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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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 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 To my beloved Sadguru Shirdi Sairam & Parents Smt. M. Parvathi & Shri. S.M. Vijaya Chandra 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..........................................................................
Publié le : samedi 1 janvier 2011
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Source : D-NB.INFO/1015474802/34
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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  
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
To my beloved
Sadguru Shirdi Sairam
&
Parents
Smt. M. Parvathi & Shri. S.M. Vijaya Chandra
CONTENTS 1. ZUSAMMENFASSUNG 1 2. SUMMARY. 2 3. INTRODUCTION.. 33.1 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
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 byFACS..... 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 8.2Units.................................................................................................................................678.3 Dimensions.......................................................................................................................67 8.4 Coding DNA sequence of APC....................................................................................... 68 8.5 Vector Maps.....................................................................................................................71 9. PUBLICATIONS................................................................................................................ 74 ACKNOWLEDGEMENTSCURRICULUM VITAE
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.
1
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
3
3. INTRODUCTION
function leading to an aberrant activation of the Wnt/-catenin signaling is considered as a key step in the initiation of colon adenoma formation (Fearon and Vogelstein, 1990). 3.2 The Wnt signaling pathway  The Wnt signaling pathway is highly conserved among animal species and plays important roles in cellular proliferation, differentiation, and migration. The Wnt ligands, a large family of cysteine-rich glycoproteins, are involved in regulating at least three distinct pathways: the canonical Wnt signaling pathway (-catenin dependent), planar cell polarity pathway and Ca2+pathway (Wodarz and Nusse, 1998; Veeman et al., 2003; Nelson and Nusse, 2004).  The canonical Wnt pathway has been extensively studied. This pathway mainly involves cytosolicdegradation and its regulation by Wnt-catenin phosphorylation and ligands. It controls a wide variety of processes like tissue-specific cell fate decisions including axis formation, anterior posterior patterning and development of the central nervous system during embryogenesis (reviewed in Logan and Nusse, 2004) as well as maintenance of tissue homeostasis in adults.  The key effector molecule of the canonical Wnt signaling pathway is-catenin. It is constitutively expressed and functions as a component of adherens junctions and also as a transcriptional co-activator for the TCF/LEF-1 family of proteins. In this pathway, in the absence of exogenous Wnt ligands, a multi-protein complex called the-catenin destruction complex functions in maintaining low levels of-catenin by constitutively targeting it for degradation in proteasomes (Seidensticker and Behrens, 2000; Polakis, 2000). This protein complex is assembled over the scaffold protein Axin/Conductin (Behrens et al, 1998; Hart et al, 1998) which has binding sites for-catenin, the tumor suppressor APC, and the kinases glycogen synthase kinase 3 beta (GSK-3 ) and casein kinase 1 (CK1). In this complex,-catenin is sequentially phosphorylated at its serine and threonine residues by CK1 at position Ser45, as a priming step, followed by GSK-3at positions Ser33, Ser37 and Thr41 (Peifer et al., 1994; Yost et al., 1996; Amit et al., 2002). Phophorylated-catenin is recognized and ubiquitilated by-TrCP (beta-Transducin repeat-Containing Protein), an E3 ubiquitin ligase (Marikawa et al., 1998) and subsequently degraded in proteasomes. This process maintains the cytosolic levels of-catenin to an extent that is not sufficient to permit its accumulation in the nucleus to higher levels. WithoutTCF/LEF bound to target gene promoters act as-catenin, repressors (Molenaar et al., 1996).
