordingly, to provide an integrative portrait Institute, Phoenix, Arizona; 6Department of Surgery, Iwate Medical University, School of Medicine, Uchimaru, Japan; and 7Helen Diller of E-cad regulation within and across cancer cell types, Family Comprehensive Cancer Center, San Francisco, California we used six different microarray platforms and bisulfite Corresponding Authors: William C. Reinhold, 9000 Rockville Pike, sequencing to assess eight potential E-cad regulatory fac- Building 37, Room 5056, Bethesda, MD 20892-4255. Fax: 301-496- tors in the NCI-60 human cancer cell line panel at the 9571. E-mail: [email protected] and John N. Weinstein, M.D. Anderson Cancer Center Systems Biology, 1515 Holcombe Boulevard, Houston, DNA, RNA, protein, and epigenetic levels. One very TX 77030-4009. Fax: 301-496-9571. E-mail: [email protected] practical motivation for understanding the complexities doi: 10.1158/1535-7163.MCT-09-0321 of E-cad regulation is the potential for reversing down- ©2010 American Association for Cancer Research. regulation of E-cad and restoring its function. That www.aacrjournals.org 1 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst January 12, 2010; DOI: 10.1158/1535-7163.MCT-09-0321 Reinhold et al. might, in principle, be achieved through the use of agents (18).Inbrief,genomicDNA(5μg) from each cell line that reverse promoter region methylation or by knocking was treated with sodium bisulfite, amplified using down relevant transcriptional repressors. nested polymerase chain reaction primers, and se- The NCI-60 panel consists of 60 diverse human quenced. cancer cell lines used by the National Cancer Insti- tute's Developmental Therapeutics Program to screen Quantitation of Transcript Expression Using Four compounds for anticancer activity (24). The panel in- Microarray Platforms cludes leukemias, melanomas, and cancer cells of breast, We have previously described our processing and nor- central nervous system (glioma), colon, non-small cell malization of NCI-60 transcript expression data from pin- lung, ovarian, prostate, and renal origin. It constitutes the spotted cDNA arrays (Incyte, Inc., Palo Alto, CA; refs. most comprehensively profiled set of cells in existence, hav- 27, 28), Affymetrix Hu-6800 arrays (Affymetrix, Sunny- ing been analyzed at the DNA, RNA, protein, chromosom- vale, CA; ref. 29), Affymetrix HG-U95 arrays (30), and al, metabolomic, and pharmacological levels (25). Profiling Affymetrix HG-U133 (30). The data from those and the of the NCI-60 has been considered a forerunner of The Can- other molecular profiling studies are available in a cer Genome Atlas Project8, which is restricted to the nucleic queryable relational database (CellMiner).10 Further in- acid level but in the more difficult context of clinical tu- formation for these microarrays is available at http:// mors. www.broadinstitute.org/mpr/NCI60/NCI60.html for To test whether the correlative relationships uncovered Hu-6800, and at the Gene Expression Omnibus11 for are causal at the molecular level and whether they pro- the cDNA array, HG-U95, and HG-U133 (identifiers vide the basis for strategies to up-regulate E-cad on a cell GDS1761, GSE5949, and GSE5720, respectively). type-specific basis, we followed up with siRNA knock- down and 5-azacytidine demethylation experiments. Quantitation of E-cad Protein Expression Using This overall integromic (26) approach, supported by Reverse-Phase Lysate Arrays functional data, yields a picture of the multifactorial reg- Our methods for quantitation of proteins using re- ulation of E-cad expression. It provides the ability to pre- verse-phase lysate arrays have been described previously dict rationally and prospectively, independent of cancer (31). Further information for this array is available at the tissue of origin type, whether E-cad will be successfully Gene Expression Omnibus, identifier GSE5501. up-regulated by a given treatment. Comparative Genomic Hybridization Array The arrays comprised 450 cancer-related BAC, PAC, Materials and Methods and P1 clones printed in quadruplicate (32). The clones and their genomic locations have been defined prev- 12 Cell lines and Cell Culture iously. Further information for this array is available at The NCI-60 cells were obtained from the National Array Express at http://www.ebi.ac.uk/microarray-as/ Cancer Institute Frederick Cancer DCTD Tumor/Cell ae/browse.html?keywords=e-geod-5720. 9 Line Repository andculturedasdescribedpreviously 5-Aza-2′-deoxycytidine Inhibition of DNA (18, 27). All culture flasks were examined by microscope ∼ Methylation for anomalies, and the cells were harvested at 80% We modified a previously described protocol (33) to confluence. It is important to note that all cell culture, study inhibition of DNA methylation by 5-AC. Briefly, harvests, and purifications were performed by a single re- 4,000 to 5,000 cells were seeded in 96-well cell culture searcher to maximize interoperability of the data. clusters on day 0, treated with 0.1 to 2 mg/mL 5-aza- RNA and DNA Isolation 2′-deoxycytidine (5-AC) (Sigma, St. Louis, MO) on days RNA was isolated as we have described previously (18, 1, 3, and 5, and washed to remove the drug on days 2 27). Briefly, total RNA was purified using the RNeasy and 4. The cells were lysed on day 6 using Lysis mixture Midi Kit (Qiagen Inc., Valencia, CA) according to the (Panomics, Inc., Fremont, CA). manufacturer's instructions. Genomic DNA was puri- fied using either the QIAamp DNA Blood Maxi Kit or RNA Interference the Blood & Cell Culture DNA Maxi Kit (Qiagen) ac- We used the synthetic siRNAs siTCF8.1, siTCF8.2, cording to manufacturer's instructions (18). siSNAI2.1, siSNAI2.2, siZFHX1B.1, and siZFHX1B.2 (Qiagen) to inhibit expression of the transcription fac- Sodium Bisulfite DNA Modification, Polymerase tors TCF8, SNAI2, and ZFHX1B, as described previous- Chain Reaction Amplification, and Sequencing ly (34). Our bisulfite-sequencing protocol for the minimal promoter region of E-cad has been described previously 10 See http://discover.nci.nih.gov/. 8 http://cancergenome.nih.gov/ 11 http://www.ncbi.nlm.nih.gov/geo/ 9 See http://dtp.nci.nih.gov/. 12 See http://cc.ucsf.edu/gray/public.
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