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IDENTIFYING the MOLECULAR MECHANISMS of EARLY CACHEXIA USING WHOLE TRANSCRIPTOME SEQUENCING in MUSCLE and FAT BIOPSIES from CANCER PATIENTS Amal Hussain Al Hadad

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IDENTIFYING the MOLECULAR MECHANISMS of EARLY CACHEXIA USING WHOLE TRANSCRIPTOME SEQUENCING in MUSCLE and FAT BIOPSIES from CANCER PATIENTS Amal Hussain Al Hadad United Arab Emirates University Scholarworks@UAEU Dissertations Electronic Theses and Dissertations Summer 6-1-2015 IDENTIFYING THE MOLECULAR MECHANISMS OF EARLY CACHEXIA USING WHOLE TRANSCRIPTOME SEQUENCING IN MUSCLE AND FAT BIOPSIES FROM CANCER PATIENTS Amal Hussain Al Hadad Follow this and additional works at: https://scholarworks.uaeu.ac.ae/all_dissertations Part of the Medical Physiology Commons Recommended Citation Al Hadad, Amal Hussain, "IDENTIFYING THE MOLECULAR MECHANISMS OF EARLY CACHEXIA USING WHOLE TRANSCRIPTOME SEQUENCING IN MUSCLE AND FAT BIOPSIES FROM CANCER PATIENTS" (2015). Dissertations. 5. https://scholarworks.uaeu.ac.ae/all_dissertations/5 This Dissertation is brought to you for free and open access by the Electronic Theses and Dissertations at Scholarworks@UAEU. It has been accepted for inclusion in Dissertations by an authorized administrator of Scholarworks@UAEU. For more information, please contact [email protected]. Title United Arab Emirates University College of Medicine and Health Sciences IDENTIFYING THE MOLECULAR MECHANISMS OF EARLY CACHEXIA USING WHOLE TRANSCRIPTOME SEQUENCING IN MUSCLE AND FAT BIOPSIES FROM CANCER PATIENTS Amal Hussain Ibrahim Al Haddad This dissertation is submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy Under the Supervision of Professor Thomas E. Adrian June 2015 ii Declaration of Original Work I, Amal Hussain Ibrahim Al Haddad, the undersigned, a graduate student at the United Arab Emirates University (UAEU), and the author of this dissertation entitled “Identifying the Molecular Mechanisms of Early Cachexia Using Whole Transcriptome Sequencing in Muscle and Fat Biopsies from Cancer Patients”, hereby, solemnly declare that this dissertation is an original research work that has been done and prepared by me under the supervision of Professor Thomas E. Adrian, in the College of Medicine and Health Sciences at UAEU. This work has not been previously formed as the basis for the award of any academic degree, diploma or a similar title at this or any other university. The materials borrowed from other sources and included in my dissertation have been properly cited and acknowledged. Signature Date iii Copyright Copyright © 2015 Amal Hussain Ibrahim Al Haddad All Rights Reserved iv Approval of the Doctorate Dissertation This Doctorate Dissertation is approved by the following Examining Committee Members: 1) Advisor (Committee Chair): Thomas E. Adrian Title: Professor Department of Physiology College of Medicine and Health Sciences Signature Date 2) Member: Milos Ljubisavljevic Title: Professor Department of Physiology College of Medicine and Health Sciences Signature Date 3) Member: Samir Attoub Title: Associate Professor Department of Pharmacology and Therapeutics College of Medicine and Health Sciences Signature Date 4) Member (External Examiner): Jörgen Larsson Title: Professor, Dean Emeritus, CLINTEC [Division