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3. INTRODUCTION
Figure 1.signaling pathway. Left panel; In the absence of Wnt ligands, theThe Canonical Wnt β-catenin destruction complex is active and promotes GSK-3β and CK1 mediated phosphorylation ofβits degradation in the proteasome. Right panel; In the-catenin, leading to presence of Wnt ligands, theβ-catenin destruction complex is disrupted resulting in the stabilization of cytoplasmicβ-catenin, which translocates to the nucleus and in association with the TCT/LEF transcription factors triggers a mitogenic effect.  In the presence of Wnt ligands, the destruction complex is deactivated, leading to the stabilization ofprocess involves the binding of the secreted cysteine-rich Wnt-catenin. This ligands to the seven-pass transmembrane receptor Frizzled (Fz) (Bhanot et al., 1996; Yang-Snyder et al., 1996; He et al., 1997) and co-receptor, the low-density lipoprotein receptor related protein 5 or 6 (LRP5/6) (He et al., 2004) forming a ternary complex which recruits the scaffolding protein Dishevelled (Dvl) at the plasma membrane (Wong et al., 2003). Then Axin and GSK-3 get recruited to this Dvl assembly which facilitates the phosphorylation of LRP5/6 by GSK-3 (Zengresults in the disruption of the et al., 2008). This -catenin destruction complex and release and stabilization of-catenin which enters the cell nucleus and in association with the TCF/LEF-1 transcription factors activates the transcription of Wnt target genes (Behrens et al., 1996; Molenaar et al., 1996).
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3. INTRODUCTION
 Since Wnt/-catenin signaling controls diverse functions, its target genes for transcription are also diverse and cell- and context-specific (Logan and Nusse, 2004). This is achieved by the capacity of-catenin to associate with a plethora of co-activators including BCL9 and Pygopus, Mediator (for transcription initiation), p300/CBP and TRRAP/TIP60 histone acetyltransferases, MLL1/2 histone methyltransferases, the SWI/SNF family of ATPases for chromatin remodeling and PAF1 complex for transcription elongation and histone modifications (Mosimann et al., 2009; Willert and Jones, 2006). To mention some of the well know target genes: c-myc (He et al., 1998) and cyclin D1 (Tetsu and McCormick, 1999) regulate cell cycle progression, Survivin, Serum glucocorticoid kinase 1 (SGK1) (Zhang et al., 2001; Dehner et al., 2008) are involved in apoptosis regulation.-catenin also regulates the expression of Wnt signaling components resulting in an autoregulation of the pathway. It induces the expression of Axin2/Conductinn, Dikkopf 1 (Dkk1) and Naked (a Dvl antagonist) and suppresses the expression of Fz and LRP6 exerting a negative feedback loop to weaken Wnt signaling (Behrens et al., 1998; Chamorro et al., 2005; Kazanskaya et al., 2004; Khan et al., 2007) while its induction of Rspo and TCF/LEF genes causes a positive feed-forward effect to strengthen Wnt signaling (Hoppler and Kavanagh, 2007).  Mutations in the components of Wnt signaling pathway can result in its constitutive activation and this is associated with many hereditary disorders, cancer and other diseases (MacDonald et al., 2009). Deregulated Wnt/-catenin signaling has been strongly associated with cancer, in particular with colorectal cancer (Polakis, 2007) where the gene for-catenin (CTNNB1) or tumor suppressor gene APC is most frequently mutated with rare reports of mutations in Axin1 and Axin2 (Liu et al., Nature Genetics 2000; Segditsas and Tomlinson, 2006). Mutations in the CTNNB1 gene that account for up to 7% of sporadically occurring colorectal cancers, affect the Ser/Thr residues in its N-terminal region, those are targets for phosporylation by the-catenin destruction complex, resulting in stabilization of-catenin and constitutive activation of the pathway (Ilyas et al., 1997 and Morin et al., 1997). However, truncating mutations in the APC gene are the most common, that account for ~80% of sporadic human colon cancers and is the cause of hereditary form of colon cancer called Familial Adenomatous Polyposis (FAP) (Fodde 2002; Su et al., 1992). 3.3 Adenomatous Polyposis Coli  The search for the genetic basis of FAP lead to the initial discovery of the APC gene locus, showing its localization to human chromosome position 5q21 (Herrera et al., 1986; Joslyn et al., 1991; Kinzler et al., 1991; Nishisho et al., 1991), and its identification and
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