Functional analysis of truncated APC protein in human colorectal cancers [Elektronische Ressource] = Funktionelle Analyse von verkürztem APC Protein in humanem kolorektalen Krebs / Shree Harsha Vijaya Chandra. Betreuer: Jürgen Behrens

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• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2011
• Nombre de lectures: 44
• Poids de l'ouvrage: 1 Mo

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   Functional analysis of truncated APC protein in human colorectal cancers

Funktionelle Analyse von verkürztem APC Protein in humanem kolorektalen Krebs       Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat.       vorgelegt von  Shree Harsha Vijaya Chandra  aus Bangalore, India        

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 Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg          Tag der mündlichen Prüfung: 28.07.2011 Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink Erstberichterstatter: Prof. Dr. Jürgen Behrens Zweitberichterstatter: Prof. Dr. Thomas Winkler

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To my beloved

Sadguru Shirdi Sairam

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Parents

Smt. M. Parvathi & Shri. S.M. Vijaya Chandra

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CONTENTS 1. ZUSAMMENFASSUNG 1 2. SUMMARY. 2 3. INTRODUCTION.. 3  3.1 Colorectal cancer. 3 3.2 The Wnt signaling pathway. 4 3.3 Adenomatous Polyposis Coli... 6 3.4 Mutations of APC in colorectal cancers.. 8 3.5 Aim of the project 10 4. RESULTS............................................................................................................................ 11 4.1 Analysis of the requirement of truncated APC for proliferation and tumor forming ability of CRC cells..11 4.1.1 Truncated APC controls proliferation of CRC cell lines 11 4.1.2 Truncated APC controls tumor development. 14 4.2 Analysis of the molecular functions of truncated APC of human CRC cells.. 17 4.2.1 Functional analysis of the activity of-catenin inhibitory domain (CID) in different CRC cell lines  ... 17 4.2.2 Down-regulation of endogenous truncated APC and its effects on-catenin levels and transcriptional activity in CRC cells.. 20 4.2.2.1 Truncated APC controls-catenin levels / wnt signaling in CRC cells........ 20 4.2.2.2 Truncated APC controls phosphorylated 23-catenin levels in CRC cells... 4.3 Allele-specific RNA interference to target truncated APC in CRC.... 25 4.3.1 Dicer-substrate RNA targeting APC mutation in DLD1 cell line.. 25 4.3.2 Luciferae reporter-based assay to screen and validate allele-specific shRNA... 27 5. DISCUSSION.. 31 6. MATERIALS AND METHODS... 37 6.1 MATERIALS...37 6.1.1 Chemicals37 6.1.2 Enzymes.. 37 6.1.3 Buffers.38 6.1.4 Commercial Kits. 40 6.1.5 Antibodies  ... 40 6.1.6 Organisms and Media. 40 6.1.7 Oligonucleotides. 41

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6.1.8 Plasmids.. 46 6.1.9 Technical equipment & Other Material.. 47 6.2 METHODS.. 48 6.2.1 Standard molecular biology procedures  .. 48 6.2.2 Cloning48 6.2.3 Cell culture..50 6.2.4 Production of Lentiviral supernatants. 51 6.2.5 Transduction of CRC cell lines & sorting of EGFP expressing cells by  FACS..... 51 6.2.6 Transient transfections 52 6.2.7 Reporter assays... 52 6.2.8 Cell proliferation assay... 54 6.2.9 Preparation of cell extracts and western blotting. .. 55 6.2.10 Xenograft experiments..55 6.2.11 Semi-quantitative RT-PCR... 56 7. LITERATURE.................................................................................................................... 58 8. ANNEX................................................................................................................................ 66 8.1 Abbreviations................................................................................................................... 66 8.2Units.................................................................................................................................67 8.3 Dimensions..................................................................................................................... .. 67 8.4 Coding DNA sequence of APC....................................................................................... 68 8.5 Vector Maps.....................................................................................................................71 9. PUBLICATIONS................................................................................................................ 74 ACKNOWLEDGEMENTS CURRICULUM VITAE         