of Surgery] Director, International Programs, Karolinska Institute Department of Development and Innovation Institution: Karolinska University Hospital, Stockholm, Sweden Signature Date v This Doctorate Dissertation is accepted by: Dean of the College of Medicine and Health Sciences: Professor Dennis Templeton Signature Date Dean of the College of the Graduate Studies: Professor Nagi T. Wakim Signature Date Copy ____ of ____ vi Abstract Cancer cachexia is responsible for one third of cancer–related deaths and contributes to the death of many others. More than 80% of cancer patients are cachectic towards the end of life. Despite intensive research, the mechanisms of cancer cachexia are still poorly understood. It is our hypothesis that identification of early changes in gene expression in cachexia will lead to an improved understanding of the mechanism that trigger this important problem in cancer patients. Thus, to shed light on the mechanisms involved in the major cachexia target tissues, we investigated the entire transcriptome in muscle and fat to identify altered expression of genes in cancer patients with and without cachexia. Samples of rectus abdominis muscle and visceral fat were collected at surgery from patients exhibiting 5-10% weight loss prior to surgery, compared with stable-weight patients. Analysis of all expressed genes was carried out using next generation sequencing (Illumina HiSeq 2500). Also, selected differentially expressed genes were confirmed using real time RT-PCR. In muscle, 30 genes showed highly significant changes in expression (25 downregulated and 5 upregulated: P<0.0005 - P<0.00001, FDR 0.2). Analysis of the 25 downregulated genes involved included 7 that are involved with metabolism (5 of which are mitochondrial); 4 with signaling; 4 with ubiquitination; and 3 with intracellular trafficking. There was marked downregulation of multiple genes involved in glycogen metabolism which correlates with the lack of glycogen, muscle weakness, and fatigue; characteristic of cachexia. The 5 upregulated genes include 2 involved with calcium signaling and 2 with cell matrix interactions. Expression of genes previously thought to be important in cachexia, including several inflammatory cytokines, was not significantly different. FBXO32, which encodes atrogin-1, upregulated in an in vitro cachexia model, was actually downregulated. No transcripts for the dermicidin gene, which contains the sequence that codes for the backbone peptide of proteolysis-inducing factor, were detected. Expression of myostatin was significantly decreased as was its receptor (ACVR2B), possibly reflecting end organ adaptation to tumor produced myostatin. vii In visceral fat, expression of 6 genes were downregulated and 10 upregulated with high statistical significance (P<0.001-0.0002). Several of these encode metabolic enzymes. Of genes in fat previously implicated in cachexia, such as hormone sensitive lipase and adipose tissue triglyceride lipase, were unchanged. In contrast, leptin was significantly downregulated and the zinc-α-2-glycoprotein (lipid mobilizing factor) was significantly upregulated as expected. These studies confirmed that for a multifactorial condition, genome wide transcriptome analysis is the method of choice to explore the disease complexity. They explain some documented