Author Manuscript Published OnlineFirst on March 7, 2019; DOI: 10.1158/1078-0432.CCR-18-1515 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Multi-omics integration analysis robustly predicts high-grade patient 2 survival and identifies CPT1B effect on fatty acid metabolism in Bladder 3 Cancer 4 Venkatrao Vantaku1^, Jianrong Dong1^, Chandrashekar R Ambati2^, Dimuthu Perera2^, 5 Sri Ramya Donepudi2, Chandra Sekhar Amara1, Vasanta Putluri2, Ravi, Shiva 6 Shankar1, Matthew J. Robertson2, Danthasinghe Waduge Badrajee Piyarathna1, 7 Mariana Villanueva2, Friedrich-Carl von Rundstedt3, Balasubramanyam Karanam4, 8 Leomar Y Ballester5, Martha K Terris6, Roni J Bollag6, Seth P. Lerner7, Apolo B. 9 Andrea8, Hugo Villanueva2, MinJae Lee10, Andrew G. Sikora9, Yair Lotan11, Arun 10 Sreekumar1, 12, Cristian Coarfa1,2,13$, Nagireddy Putluri1# 11 1Department of Molecular and Cell Biology, Baylor College of Medicine, Houston, TX, USA; 12 2Dan L. Duncan Cancer Center, Advanced Technology Core, Alkek Center for Molecular 13 Discovery, Baylor College of Medicine, Houston, TX, USA; 3Department of Urology, Jena 14 University Hospital, Friedrich-Schiller-University, Jena, Germany; 4Department of Biology and 15 Center for Cancer Research, Tuskegee University, Tuskegee, AL, USA; 5Pathology & 16 Laboratory Medicine, Neurosurgery, University of Texas Health Science center, Houston, 17 6Augusta University, Augusta, GA, USA; 7Scott Department of Urology, Baylor College of 18 Medicine, Houston, TX, USA; 8Center for Cancer Research, National Cancer Institute, Bethesda, 19 MD, USA; 9Otolaryngology-Head & Neck Surgery, Baylor College of Medicine, Houston, TX, 20 USA; 10Division of Clinical and Translational Sciences, Department of Internal Medicine, 21 McGovern Medical School at The University of Texas Health Science Center, Houston, TX, 22 USA; 11Department of Urology, University of Texas Southwestern, Dallas, TX, USA; 12Verna and 1 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 7, 2019; DOI: 10.1158/1078-0432.CCR-18-1515 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 23 Marrs McLean Department of Biochemistry and Molecular Biology, 13Center for Precision 24 Environmental Health, Baylor College of Medicine, Houston, TX, USA; ^authors contributed 25 equally. 26 #Primary Corresponding author: 27 Nagireddy Putluri, Ph.D. 28 Department of Molecular and Cellular Biology 29 Baylor College of Medicine, Houston, TX 77030, Tel.: (713) 798 3139 30 Email: [email protected] 31 32 $Correspondence for Bioinformatics: 33 Cristian Coarfa, Ph.D. 34 Dan L Duncan Comprehensive Cancer Center 35 Center for Precision Environmental Health 36 Department of Molecular and Cellular Biology 37 Baylor College of Medicine, Houston, TX 77030 38 E-mail: [email protected] 39 Conflict of interest: Authors do not have any conflict of interest. 40 Key words: Multi-omics integration analysis, Metabolomics and Lipidomics, CPT1B, 41 Fatty acid oxidation. 42 Running title: OMICS: Identification of CPT1B role in Bladder Cancer 43 Funding support: Supported by American Cancer Society (ACS) Award 127430-RSG- 44 15-105-01-CNE (N.P.), NIH/NCI R01CA220297 (N.P), and NIH/NCI R01CA216426 45 (N.P.). 46 Translational Relevance 47 Cancer metabolism varies depending on the tumor grade. Currently, there is an absence of 48 multi-OMICs data integration to predict bladder cancer (BLCA) survival. To fill in this gap, we 49 performed unbiased metabolomics and lipidomics analyses of matched BLCA tissues and 50 integrated them with BLCA transcriptomics analyses to generate an integrated gene signature 51 that was associated with patient survival in multiple BLCA cohorts. Our results revealed altered 52 metabolic differences between high-grade BLCA and low-grade BLCA and suggest that 53 impaired fatty acid β-oxidation (FAO) due to the downregulation of CPT1B plays a crucial role 54 in the progression of low grade to high grade BLCA. CPT1B overexpression in high-grade 55 BLCA cell lines reduced cell proliferation, epithelial-mesenchymal transition, and metastasis to 56 the liver in vivo by increasing FAO. Our metabolic-centered multi-OMICs based integrative 57 analysis provides a system-level perspective on BLCA and potential targets for novel 58 therapeutics against high-grade BLCA. 2 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 7, 2019; DOI: 10.1158/1078-0432.CCR-18-1515 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 59 Abstract 60 Purpose: The perturbation of metabolic pathways in high-grade BLCA has not been 61 investigated. We aimed to identify a metabolic signature in high-grade BLCA by 62 integrating unbiased metabolomics, lipidomics, and transcriptomics to predict patient 63 survival and to discover novel therapeutic targets. 