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TCF21 Promotes Luminal-Like Differentiation and Suppresses Metastasis in Bladder Cancer Sharada Mokkapati1, Sima P

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TCF21 Promotes Luminal-Like Differentiation and Suppresses Metastasis in Bladder Cancer Sharada Mokkapati1, Sima P Published OnlineFirst March 2, 2020; DOI: 10.1158/1541-7786.MCR-19-0766 MOLECULAR CANCER RESEARCH | CANCER GENES AND NETWORKS TCF21 Promotes Luminal-Like Differentiation and Suppresses Metastasis in Bladder Cancer Sharada Mokkapati1, Sima P. Porten2, Vikram M. Narayan1, Amy H. Lim1, Isuru S. Jayaratna3, Beat Roth4,5, Tiewei Cheng1, Neema Navai1, Matthew Wszolek6, Jonathan Melquist7, Ganiraju Manyam8, Woonyoung Choi9, Bradley Broom8, Shanna Pretzsch1, Bogdan Czerniak10, David J. McConkey9,and Colin P. N. Dinney1 ABSTRACT ◥ Little is known regarding the subclone evolution process in differentially expressed in primary bladder cancer and correspond- advanced bladder cancer, particularly with respect to the genomic ing LN metastasis pairs at an FDR cutoff of 0.1 and a fold change of alterations that lead to the development of metastatic lesions. In this 1. Among those significantly altered, expression of TCF21 was project, we identify gene expression signatures associated with higher in the primary tumor compared with LN metastasis. We metastatic bladder cancer through mRNA expression profiling of validated this finding with qPCR and IHC on patient samples. RNA isolated from 33 primary bladder cancer and corresponding Moreover, TCF21 expression was higher in luminal cell lines and lymph node (LN) metastasis samples. Gene expression profiling knockdown of TCF21 increased invasion, tumor cell dissemination, (GEP) was performed on RNA isolated using the Illumina DASL and metastasis. In contrast, overexpression of TCF21 in highly platform. We identified the developmental transcription factor metastatic basal bladder cancer cell lines decreased their invasive TCF21 as being significantly higher in primary bladder cancer and metastatic potential. compared with LN metastasis samples. To elucidate its function in bladder cancer, loss- and gain-of-function experiments were Implications: TCF21 is differentially overexpressed in primary conducted in bladder cancer cell lines with high and low expression bladder cancer compared with matched LN metastasis, with of TCF21, respectively. We also performed GEP in bladder cancer in vitro and in vivo studies demonstrating a metastasis suppressor cell lines following TCF21 overexpression. We identified 2,390 genes function of this transcription factor. Introduction of patients diagnosed with bladder cancer are diagnosed with urothelial carcinoma, a heterogenous epithelial malignancy with Bladder cancer accounts for nearly 200,000 deaths worldwide and a high somatic mutation rate (4, 5). is a source of significant morbidity, with over 500,000 new cases Large-scale mRNA expression profiling efforts of MIBC have diagnosed each year. Among all malignancies in the United States, led to the development of several genomic taxonomies for bladder cancer is the fifth most common overall (1). It is estimated bladder cancer, classifying the disease into molecular subtypes that up to one-third of patients present with muscle-invasive to better elucidate disease pathology and potentially guide treat- bladder cancer (MIBC), which can result in mortality within 2 years ment decisions (4, 6, 7). Patients with metastatic bladder cancer of diagnosis in 85% of cases when left untreated (2, 3). The majority have a poor prognosis, and lymph node (LN) positive disease is an independent predictor of worse survival (8, 9). To date, little is known regarding the subclone evolution process in advanced 1Department of Urology, University of Texas MD Anderson Cancer Center, bladder cancer, particularly with respect to the genomic altera- Houston, Texas. 2Department of Urology, University of California San Francisco, tions that lead to the development of metastatic lesions. Previous San Francisco, California. 3Department of Urology, Icahn School of Medicine at 4 reports have suggested that genomic assessments originating Mount Sinai, New York, New York. Department of Urology, University Hospital from the primary tumor alone may underestimate the true of Bern, University of Bern, Bern, Switzerland. 5Department of Urology, University Hospital of Lausanne (CHUV), University of Lausanne, Lausanne, mutational burden that exists within heterogenous tumors (10). Switzerland. 6Department of Urology, Massachusetts General Hospital, Boston, Phylogenetic reconstructions have demonstrated that tumors can Massachusetts. 7Department of Urology, Baptist MD Anderson Cancer Center, exhibit an evolutionary pattern of growth, with metastatic lesions Jacksonville, Florida. 8Department of Bioinformatics and Computational demonstrating greater intratumoral heterogeneity than the pri- Biology, University of Texas MD Anderson Cancer Center, Houston, Texas. 