evidence in cachexia pathogenesis, highlight ambiguous data from animal models, and reveal unexpected changes in gene expression that underlie the pathophysiology of the cachectic state in cancer. These results bring reliable, representable, and consistent data from the clinic and back to the bench with more focused insights to be investigated and verified. Keywords: Cancer cachexia, wasting, skeletal muscle, adipose tissue, RNA sequencing, real-time RT-PCR. viii Title and Abstract (in Arabic) تحديد اﻵليبت الجزيئية للمرحلة اﻷولية للهزال بدراسة تسلسل كبمل التعبيرات الجينية مه خزعبت عضلية ودهنية مه مرضى مصببيه ببلسرطبن الملخص الهزال أو الدنف المصاحب للسرطان مسإول بشكل مباشر عن ثلث وفٌات السرطان، وهو أحد المسببات لباقً الوفٌات. ٌعانً أكثر من 80% من مرضى السرطان من الهزال فً أٌامهم اﻷخٌرة. وبالرغم من اﻷبحاث المكثفة، فإن آلٌات الهزال لمرضى السرطان ﻻ زالت مجهولة. لقد تم بناء هذه الدراسة على فرضٌة أن تحدٌد متغٌرات التعبٌر الجٌنً فً المراحل اﻷولٌة للهزال ستساهم فً تطوٌر الفهم لﻵلٌات المحفزة لهذه المشكلة عند مرضى السرطان. لذلك، فإن الهدف من هذه اﻷطروحة هو تسلٌط الضوء على اﻵلٌات المسببة للدنف فً اﻷنسجة المستهدفة، عن طرٌق دراسة كامل التعبٌرات الجٌنٌة "الترانسكرٌبتوم" )Transcriptome( فً اﻷنسجة العضلٌة والدهنٌة، بهدف تحدٌد الجٌنات المتماٌزة تعبٌرٌا لمرضى السرطان اللذٌن ٌعانون من الهزال، ومقارنتهم بمرضى سرطان ﻻ ٌعانون منه. تم جمع عٌنات من العضﻻت القطنٌة )rectus abdominis) والدهون الحشوٌة )visceral fat( أثناء العملٌات المجدولة للمرضى الذٌن خسروا 5-10% من أوزانهم قبل إجراء العملٌة، وتمت مقارنتها بآخرٌن من الذٌن بقٌت أوزانهم ثابتة. تم تحلٌل مستوى التعبٌر الجٌنً بدراسة تسلسل كافة الجٌنات بواسطة تقنٌات عالٌة اﻹنتاجٌة )Next generation sequencing( باستخدام جهاز ) Illumina HiSeq 2500(. كما وتم تؤكٌد النتائج على مجموعة مختارة من الجٌنات باستخدام تفاعل البولٌمٌراز المتسلسل اللحظً (Real-time RT-PCR). أظهرت نتائج الدراسة فً اﻷنسجة العضلٌة تغٌرات هامة للغاٌة فً التعبٌرات ل 30 جٌن )25 منها مثبط، بٌنما 5 منها مفعل: القٌمة اﻻحتمالٌة (FDR 0.2, P˂0.0005-0.00001(. تحلٌل ال 25 جٌن المثبط أظهر أن: 7 منها ٌساهم فً عملٌات اﻷٌض )metabolism( )5 من هذه السبعة عملٌات أٌض فً المٌتوكوندرٌا(، 4 فً تؤشٌر الخلٌة )signaling(، 4 فً عملٌات التحلٌل البروتٌنً )ubiquitination(، و 3 فً حركة سٌر البروتٌنات داخل الخﻻٌا )intracellular trafficking(. كما أظهرت الدراسة تثبٌطا واضحا للعدٌد من الجٌنات المشاركة بؤٌض الجﻻٌكوجٌن، والتً بدورها تفسر سبب نقص الجﻻٌكوجٌن بالعضﻻت وما ٌصاحبه من ضعف وإنهاك، وهً أعراض أساسٌة مصاحبة للهزال. فً المقابل، فإن تحلٌل ال 5 جٌنات المفعلة أظهر أن: 2 منها ٌساهم فً تؤشٌر الكالسٌوم، و 2 بتفاعﻻت نسٌج الخلٌة الخارجً. من ناحٌة أخرى، فإن بعض التعبٌرات الجٌنٌة للسٌتوكٌنات المسببة لﻻلتهاب – والتً كان ٌُعتقد حسب دراسات سابقة أن لها تؤثٌرا فً الهزال – لم تظهر أي تغٌٌر ٌُذكر. كما أن الجٌن المﻻحظ تفعٌله بالعدٌد من الدراسات المختبرٌة )in vitro( وهو )FBXO32; atrogin-1( وجد مثبطا. كما لم ٌتم الكشف عن أي تعبٌر جٌنً لجٌن ال )dermicidin(. كما أن التعبٌر الجٌنً لجٌن ال )myostatin( باﻹضافة للمستقبل الذي ٌعمل علٌه )ACVR2B(، وجدا مثبطٌن على غٌر المتوقع، مما قد ٌعكس احتمالٌة وجود تكٌف من اﻷنسجة العضلٌة لتؤثٌر ال )myostatin( المنتج من الورم السرطانً. ix أما بخصوص النسٌج الدهنً الحشوي ، فقد أظهرت نتائج الدراسة تغٌرات هامة للغاٌة فً التعبٌرات ل 16 جٌن )6 منها مثبط، بٌنما 10 منها مفعل: القٌمة اﻻحتمالٌة (P˂0.001-0.0002(. تحلٌل هذه الجٌنات أظهر مشاركتها اﻹنزٌمٌة فً العملٌات اﻷٌضٌة. مقارنة نتائج هذه
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