miRNAs in control of oncogenic signaling in breast cancer cells

miRNAs in control of oncogenic signaling

in breast cancer cells

Von der Fakultät Energie-, Verfahrens- und Biotechnik der Universität

Stuttgart zur Erlangung der Würde eines Doktors der

Naturwissen-schaften (Dr. rer. nat.) genehmigte Abhandlung

Vorgelegt von

Annabell Bischoff

aus Segnitz

Hauptberichter: Prof. Dr. Monilola Olayioye

Mitberichter: Prof. Dr. Klaus Pfizenmaier

Tag der mündlichen Prüfung: 12.11.2014

Institut für Zellbiologie und Immunologie

Universität Stuttgart

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Eidesstattliche Erklärung

Hiermit erkläre ich, Annabell Bischoff, dass ich die vorliegende Arbeit selbständig angefertigt habe. Es wurden nur die in der Arbeit ausdrücklich benannten Quellen und Hilfsmittel be-nutzt. Wörtlich oder sinngemäß übernommenes Gedankengut habe ich als solches kenntlich gemacht.

I hereby assure that I performed this work independently without further help or other materi-als than stated.

______________________ ______________________

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Table of content

1.2 Development of breast cancer ...17

1.3 ErbB receptors ...18

1.4 ErbB2-ErbB3 receptor dimer ...20

1.5 PI3K/Akt signaling ...22

1.6 Ras-ERK (Extracellular Signal Regulated Kinase) and PLCγ signaling ...25

1.7 Cell motility ...26

1.8 miRNAs ...27

1.8.1 miRNA biogenesis ...27

1.8.2 miRNA targeting ...28

1.8.3 Nomenclature of miRNAs ...29

1.8.4 miRNA target prediction ...30

1.8.5 miRNAs and their biological function ...31

1.8.6 miRNAs in cancer ...31

1.8.7 Aim of the thesis ...33

2 Materials and Methods ...35

2.1 Materials ...35

2.1.1 Equipment ...35

2.1.2 Chemicals and consumables ...36

2.1.3 Buffers and solutions ...37

2.1.4 Bacterial strain ...38

2.1.5 Cell lines, cell culture medium ...38

2.1.6 Animals ...39

2.1.7 Oligonucleotides ...39

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2.2 Methods ...42

2.2.1 Cell culture ...42

2.2.2 Cell transfection ...43

2.2.3 Screening workflow ...43

2.2.4 In-Cell Western analysis (ICW) ...43

2.2.5 Plasmid Constructs – QuickChange Site directed PCR Mutagenesis ...44

2.2.6 Transformation of E.coli ...44

2.2.7 Purification of plasmid DNA (Mini-Prep) ...45

2.2.8 Preparation of plasmid DNA (Midi-Prep) ...45

2.2.9 Migration/Invasion (Transwell) Assay ...45

2.2.10 Impedance measurement ...45

2.2.11 Proliferation Assay ...46

2.2.12 Life cell imaging ...46

2.2.13 Luciferase Reporter Assay ...46

2.2.14 Rac activity assay. ...46

2.2.15 Quantitative Real Time PCR ...46

2.2.16 Cell lysis, SDS-PAGE and Western Blotting ...47

2.2.17 FACS analysis ...47

2.2.18 Immunofluorescence microscopy ...48

2.2.19 Animal experiment ...48

2.2.20 Target prediction analysis for miR-149 using KEGG...48

3 Results ...49

3.1 miRNA Screen ...49

3.1.1 Establishment of a screening procedure to monitor Akt activation upon HRG stimulation ...49

3.1.2 miR-149 serves as a positive control for the screen targeting the ErbB3 3’UTR 51 3.1.3 Genome-wide miRNA screening for regulators of HRG-induced Akt activation 54 3.1.4 Identification of a miRNA-ErbB interaction network. ...58

3.1.5 Expression of miR-148b, miR-149, miR-326 and miR-520a-3p reduces ErbB3-expression and affects Erk and Akt signaling ...61

3.1.6 Overexpression of miR-148b, miR-149, miR-326 and miR-520a-3p reduces the heregulin-driven proliferation. ...63

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3.2.1 Clinical data of miR-149 ...65

3.2.3 miR-149 expression affects cell adhesion and cell spreading ...67

3.2.5 miR-149 expression affects Rac activity ...71

3.2.6 miR-149 affects migration and invasion also in the prostate cancer cell line PC3 73 3.2.7 miR-149 expression in vivo model ...74

4 Discussion ...77

4.1 miRNA Screen ...77

4.1.1 Screening for miRNAs altering the ErbB/Akt pathway ...77

4.1.2 miRNAs alter heregulin-dependent Akt activation ...78

4.1.3 Protein target network of miRNAs negatively affecting ΔpAkt ...79

4.1.4 Protein target network of miRNAs enhancing ΔpAkt ...82

4.1.5 miRNAs regulate multiple targets within the ErbB/Akt pathway ...83

4.1.6 Clinical relevance of miR-148b, miR-149 miR-326, and miR-520a-3p ...84

4.2 miR-149 functions as a tumor suppressor by controlling breast epithelial cell migration and invasion ...85

4.2.1 miR-149 is a novel tumor suppressor in basal-like breast cancer ...85

4.2.2 miR-149 affects the activity of focal adhesion proteins and focal adhesion formation ...85

4.2.3 Overexpression of miR-149 reduces Rac activity and has anti-metastatic function in vitro and in vivo ...86

4.2.4 miR-149 downregulates the Rac effector proteins Rap1A, Rap1B and Vav2 ...87

4.2.5 Conclusions and outlook ...88

List of Figures ...89

List of Tables ...91

5 List of references ...93

6 Supplements ... 107

6.1 mimic miRNA library ... 107

6.2 Screen miRNA raw data ... 112

6.3 Lists of computationally predicted miRNA targets ... 124

Acknowledgements ... 133

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Abbreviations

% percent (per hundred)

°C degree Celsius µg microgram µL microliter µm micrometer µM micromolar 2-ME 2-mercaptoethanol A adenosine aa amino acid Ab antibody