9 mary tumor (11, 12). This heterogeneity can confer challenges Greenberg Bladder Cancer Institute, Johns Hopkins University, Baltimore, in personalizing therapy, and additional investigation into Maryland. 10Department of Pathology, University of Texas MD Anderson Cancer Center, Houston, Texas. the genomic drivers of metastatic lesions in bladder cancer is required. Note: Supplementary data for this article are available at Molecular Cancer In this study, we describe the results of our efforts to use gene Research Online (http://mcr.aacrjournals.org/). expression profiling to identify differences between primary blad- Corresponding Author: Colin P.N. Dinney, University of Texas MD Anderson der cancer tissue and their corresponding metastatic lesions, Cancer Center, 1515 Holcombe Blvd, Unit 1373, Houston, TX 77030. Phone: 713- fi 792-3250; Fax: 713-794-4824; E-mail: [email protected] based on mRNA expression pro ling on matched LN tissue from human specimens. Metastatic xenograft models were then used to – Mol Cancer Res 2020;XX:XX XX confirm the role of novel candidate genes in the development of doi: 10.1158/1541-7786.MCR-19-0766 tumor metastasis, and to identify potential targets for biomarkers Ó2020 American Association for Cancer Research. and therapy. AACRJournals.org | OF1 Downloaded from mcr.aacrjournals.org on September 30, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst March 2, 2020; DOI: 10.1158/1541-7786.MCR-19-0766 Mokkapati et al. extracted from FFPE matched primary and lymph node metastasis using Materials and Methods an AllPrep DNA/RNA FFPE Kit (Qiagen GmBH). Human subjects Thirty-three well annotated clinically homogeneous patients were Gene expression profiling identified. Patients treated with neoadjuvant chemotherapywere exclud- After RNA isolation, RNA purity and integrity were measured by ed from the study. Tumor tissues from the same histologic component of a NanoDrop ND1000 and an Agilent Bioanalyzer, respectively. High- both the primary tumor and metastases were macrodissected by an quality RNA was then used for the synthesis of biotin-labeled cRNA experienced genitourinary pathologist from formalin-fixed paraffin using the Illumina RNA Amplification Kit (Ambion). Briefly, 500 ng of embedded (FFPE) sections and RNA was isolated. A schematic repre- total RNA was converted to cRNA by in vitro transcription, purified, sentation of our experimental design is shown (Fig. 1A). RNA was and 1.5 mg cRNA was hybridized to Illumina HT12 v4 chips (Illumina). Figure 1. Transcriptome analysis of primary bladder cancer (BLCA) and LN metastases. A, Schematic representation of experimental study design to perform differential expression analysis between matched primary bladder cancer and LN metastasis samples. B, PCA showing that the primary tumors and metastases form two distinct groups. C, Heatmap of hierarchical clustering showing gene expression of 64 of 2,390 genes that are differentially expressed between the primary and LN metastases at FDR cutoff of 0.1 and with a log fold change of 1. Red: increased relative expression; green: decreased relative expression. OF2 Mol Cancer Res; 2020 MOLECULAR CANCER RESEARCH Downloaded from mcr.aacrjournals.org on September 30, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst March 2, 2020; DOI: 10.1158/1541-7786.MCR-19-0766 TCF21 in the Suppression of Metastasis in Bladder Cancer The slides from Direct Hybridization (Illumina) were washed, scanned from The Jackson Laboratory and used for xenograft experiments. with a Bead Station 500 (Illumina), and the signal intensities from the Between 19 and 20 mice were used in each group. The mice were scanner were quantified using Genome Studio (Illumina). Quantile housed under specific pathogen-free conditions and all surgeries were normalization in linear models was used to normalize the data, which conducted under anesthesia by approved animal protocols. were processed by established techniques. Unsupervised analysis was Labeled (luciferase and red fluorescent protein; luc-RFP) and performed to identify outliers and assess overall similarity/differences orthotopically recycled human bladder cancer cells UM-UC-3, UM- among the samples. Differential expression analysis was performed UC13, and UM-UC-14 were used. Recycling was performed to enrich using t tests and FDR was estimated using the BUM method (13). the spontaneous metastatic phenotype as described previously (15). Cell lines were transfected with the lentiviral TCF21 shRNAi (UM- qPCR UC14) or the TCF21 overexpression pLOC construct (UM-UC3, UM- For qPCR, 20 ng of RNA was used along with the AgPAth-ID One- UC13) were injected at a concentration of 105/50 mL using HBSS fi Step RT-PCR Kit (Thermo Fisher Scienti c) with Taqman probes to (Gibco) by intramural injection into bladder wall. For shRNAi therapy detect
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