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1. ZUSAMMENFASSUNG

1. ZUSAMMENFASSUNG  Mutationen im APC(Adenomatöse Polyposis Coli)-Gen sind ein Markenzeichen beim kolorektalen Krebs (KRK). Diese sind ein frühes Ereignis während der Tumorigenese und tragen durch eine fehlgesteuerten Aktivierung des Wnt--Catenin-Signalwegs zu einer unkontrollierten Proliferation der Kolonepithelzellen bei. APC ist ein großes Protein mit zahlreichen Domänen. Als ein multifunktionelles Protein ist es an einer Vielzahl von zellulären Prozessen z.B. der Regulation der Zellproliferation, der Zelladhäsion, der Zellmigration, der Organisation des Zytoskeletts und der Stabilität von Chromsomen beteiligt. APC-Mutationen sind biallelisch, trotzdem führen diese Mutationen nicht zu einem totalen Verlust des Proteins, sondern alle kolorektalen Tumore exprimieren noch ein verkürztes N-terminales APC-Protein. Der Grund für die Erhaltung dieses N-terminalen APC-Fragmentes in den KRK ist bisher noch nicht vollständig verstanden. Das Ziel dieser Arbeit ist es, die funktionelle Relevanz dieses verkürzten APC- Proteins beim KRK herauszufinden. Mittels RNA-Interferenz wurden die verkürzten APC- Fragmente in den kolorektalen Krebszelllinien herunterreguliert. Dabei konnte ich zeigen, dass verkürztes APC essentiell für die Proliferation dieser Zelllinienin vitro ist. Tatsächlich konnte dies bei mehreren, unterschiedlichen kolorektalen Krebszelllinien demonstriert werden, welche unterschiedliche Varianten des verkürzten APCs tragen. Noch bedeutsamer ist die Tatsache, dass ich durch Xenotransplantations Experimente in Nacktmäusen nachweisen konnte, dass ein verkürztes APC für die Bildung von Tumoren durch kolorektale Tumorzellen benötigt wird. Zusätzlich analysierte ich die Funktion einer neu identifizierten APC-Domäne mit dem Namen ß-catenin inhibitory domain (CID) in verschiedenen kolorektalen Krebszellen und fand heraus, dass es eine Gegenselektion für diese Domäne in KRK gibt. Außerdem konnte ich zeigen, dass die verkürzten APC-Isoformen in KRK-Zellen immer noch die Fähigkeit besitzen, die Menge an-Catenin und seine transkriptionelle Aktivität negativ zu regulieren. Dieser Befund unterstützt die "just right signaling-Hypothese, welche davon ausgeht, dass verkürzte APC-Mutationen dahingehend selektioniert werden, eine optimale Aktivität von-Catenin zu gewährleisten, welche der Tumorentwicklung förderlich ist.  Diese Ergebnisse verdeutlichen eine funktionelle Relevanz für die Erhaltung des verkürzten APCs in KRK und machen es zu einem Ziel einer möglichen therapeutischen Intervention. Mit dieser therapeutischen Perspektive habe ich daher shRNAs entworfen und getestet, die gegen spezifische APC-Mutationen gerichtet waren, welche in verschiedenen kolorektalen Krebszellen gefunden wurden. Tatsächlich konnte ich nachweisen, dass es möglich ist eine Allel-spezifische RNA-Interferenz gegen verkürztes APC zu erreichen.