Ago argonaute protein

Akt/PKB protein kinase B

AP alkaline phosphatase

APS ammonium persulfate

ATP adenosine triphosphate

BH3 Bcl-2 homology domain 3

bp base pairs

BSA bovine serum albumin

C cytosine

C. elegans caenorhabditis elegans

ca catalytically active

caspase cysteine-aspartic protease

Cdc24 cell division control protein 42 homolog

cDNA complementary deoxyribonucleic acid

CO2 carbon dioxide

conc. concentration

Cq quantification cycle

DAG diacylglycerol

DMEM Dulbecco's Modified Eagle Medium

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

dNTP deoxynucleotide triphosphate

DTT dithiothreitol

E. coli escherichia coli

e.g. for example (example given)

ECL enhanced chemiluminescence

EDTA ethylenediaminetetraacetic acid

EGF epidermal growth factor

eGFP enhanced green fluorescent protein

EGFR epidermal growth factor receptor

EMT epithelial mesenchymal transition

ERK extracellular regulated kinase

EtBr ethidium bromide

Exp-5 exportin-5

FA focal adhesion

FACS fluorescence activated cell sorting

FAK focal adhesion kinase

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FCS fetal calf serum

FOXO forkhead transcription factors

g gravitational acceleration

G guanine

GAP GTPase activating protein

GAPDH glyceraldehyde 3-phosphate dehydrogenase

GDI guanosine nucleotide dissociation inhibitors

GDP guanosine diphosphate

GEF guanine nucleotide exchange factor

Grb2 growth factor receptor-bound protein-2

GTP guanosine triphosphate

h hour

HEK293 human embryonic kidney cells 293

HER2 human epidermal growth factor receptor 2 (ErbB2)

HRG heregulin

HRP horseradish peroxidase

hsa homo sapiens

hsa-miR homo sapiens microRNA

IF Immunofluorescence

kDa kilo Dalton

LacZ beta-galactosidase gene in E. coli

mA milliampere

mAb monoclonal antibody

MAPK mitogen-activated protein kinase

max maximal

MEK mitogen-activated protein kinase kinase (MAPKK)

mg milligram

min minute

miR- mature miRNA

miRISC microRNA induced silencing complex

miRNA microRNA (ribonucleic acid)

ml milliliter

mM millimolar

mRNA messenger ribonucleic acid

mTOR mammalian target of rapamycin

MW molecular weight

n.s. non significant

Na3VO4 sodium orthovanadate

NaDoc sodium deoxycholate

NaF sodium fluoride

ng nanogram

nt nucleotide

OD optical density

ORF open reading frame

PBS phosphate buffered saline

PCR polymerase chain reaction

PDK1 3-phosphoinositide-dependent protein kinase 1

PFA paraformaldehyde

pH potential of hydrogen

PI3K phosphoinositide 3-kinase

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PI4P phosphatidylinositol 4-phosphate

PIP2 phosphatidylinositol 4,5-bisphosphate

PIP3 phosphatidylinositol 3,4,5-trisphosphate

PKC protein kinase C

PLCγ phosphoinositide phospholipase C γ

PMSF phenylmethylsulfonylfluorid

pre-miRNA precursor microRNA

pri-miRNA primary microRNA

PTEN phosphatase and tensin homolog

PVDF polyvinylidene difluoride

qRT-PCR quantitative real time polymerase chain reaction

Rho proteins Rho GTPase proteins (e.g. RhoA, Rac1 and Cdc42)

RIPA radio immuno precipitation assay

RISC RNA induced silencing complex

RNA ribonucleic acid

RNA pol II RNA polymerase II

RNAi RNA interference

RNase ribonuclease

rpm rotations per minute

RPMI Roswell Park Memorial Institute medium

RT room temperature

RTK receptor tyrosine kinase

s second

SDS sodium dodecyl sulfate

SDS-PAGE SDS- polyacrylamide gel electrophoresis

SEM standard error of the mean

Ser serine

siRNA small interfering RNA

SOS son of Sevenless

Src Src proto-oncogene tyrosine-protein kinase Src

T thymine

TAE tris-acetate-EDTA

TEMED N,N,N',N'-Tetramethylethylendiamin

Thr threonine

TNBC triple-negative breast cancer

Tris tris-(hydroxylmethyl)-amino methane

Tyr tyrosine

U uracil

UTR untranslated region

v/v volume/volume

w/v weight/volume

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Abstract

microRNAs (miRNAs) are 17-nt to 24-nt short non-coding RNAs that have emerged as criti-cal regulators of gene expression in almost all forms of life. miRNAs act by partial comple-mentary binding usually within the 3’-untranslated region (3’UTR) of the mRNA target result-ing in translational repression and/or mRNA degradation. Microarray and proteomic experi-ments have demonstrated the impact of a single miRNA on fine-tuning expression of a hun-dred of targets affecting a multitude of biological processes such as development, prolifera-tion and apoptosis. Deregulaprolifera-tion of miRNA funcprolifera-tion is also implicated in various diseases including the development of cancer. Furthermore, recent miRNA profiling studies conducted on different tumor types have identified sets of miRNAs that have altered expression in tumor and normal tissue, making them attractive targets for therapeutic intervention or as diagnos-tic markers. Nevertheless, target identification and detailed knowledge of miRNA functions is the key for the correct selection of miRNAs causally involved in the specific disease process.