64 Experimental design: We performed high-resolution liquid chromatography mass 65 spectrometry (LC-MS) and bioinformatic analysis to determine the global metabolome 66 and lipidome in high-grade BLCA. We further investigated the effects of impaired 67 metabolic pathways using in vitro and in vivo models. 68 Results: We identified 519 differential metabolites and 19 lipids that were differentially 69 expressed between low-grade and high-grade BLCA using the NIST MS metabolomics 70 compendium and lipidblast MS/MS libraries, respectively. Pathway analysis revealed a 71 unique set of biochemical pathways that are highly deregulated in high-grade BLCA. 72 Integromics analysis identified a molecular gene signature associated with poor patient 73 survival in BLCA. Low expression of CPT1B in high-grade tumors was associated with 74 low FAO and low acyl carnitine levels in high-grade BLCA which were confirmed using 75 tissue microarrays. Ectopic expression of the CPT1B in high-grade BLCA cells led to 76 reduced EMT in in vitro, and reduced cell proliferation, EMT, and metastasis in vivo. 77 Conclusions: Our study demonstrates a novel approach for the integration of 78 metabolomics, lipidomics, and transcriptomics data, and identifies a common gene 79 signature associated with poor survival in BLCA patients. Our data also suggest that 80 impairment of FAO due to down-regulation of CPT1B plays an important role in the 81 progression towards high-grade BLCA and provide potential targets for therapeutic 82 intervention. 83 84 85 86 87 88 89 3 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 7, 2019; DOI: 10.1158/1078-0432.CCR-18-1515 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 90 Introduction 91 Bladder cancer (BLCA) causes significant morbidity and mortality worldwide. In 92 the United States, there were about 79,000 cases and 17,000 deaths due to BLCA in 93 20171. In the past few decades advances in cancer genomics, transcriptomics, 94 proteomics, and metabolomics resulted in the discovery of potential biomarkers for 95 cancer2,3. Unfortunately, most of the biomarkers have failed to demonstrate superior 96 performance characteristics compared with existing clinical tests. Recent advancements 97 in OMICs technologies have ushered in a new era of targeted cancer therapies by 98 improving our understanding of molecular carcinogenesis. OMICs studies have 99 identified therapeutic targets and resulted in the successful development of new drugs 100 to treat various solid tumors like breast cancer and lung cancer4,5. For BLCA, 101 prognostication and treatment still depend mainly on pathological and clinical 102 characteristics6. Patients diagnosed with high-grade invasive disease are difficult to 103 treat effectively and have a relatively low life-expectancy despite available multimodal 104 therapies. Therefore, novel prognostic and therapeutic targets against BLCA are 105 needed. 106 Tumor progression relies on the reprogramming of cellular metabolism7. Our 107 previous metabolomic and lipidomic studies highlighted the significance of altered 108 xenobiotic and fatty acid metabolism in BLCA development8-10. However, we still know 109 little about the altered metabolic pathways during the transition from low grade to high 110 grade BLCA. The integration of OMICS facilitates the study of interactions among all 111 classes of biomolecules that can occur in cell, which in turn, determines the cellular 112 physiology and behavior. Treatment for high-grade muscle invasive bladder cancer 113 (MIBC) has not advanced beyond cisplatin-based combination chemotherapy in the last 114 three decades and very few drugs for the disease have been approved recently11. The 115 identification of markers to predict poor outcomes among patients with BLCA will 116 improve our ability to identify patients who might benefit from adjuvant therapy. 117 Furthermore, a greater understanding of the metabolic molecular mechanisms of BLCA 118 progression and the identification of novel therapeutic targets will improve outcomes for 119 patients with high-grade BLCA. 4 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 7, 2019; DOI: 10.1158/1078-0432.CCR-18-1515