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2. SUMMARY

2. SUMMARY  Mutations of the Adenomatous Polyposis Coli (APC) gene is a hallmark of colorectal cancers (CRC). This is an early event in the process of tumorigenesis that contributes to an uncontrolled proliferation of the colonic epithelial cells by abberantly activating the Wnt- catenin signaling pathway.  APC is a large protein with multiple domains and serves as a multifunctional protein involved in a variety of cellular processes such as regulation of cell proliferation, cell adhesion, cell migration, cytosketal organization and chromosomal stability.APCmutations in colorectal tumors are biallelic, however, these mutations do not lead to the complete absence of the protein. Invariably almost all of the colorectal tumors express truncated N-terminal portion of the APC protein. For what reasons the N-terminal portion of the APC protein is retained by colorectal tumors is not completely understood. This project aimed to understand the functional significance of truncated APC in colorectal cancers. Using RNA interference techniques to down-regulate truncated APC in CRC cell lines I could show that truncated APC is required for the proliferation of colorectal cancer cell linesin vitro. In fact this was demonstrated in a set of distinct CRC cell lines harbouring different kinds of truncating APC mutations. More significantly, it could be shown that truncated APC is required for tumor formation by colorectal cancer cells, as demonstrated by the Xenograft experiments in nude mice.  In addition to this, I analyzed the function of a newly identified domain of APC called the (CID) in different CRC cell lines and found that there is a-catenin inhibitory domain counter selection for this domain in colorectal cancers. Furthermore, I could show that truncated APC isoforms in CRC cells still retains the ability to negatively regulate the levels and transcriptional activity of-catenin. This finding is supportive of the just right signaling hypothesis which advocates that truncating APC mutations are selected to maintain an optimal level of-catenin transcriptional activity that is conducive for tumor development.  These results reveal a functional significance for the retention of truncated APC in CRC and hence project it as a possible therapeutic target. Therefore, with a therapeutic perspective, I designed and tested shRNAs to specifically target APC mutations found in different CRC cell lines. In fact it could be demonstrated that it is possible to achieve an allele-specific RNA interference targeting truncated APC.    

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3. INTRODUCTION

3. INTRODUCTION  Cancer is a major cause of death worldwide. It is caused by a series of mutations in multiple genes followed by the selective outgrowth of the mutant progeny cells with proliferative and survival advantage. Colon cancer is one of the leading causes of deaths in the western countries although it can be easily and effectively treated if detected early, while chemoprevention has been moderately successful in reducing the disease. (Gustin & Brenner 2002). A hallmark of colon cancer is that most tumors have mutations in a single gene which encodes for the tumor suppressor Adenomatous Polyposis Coli (APC) protein. These are truncating mutations found in a majority of sporadic colon cancers and are also the cause of an inherited form of colon cancer called Familial Adenomatous Polyposis (FAP) (Gorden et al., 1991; Kinzler et al. 1991).  3.1 Colorectal cancer  The inner surface of a healthy human colon is lined by an epithelium that invaginates frequently to form pit-like structures called crypts. The bottom of the crypts harbour stem cells that divide actively giving rise to transit-amplifying cells that further proliferate under stimulation from Wnt family growth factors present around the crypt bottom and migrate upwards towards the lumen of the colon. Before they reach the neck of the crypt, they stop proliferating, as they could be far away from the Wnt source, while they differentiate. The differentiated cells reside in the epithelium only for 3-5 days after which they apoptose and are exfoliated, making room for newer cells. In colorectal cancer the epithelial cells acquire mutations that confer them with inappropriate proliferative and/or survival capacity thus disrupting the balance between proliferation and apoptosis. This uncontrolled proliferation of the mutated epithelial cells leads to the initial formation of small bud-like protrusions called adenomatous polyps, which could further progress into invasive tumors (adenocarcinomas) and finally metastasize (Reya and Clevers, 2005; Potten and Loeffler, 1987).  Studies to understand the genetic basis of colorectal tumor development emphasise a temporal series of genetic alterations involving several tumor suppressor genes and oncogenes as well as epigenetic changes (Vogelstein et al., 1988; Kinzler and Vogelstein, 1996). The adenomatous polyposis coli (APCsuppressor gene involved in the negative) gene, a key tumor regulation of the Wnt/-catenin signaling pathway, is found to be mutated in familial adenomatous polyposis (FAP) (Gorden et al., 1991; Kinzler et al. 1991) and also in sporadic colorectal cancers and is an early event in tumorigenesis (Powell et al. 1992). Loss of APC

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