This thesis focuses especially on the role of miRNAs in two processes that are of major in-terest to breast cancer research; the ErbB2/ErbB3/Akt signaling pathway and cancer cell motility. Prolonged ErbB2/ErbB3/Akt signaling is frequently reported in various cancers and enables the cell to bypass targeting therapies as it favors cell survival. In the case of breast cancer, this is particularly achieved by increased ErbB2/ErbB3 receptor activation. In order to investigate the extent by which miRNAs modulate the ErbB receptor signaling pathway, we performed a genome-wide screen in the breast cancer cell line MCF7 based on Akt phos-phorylation as a read-out. We identified 43 miRNAs that specifically regulate heregulin (HRG)-induced Akt activation, either positively or negatively, and revealed the complexity of coordinated miRNA-target interactions within the ErbB signaling pathway. We further validat-ed four miRNAs, miR-149, miR-148b, miR-326, and miR-520a-3p, with potential tumor sup-pressive function as novel regulators of ErbB3 transcript and protein levels. But also the ex-pression levels of other key components within the ErbB/Akt pathway were affected either on the protein or mRNA level, like Erk1/2 and PIK3CA. The selected miRNAs further efficiently blocked ErbB signaling and HRG-dependent proliferation, supporting their tumor-suppressive role. In the second part, we focused on the role of the screen hit miR-149 in regulating can-cer cell motility, a process that is of pivotal importance for the formation of metastasis espe-cially in later stages of breast cancer. Clinical data revealed that miR-149 is downregulated in various tumor types, including the basal-like breast cancer subtype. Using the basal-like MDA-MB-231 cell line as a model system and a combination of biochemical and cellular as-says, we showed that miR-149 interfered with signaling downstream of integrin receptors at multiple levels, impairing Rac activation and efficiently blocking cell migration and invasion both in vitro and in vivo. Furthermore, Rap1a, Rap1b and Vav2 were identified as potential targets of miR-149. In addition, we confirmed the observed phenotype in other basal-like breast cancer cell lines, therefore providing evidence that miR-149 has broader tumor-suppressive function.

Taken together, this study demonstrates a new role for several miRNAs in regulating cancer-associated pathways and further broadens our knowledge of their functions.

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Zusammenfassung

microRNAs (miRNAs) sind 17 – 24nt kurze, nicht-kodierende RNAs, welche einen

wesentli-chen Beitrag zur Genregulation in nahezu allen Lebewesen ausüben. Durch die Anbindung an die entsprechende Ziel-mRNA können miRNAs entweder die mRNA Degradation imitie-ren oder die mRNA Translation reprimieimitie-ren. Dabei erfolgt die Bindung über eine möglichst komplementäre Basenpaarung der miRNA mit der untranslatierten 3’-Region der Ziel-mRNA. Protein- und RNA-basierte Analysen haben gezeigt, dass eine miRNA die Expression von hunderten von Genen beeinflussen kann. Dementsprechend sind miRNAs auch in der Regu-lation von nahezu allen biologischen Prozessen involviert, wie zum Beispiel bei der Zellent-wicklung, der Proliferation und der Apoptose. Allerdings ist die Funktion einiger miRNAs auch eng mit verschiedenen Krankheiten wie Krebs verknüpft, und etliche Studien haben bereits deutlich veränderte Expressionsraten einiger miRNAs in den Expressionsprofilen von gesunden und kanzerogenen Geweben gezeigt. Dies veranschaulicht nicht zuletzt die hohe Relevanz von miRNAs für die Anwendung als Biomarker beziehungsweise für den Einsatz als Therapeutikum. Allerdings erfordert eine erfolgreiche klinische Anwendung von miRNAs die Identifikation relevanter Zielgene sowie eine detaillierte Aufklärung der biologischen Funktionen und beteiligten Prozesse.

Diese Arbeit beschäftigt sich mit der Rolle von miRNAs in zwei Prozessen, die wesentlich zur Entstehung von Brustkrebs beitragen. Dabei handelt es sich zum einen um den ErbB2/ErbB3/Akt-Signalweg und zum anderen um die Zellmotilität von Krebszellen. Eine er-höhte Aktivität des Akt-Signalwegs ist eng mit der Entstehung von therapieresistenten Krebszellen verbunden, da Akt das Überleben der Zellen unterstützt. In Brustkrebszellen wird dies häufig durch eine erhöhte Aktivität des ErbB2/ErbB3-Rezeptors hervorgerufen. Ba-sierend auf der heregulininduzierten Akt-Phosphorylierung wurde ein genomweiter miRNA-Screen in der luminalen Brustkrebszelllinie MCF7 durchgeführt. Dabei wurden 43 miRNAs identifiziert, welche signifikant die heregulininduzierte Akt-Phosphorylierung positiv oder ne-gativ regulierten. Die bioinformatische Analyse lieferte einen ersten Hinweis auf das komple-xe Zusammenspiel der miRNA und deren potentieller Zielgene im ErbB-Signalweg, wobei für die vier ausgewählten miRNAs, miR-148b, miR-149, miR-326 und miR-520a-3p, die Negativ-Regulation von ErbB3 auf Transkript und Protein-Ebene experimentell bestätigt wurde. Aber auch andere Komponenten des ErbB-Signalweges, wie Erk1/2 oder PIK3CA, wiesen ein deutlich reduziertes Protein- oder mRNA Level auf. Die tumorsuppresiven Eigenschaften dieser miRNAs wurden ferner gestützt durch die verminderte Aktivität des ErbB-Signalweges und der inhibierten heregulinabhängigen Proliferation der MCF7-Brustkrebszellen.

Im zweiten Teil der Arbeit wurde vor allem die Rolle von miR-149 in der Regulation von Zell-motilität behandelt, welche ein entscheidender Faktor bei der Entstehung von Metastasen ist. Klinische Daten weisen eine deutliche Reduktion von miR-149 in verschiedenen Krebstypen auf, wobei in Brustkrebs die miR-149-Level vorwiegend im basalen Subtyp reduziert sind. Durch verschiedenste biologische und biochemische Experimente konnte gezeigt werden, dass miR-149 in Zelllinien des basalen Subtyps, wie MDA-MB-231, die Ausbildung und Akti-vität fokaler Adhäsionen hemmt, Rac-Aktivierung inhibiert, und Zellmigration und -invasion

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deutlich reduziert, in vitro und in vivo. Als potentielle Zielgene konnten Rap1a, Rap1b und Vav2 identifiziert werden. Zudem konnte der beobachtete Phänotyp auch in weiteren Zellli-nien reproduziert werden, was nicht zuletzt auf eine konservierte und zelltypunabhängige Funktion von miR-149 als Tumorsuppressor hindeutet.

Zusammengefasst erweitert diese Arbeit die bisherigen Kenntnisse über die miRNA-vermittelte Regulation von brustkrebsrelevanten Signalwegen und liefert darüber hinaus neue Details über deren Zielgene.

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1 Introduction

1.1 Breast Cancer

Cancer is a universal term for a wide group of diseases that can affect almost every part of the human body and is one of the leading causes of deaths worldwide, accounting for 8.2 million deaths in 2012 (World Health Organization). Overall, there are more than 100 differ-ent types of cancer, and each is categorized by the type of cell that is initially affected. Also the factors contributing to the development of cancer are very diverse, ranging from envi-ronmental influences, genetic alterations up to viral infections.

Among women, breast cancer is the most frequent type of cancer, with an estimated 1.67 million new cancer cases diagnosed in 2012, and accounts for approximately 25% of all can-cers in women. But despite the fact that therapy prognoses are relatively good, breast cancer is still the second leading cause of cancer mortality in women (522,000 deaths in 2012) (World Health Organization). Like other cancers, breast cancer is a heterogeneous disease with diverse morphological and molecular features. Among parameters such as tumor size, histological grade, lymph node involvement, hormone receptor status and metastases for-mation it is currently classified into five main molecular classes: luminal A, luminal B, basal-like, ErbB2-positive and unclassified breast cancer subtypes. The basal-like subtype consti-tutes approximately 20% of all breast cancers and is also referred to as triple-negative breast cancer because it frequently lacks expression of estrogen, progesterone and ErbB2/HER2 receptors. While the presence of estrogen and progesterone receptors allows a better clinical prognosis, as those cancers respond to hormone therapy, the basal-like and ErbB2-positive subtypes are more aggressive and are characterized by a higher risk of early relapse and a higher metastatic potential (Patel et al., 2007; Sørlie, 2007; Nishimura and Arima, 2008). De-pendent on the cancer subtype and the state of disease progression, cancer treatment com-prises one or the combination of the following options: surgery, radiation, chemotherapy, im-munotherapy, hormone therapy and gene therapy. Nevertheless the prerequisite for suc-cessful treatment is the detection of cancer in early stages and furthermore a profound un-derstanding of the underlying molecular mechanisms to refine therapeutic strategies. In this context, the field of miRNAs is of increasing interest as miRNAs function as key regulators of gene expression and are frequently subject to change during the development of human dis-eases, including cancer. Consequently, they have led to the discovery of a completely new repertoire of promising tools for diagnostic and therapeutic purposes.

1.2 Development of breast cancer

Breast cancer development is a complex multistep process which is usually initiated by un-controlled cell growth followed by sustained cell survival, extravasation of primary tumor cells, infiltration of new tissue, and finally the formation of secondary tumors (metastases) in distant parts of the body (Figure 1).

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Figure 1: Cancer development. The general steps involved in cancer progression are increased cell proliferation, sustained survival, extravasation, invasion, and infiltration of other parts of the human body resulting in the formation of metastases. The figure is reproduced from “What is Cancer?” (What is Cancer?, 2012).

In healthy tissue, cell growth and tissue homeostasis is strictly controlled and furthermore, cells are conditioned to commit suicide (apoptosis) if proper cell function is no longer guaran-teed. This protective system is made up of various intracellular and extracellular signaling check-points (Kastan and Bartek, 2004; Collado et al., 2007). To overcome these protective systems, cells have to accumulate various genetic alterations, resulting in the loss of tumor suppressor activity and/or an increase in oncogenic processes. Signaling processes fre-quently associated with sustained survival and an enhanced proliferation of cancer cells are the PI3K/Akt pathway and the RAS/MEK/ERK pathway (Cantley and Neel, 1999; Vivanco and Sawyers, 2002). Deregulation of these pathways is reported on multiple levels such as the hyperactivation or overexpression of upstream effectors such as growth factor receptors, the loss of negative regulators like PTEN or activating mutations of members within the pathway, as it is reported for Ras (Cantley and Neel, 1999; Downward, 2003). Consequently, cells grow beyond their normal border limits and sooner or later cells acquire the capacity to escape from their usual environment, a process referred to as epithelial-mesenchymal transi-tion (EMT), in which cells lose their polarity and their cell-cell adhesion and gain migratory and invasive properties. This is the driver for the development of metastasis, a process that accounts for approximately 90% of all cancer-related deaths (Parkin et al., 2005; Fantozzi and Christofori, 2006).

1.3 ErbB receptors

The ErbB (Erythroblastic Leukemia Viral Oncogene Homolog) family of transmembrane re-ceptor tyrosine kinases plays an important role in the pathogenesis of many cancers and is one of the main targets of anticancer therapy. Members of the ErbB family contribute to a wide range of cellular signaling processes including proliferation, apoptosis, survival and dif-ferentiation. The ErbB (also known as HER) family consists of four structurally related type 1

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receptor tyrosine kinases (RTK): epidermal growth factor receptor (EGFR; also known as Her1 or ErbB1), ErbB2 (Her2), ErbB3 (Her3) and ErbB4 (Her4) (Figure 2).

Figure 2: ErbB receptor family. (A) Structure of the four members of the ErbB receptor family. Each

receptor has an extracellular domain for ligand binding, an α-helical transmembrane domain and an

intracellular domain comprising the tyrosine kinase activity and binding motifs for the interaction with intracellular signaling molecules (Olayioye et al., 2000). In the absence of an external ligand, EGFR, ErbB3 and ErbB4 exist in a closed (tethered) conformation where the dimerization domain is hidden and therefore not accessible for the interaction with the other receptors. ErbB2 is always in an active (open) position and therefore permanently available for dimerization. Furthermore, while ErbB2 has no known external ligand, ErbB3 has an impaired intracellular kinase domain (Burgess et al., 2003). (B) Mechanism of receptor activation. Ligand binding induces a conformational change, thereby exposing the dimerization domain which is now available for dimerization. After the formation of a receptor di-mer, the intracellular domains are transphosphorylated and activated. The figure is reproduced from Baselga and Swain (Baselga and Swain, 2009).

The four ErbB receptors are composed of an extracellular ligand binding domain, an α-helical transmembrane domain and an intracellular domain which contains the protein tyrosine ki-nase and further phosphotyrosine binding motifs for intracellular signaling molecules (Olayioye et al., 2000). In the absence of an external ligand, EGFR, ErbB3 and ErbB4 exist

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in a closed (tethered) conformation where its external dimerization domain is hidden to pre-vent dimerization. Ligand binding induces a conformational change unveiling its dimerization domain and making it accessible for the interaction with other active ErbB receptors. By con-trast, ErbB2 always exists in an open (extended) conformation and is permanently available for dimerization and is thus the preferred interaction partner for the other ErbB receptors. Nevertheless, ErbB2 lacks an external ligand. Therefore, homodimerization is very unlikely and happens only upon an unphysiologically high overexpression of ErbB2. After the for-mation of a dimer, the intracellular domains are in close proximity leading to the transphosphorylation and activation of each tyrosine kinase. ErbB3 has an impaired kinase domain lacking catalytic function and thus can only be phosphorylated within a heterodimeric complex (Burgess et al., 2003; Berger et al., 2004; Hynes and Lane, 2005). The activation of the kinase initiates further phosphorylation events in the intracellular domain of the receptors. This triggers the recruitment and activation of downstream proteins which initiate further downstream signaling cascades (Olayioye et al., 2000). Major signaling pathways activated by ErbB receptors include the phosphatidylinositol 3-kinase/Akt (PKB) pathway, the Ras/Raf/MEK/ERK1/2 pathway, and the phospholipase C γ (PLCγ) pathway, which stimulate cellular responses such as survival, proliferation, and migration. The identities of the activat-ed downstream pathways are dependent on the ligand itself and the individual dimer partners due to their ability to bind distinct effector proteins (Baselga and Swain, 2009). The expres-sion levels of the ErbB receptors are dynamically regulated via transcriptional and transla-tional mechanisms, whereas the amplitude of ErbB signaling is determined by the amount of the ligand and by the abundance of proximal ErbB receptors. Additionally, signaling duration is regulated by the engagement of positive and negative effectors mediating membrane lo-calization, protein stabilization and protein dephosphorylation (Fry et al., 2009). In this con-text the family of cytohesins are reported to function as conformational activators, facilitating the formation of EGFR dimers after ligand binding, while negative effectors, such as Nrdp1 or LRIG-1 increase the ubiquitination of the receptors leading to enhanced proteasomal degra-dation (Fry et al., 2009; Bill et al., 2010).

1.4 ErbB2-ErbB3 receptor dimer

Among all signaling dimers, the ErbB2-ErbB3 heterodimer is considered as the most potent signaling complex in terms of cell growth and transformation. This might be surprising at first sight as ErbB2 lacks an external ligand and ErbB3, on the other hand, has an impaired ki-nase domain. But upon the activation of ErbB3 by the binding of neuregulin ligands such as heregulin β-1 (HRG), it can readily dimerize with its preferred partner ErbB2 (Campbell et al., 2010). Heterodimerization with the catalytically active ErbB2 permits the tyrosine phosphory-lation of several docking sites within the carboxyterminal domain of each partner, which facili-tates the recruitment of different signaling molecules (Songyang et al., 1993) (Figure 3). The main downstream pathway includes the PI3K/Akt pathway and the Ras/Raf/MEK/ERK1/2 mediating diverse biological processes including proliferation and survival. The PI3K/Akt pathway is mainly mediated by ErbB3, which harbors six binding motifs for the regulatory subunit (p85) of PI3K in its carboxyterminal domain. Although of minor importance, activation

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of the PI3K/Akt pathway can also be mediated through the adaptor protein Grb2 which is present in both receptors (further information is provided in section 1.5). Both ErbB2 and ErbB3 contribute to the activation of the MAPK pathway as they both have docking sites for the adapter proteins Grb2 and Shc. Binding of these adaptors to the receptors activates the Ras/Ref/MEK/ERK1/2 signaling module (detailed information is provided in section 1.6).

Figure 3: Signaling downstream of the ErbB2-ErbB3 dimer. (A) Depicted is the carboxyterminal part of the receptors including the tyrosine phosphorylation sites and the corresponding binding mole-cules. Residue numbers correspond to the nascent protein including the signal peptides. The figure is reproduced from Wilson et al. (Wilson et al., 2009). (B) Main downstream modules of the ErbB2-ErbB3 receptor dimer. GRB2, Shc and regulatory subunit (p85) of PI3K bind to their phosphorylated tyrosine consensus site residues within the carboxyterminal parts of the receptors initializing the Ras/Ref/MEK/ERK1/2 and PI3K/Akt signaling cascades. The figure is reproduced from Baselga and Swain (Baselga and Swain, 2009).

Many studies have revealed that the mitogenic potential of the ErbB2-ErbB3 dimer is particu-larly caused by the very potent direct interaction of ErbB3 and phosphoinositide 3-kinase (PI3K). Furthermore, PI3K signaling is critical for ErbB3-driven breast cancer cell motility and metastasis. Deleting the direct binding of the p85 subunit of PI3K to the receptor by mutating the tyrosine residues decreased tumor cell motility and metastic burden in vivo (Smirnova et al., 2012). Furthermore, it has been shown that ErbB2 overexpression alone is insufficient to promote cell growth of breast cancer cells. For proper proliferation, additional signaling of ErbB3 is required as it couples active ErbB2 to the PI3K signaling (Holbro et al., 2003). Moreover, while aberrant ErbB2 function can be successfully blocked by the use of monoclo-nal antibodies such as Herceptin/Trastuzumab targeting the extracellular part of ErbB2, a certain number of tumors eventually become resistant to this treatment (Roskoski, 2014).

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The acquired resistance to targeting therapies in ErbB2 overexpressing breast cancers can be partially attributed to the PI3K pathway by the activation of ErbB3 (Fiddes et al., 1998). Furthermore, although the ErbB3 gene is rarely amplified in human cancers, ErbB2 overex-pression, which is usually caused by gene amplification, is frequently associated with the upregulation of ErbB3 protein levels (Slamon et al., 1987; DiGiovanna et al., 2002; Sithanandam and Anderson, 2008). This upregulation of ErbB3 might be a mechanism to compensate for the inhibition of the other family members (Sergina et al., 2007). For exam-ple, the ErbB3-dependent activation of the Ras/Raf/MEK/Erk and PI3K-Akt pathways has also been implicated in the development of Gefitinib resistance in non-small-lung carcino-mas, which are driven by activating EGFR mutations. In these cells, elevated ErbB3 signal-ing was caused by an amplification of the Met receptor tyrosine kinase which promotes the activation of the ErbB3 pathway (Engelman et al., 2007). Beside ErbB3 upregulation, over-expression of its ligand heregulin was also reported to increase tumorigenicity and metasta-sis whereas blocking heregulin expression reversed these effects (Atlas et al., 2003). Due to the fact that ErbB3 has a key role in driving oncogenic proliferation and survival in several human tumors, it is of special interest for the use of targeting therapies. MM-121, a therapeu-tic anti-ErbB3 antibody, was shown to block ligand-dependent activation of ErbB3 and using a lung cancer mouse model with an activating EGFR mutation resistant to cetuximab, an antagonistic anti-EGFR antibody, concomitant cetuximab treatment with MM-121 blocked reactivation of ErbB3 and resulted in a sustained and durable response (Schoeberl et al., 2010).

1.5 PI3K/Akt signaling

The key player of the PI3K/Akt signaling pathway is the serine/threonine kinase Akt, also known as protein kinase B (PKB). Since its discovery as an oncogene, Akt has gained great attention because of its impact on cancer cell growth, survival, motility and metabolism. The family of Akt proteins consists of three highly homologous isoforms: Akt1, Akt2, and Akt3. These isoforms differ slightly in their tissue-specific expression, their substrate specificity and their localization. While Akt1 and Akt2 are the predominantly expressed isoforms, Akt3 is usually expressed at the lowest level and is absent in several tissues. Furthermore, Akt1 was reported to be primarily localized in the cytoplasm, Akt2 was colocalized with the mitochon-dria and Akt3 in the nucleus (Santi and Lee, 2010). This distinct localization may also con-tribute to their slightly different target spectrum. Nevertheless it was shown that these isoforms are able to partly compensate for each other, as the single knockout of Akt mem-bers in mice resulted in a rather mild but viable phenotype. While Akt1 deficient mice showed growth retardation of all organs and increased apoptosis (Chen et al., 2001), knockout of Akt2 was accompanied by insulin resistance (Cho et al., 2001), and mice lacking Akt3 ex-pression exhibited a selective decrease in brain size (Easton et al., 2005). Furthermore it was shown that Akt1 is probably the most essential isoform within the family as the Akt1/Akt2 and Akt1/Akt3 double-knockout were lethal (Peng et al., 2003; Yang et al., 2005), whereas mice lacking Akt2/Akt3 expression were viable (Dummler et al., 2006). Furthermore, elevated Akt levels or Akt activity have been reported in a variety of human cancers. For instance,

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Akt1 is reported to be involved in tumor growth, invasion and chemoresistance, Akt2 is relat-ed to invasion and survival, while Akt3 is implicatrelat-ed in tumor growth and drug resistance (Fortier et al., 2011).

Figure 4: PI3K/Akt signaling pathway. Activated receptor tyrosine kinases (RTKs), like growth factor receptors, activate class I phosphatidylinositol 3-kinase (PI3K) through direct binding or through tyro-sine phosphorylation of scaffolding adaptors which then bind and activate PI3K. Active PI3K catalyzes the phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3). Direct binding of PIP3 recruits Akt and PDK1 to the plasma membrane where Akt is phosphorylated on T308 by PDK1 and on S473 by mTOR complex 2 (mTORC2). Termination of the signaling cascade is mediated by PTEN by dephosphorylation of PIP3, and by PHLPP mediated dephosphorylation of Akt on S473. After its activation, Akt translocates to different subcellular com-partments where it triggers the phosphorylation of its substrates. Akt activates mTORC1 indirectly starting with the multisite phosphorylation of the TSC1-TSC2 complex. This blocks the ability of TSC2 to inhibit Rheb, thereby allowing Rheb-GTP to accumulate. Rheb-GTP activates mTORC1 which phosphorylates its downstream target 4E-BP1 and S6 kinases (S6K). Among the other targets of Akt are many transcription factors and kinases contributing to survival, growth, proliferation, glucose up-take, metabolism, and angiogenesis (Courtney et al., 2010; Manning and Cantley, 2007). The figure is reproduced from Baselga and Swain (Baselga and Swain, 2009).

Akt signaling can be activated by an extremely versatile spectrum of different stimuli includ-ing receptor tyrosine kinases, integrins, B and T cell receptors, cytokine receptors, G-protein-coupled receptors and many others. The initial step of pathway activation is the production of phosphatidylinositol 3,4,5-trisphosphate (PIP3) by phosphoinositide 3-kinase (PI3K) and in the case of receptor tyrosine kinase activation this is mediated by class IA phosphoinositide 3-kinase (PI3K) (Figure 4) (Manning and Cantley, 2007). Class IA PI3Ks are composed of a p110 catalytic and a p85 regulatory subunit. These subunits exist as different isoforms

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coded by the genes PIK3CA/p110α, PIK3CB/p110β, PK3CD/p110δ for the catalytic subunit and by PIK3R1/p85α, PIK3R2CB/p85β, PK3R3/p85γ for the regulatory subunit. The regulato-ry p85 subunit binds to the phosphotyrosine consensus sites on the RTK or its adaptor pro-teins. This results in the allosteric activation of the p110 catalytic subunit which then catalyz-es the phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2) to form phosphatidyl-inositol 3,4,5-trisphosphate (PIP3) (Zhao and Vogt, 2008). Furthermore, PI3K activity can also be stimulated by activated Ras or by G-protein coupled receptors which directly bind the p110 subunit (Shaw and Cantley, 2006). The lipid phosphates PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome 10) can revert PI3K activity by the hydrolysis of PIP3, thereby terminating the signal cascade. The membrane-bound PIP3 serves as a dock-ing site for Akt and Phosphoinositide-Dependent Protein Kinase-1 (PDK1). Both proteins bind to the lipid by their pleckstrin-homology (PH) domain, recruiting them to the plasma membrane where PDK1 phosphorylates Akt in its activation loop at T308. Mammalian target of rapamycin complex 2 (mTORC2) fully activates Akt by the phosphorylation of S473 which can be reverted by the phosphatase PHLPP (Cantley and Neel, 1999; Manning and Cantley, 2007). The bi-phosphorylated, fully active Akt then translocates to the different subcellular compartments where it phosphorylates its substrates, resulting in various downstream ef-fects.

Among the targets are many components regulating cell-cycle, survival or apoptosis. Akt promotes survival by the functional inhibition of the proapoptotic Bcl-2 family members BAD and BAX which bind and inactivate prosurvival Bcl-2 family members (Engelman et al., 2006). Akt also blocks cell cycle arrest by the inhibition of forkhead transcription factors (FOXO) which mediate the expression of Bcl-2 homology domain 3 (BH3)-only proteins. An-other important downstream effector molecule of Akt is Mdm2 which antagonizes p53-mediated apoptosis and Akt also impedes the negative regulation of the transcription factor NF-κB to enhance the transcription of antiapoptotic and prosurvival genes (Duronio, 2008). The role of Akt in promoting cell growth is predominantly mediated through its effect on mTOR complex 1 (mTORC1) which is a critical regulator of protein biosynthesis. Akt acti-vates mTORC1 indirectly by inhibiting TSC2, thereby allowing Rheb-GTP to activate mTORC1 signaling (Manning and Cantley, 2007). mTORC1 then triggers downstream signal-ing via phosphorylation of its effector molecules eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and the ribosomal protein S6 kinase (S6K1). S6K acts in a feed-back mechanism as it can phosphorylate the adaptor protein insulin receptor substrate 1, thereby inhibiting insulin-like growth factor 1–mediated PI3K activation. Moreover, activation of mTORC1 also contributes to an enhanced cell proliferation, which is also mediated by the phosphorylation and inactivation of p27Kip1 cyclin-dependent kinase inhibitor (Liang et al., 2002).

The PI3K/Akt signaling is activated in a wide range of human cancers via several mecha-nisms. Beside the aberrant activation of an upstream receptor pathway, loss of PTEN activity resulting in enhanced PIP3 levels is a common mechanism for increased Akt signaling. Loss of PTEN is caused by genetic loss-of-function mutations, epigenetic alterations or promoter hypermethylation, respectively (Cantley and Neel, 1999; Vivanco and Sawyers, 2002).

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thermore, mutational activation or amplification of the genes encoding key components of the PI3K pathway is also reported. For example, the genes encoding the catalytic p110α and the regulatory p85 subunit of class IA PI3K, PIK3CA and PIK3R1, are frequently implicated to carry somatic mutations in human cancer (Philp et al., 2001; Mizoguchi et al., 2004; Samuels and Velculescu, 2004; Ikenoue et al., 2005; Yuan and Cantley, 2008). Mutations are also reported within the genes of the Akt family. A mutation within the PH domain of Akt1 leads to its constitutive membrane localization also in the absence of PIP3 (Carpten et al., 2007). Be-cause genomic aberrations can predict responsiveness to targeted therapies, and beBe-cause multiple PI3K pathway members are frequently aberrant in breast tumors, targeting this pathway may provide a highly effective therapeutic approach (Samuels et al., 2004; Hen-nessy et al., 2005).

1.6 Ras-ERK (Extracellular Signal Regulated Kinase) and PLCγ

signaling

Beside the PI3K/Akt pathway, the Ras/Raf/MEK/ERK1/2 and PLCγ pathways are two other major pathways activated by the ErbB2-ErbB3 mediated signaling.

Mitogen-Activated Protein Kinases (MAPKs) are Ser/Thr kinases and are among the best-studied signal transduction components. In mammals, 14 different MAPKs have been identi-fied and downstream signaling of ErbB2-ErbB3 is mainly mediated by MAPK1/2, also called Erk1/2. The Erk1/2 pathway is initiated by the adaptor proteins GRB2 (Growth Factor Recep-tor-Bound Protein-2) or SHC which bind to phosphotyrosine consensus sites within the ErbB receptors. GRB2 is then recognized by the guanine exchange factor SOS (Son of Sevenless), which is recruited from the cytosol to the plasma membrane where it activates Ras by stimulating the exchange of GDP to GTP. Active-GTP-bound Ras then activates Raf which then in turn stimulates the dual-specificity kinases MEK1/2 and finally, MEK1/2 phos-phorylates Erk1/2. Active Erk1/2 phosphos-phorylates several substrates, like the cytoplasmic ri-bosomal S6 kinase (RSK) family or members of the cytoskeleton. Furthermore, Erk1/2 also translocates to the nucleus, where it activates a number of transcription factors, including Elk1, c-Fos, c-Jun, STAT3, and the oncogenic c-Myc, known to have oncogenic potential driving proliferation (Cargnello and Roux, 2011; Roskoski, 2012). ErbB1, ErbB2, and ErbB4 possess several potential PLCγ phosphotyrosine binding sites (Wilson et al., 2009). After its activation, PLCγ catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to form inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (Kadamur and Ross, 2013). IP3 triggers the release of Ca2+ from the endoplasmatic reticulum and together with DAG acti-vates the protein-serine/threonine kinase C (PKC). The target spectrum of PKC, contributing to gene transcription, angiogenesis, cell proliferation, cell death, but also to migration and adhesion, is very diverse. Interestingly, PKC can activate the Raf/MEK/ERK1/2 pathway, thereby circumventing the involvement of Ras kinase (Basu and Sivaprasad, 2007; Roskoski, 2012).

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1.7 Cell motility

Cell motility plays a key role in many physiological processes including embryogenesis, dif-ferentiation and immune response, but also in pathological processes like cancer cell migra-tion and invasion (Le Clainche and Carlier, 2008; Petrie et al., 2009). Cell motility is a highly complex process constantly integrating and coordinating various biochemical and biome-chanical signals. It requires the coordinated remodeling of the cytoskeleton and the regula-tion of membrane dynamics as these factors are the major determinants for cell shape and motility (Le Clainche and Carlier, 2008). In general, cell motility can be subdivided into differ-ent inter-connected cellular processes, including adhesion, spreading, migration and inva-sion, with each process being characterized by a distinct set of proteins and molecules. Cell adhesions are of special importance here as they anchor the cells to the surrounding ECM and generate the traction force necessary for cell movement. They also act as important in-side-out signaling nodes as they transmit information about matrix composition, rigidity and topography inside the cells (Geiger et al., 2009). These focal adhesions are multiprotein complexes consisting of ~160 different components, including scaffolding proteins and en-zymes regulating the activity of the focal adhesion components (Zaidel-Bar et al., 2007; Gei-ger et al., 2009). Especially during migration processes these focal adhesions undergo per-manent turnover with a constant formation of new adhesions at the cell front and a release of adhesions at the cell rear pushing the cell forward. This multistep process begins at the front where cells initially scan the ECM by the lamellipodium, a small membrane protrusion which is enriched with a branched network of actin filaments generated by the (Arp2/3) complex (Small et al., 2002). The predominant class of transmembrane proteins recognizing and con-necting the ECM with the cytoskeleton are integrins, which are dimeric proteins composed of an α and a β subunit (Geiger et al., 2009). These subunits exist in different isoforms and their identity within the dimer is matrix-dependent. Fixing the cells and establishing a traction point for cell migration, cells locally recruit proteins to these focal contact sites, leading to the for-mation of several short-lived nascent adhesions, which mature into larger focal adhesions and finally become large and prominent mature focal adhesions. The initial steps of focal adhesion assembly requires proteins like talin, integrin-linked-kinase (ILK), tensin, and integ-rin-binding proteins (kindlins) which are essential for the conformational activation of the integrins and provide a first link to the actin-filaments (Nayal et al., 2004; Geiger et al., 2009). Integrin engagement also triggers the activation of the focal adhesion-linked protein kinases Src and FAK, which then recruit additional components and activate downstream pathways (Geiger et al., 2009; Eleniste and Bruzzaniti, 2012; Ross et al., 2012). Further strengthening of the focal adhesions requires the scaffolding protein vinculin, which is recruited to talin and induces the clustering of the integrin dimers (Humphries et al., 2007; Le Clainche and Carlier, 2008; Geiger et al., 2009). During the maturation process, focal adhesions increase their length and thickness. A hallmark for the mature focal adhesions is the presence of stress fibers, actin-filament bundles containing filamentous actin, α-actinin and myosin II. These stress fibers are the driver for cell contractility and migration (Geiger et al., 2009). The integrity of cell adhesion, spreading and migration is guaranteed by the coordinated activity of a wide range of regulatory proteins controlling the spatiotemporal activation and

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tion of small Rho-family GTPases, like Cdc42, Rac, and Rho. These small GTPases cycle between an active GTP-bound state and an inactive GDP-bound state and their activity is controlled by three classes of proteins; guanine nucleotide dissociation inhibitors (GDIs), guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) (Petrie et al., 2009). Local activation of Cdc42 and Rac is necessary for the formation of nascent adhesions as they are required for cell polarization and the formation of membrane exten-sions including the lamellipodium. In contrast, Rho is predominantly involved in cell migra-tion, promoting the adhesion maturation at the leading edge and adhesion disassembly at the rear of the cell (Ridley, 2001; Raftopoulou and Hall, 2004).

1.8 miRNAs

microRNAs (miRNAs) are a class of endogenous small non-coding RNAs of approximately 17 to 24 nucleotides that act as key regulators of gene expression and their discovery in 1993 by Ambros et al. has uncovered a new mechanism of gene regulation at the post-transcriptional level. Ambros and Lee et al. described a 22-nucleotide long RNA encoded by the lin-4 gene that interferes with the expression of the lin-14 transcript in C. elegans, thereby contributing to the postembryonic development (Lee et al., 1993). In the beginning, it was assumed that this mechanism of gene regulation was specific for nematodes until seven years later Pasquinelli identified let-7, a miRNA that was conserved in many species, includ-ing vertebrates, suggestinclud-ing that gene regulation by RNA interference is a widespread phe-nomenon (Pasquinelli et al., 2000). Finally, in 2001, the term miRNA was formally introduced and since then the field of miRNA research has expanded tremendously. Currently, accord-ing to the miRBASE database, a searchable database of published miRNA sequences and annotations, 2578 mature miRNAs are encoded within the human genome [Release 20: June 2013], which theoretically target more than one third of the protein-coding genes (Esteller, 2011). Moreover, there is increasing evidence that altered miRNA expression is strongly as-sociated with the development of several diseases. Consequently, miRNAs represent a promising field for basic research, biomarker discovery, and therapeutic application.

1.8.1 miRNA biogenesis

The biogenesis of miRNAs is a multi-step process starting in the nucleus where miRNAs are encoded by the genome either as single genes or as a cluster of several miRNAs which are processed as a unique transcript and are therefore often co-regulated (Rodriguez et al., 2004). Furthermore, several miRNAs, so called mirtrons, are found to be located in the intronic sequences of protein coding genes and are co-expressed with their host genes. Transcription of miRNAs is carried out by the polymerase machinery, starting with the gener-ation of a capped and polyadenylated primary miRNA (pri-miRNA) by RNA polymerase II. The pri-miRNA is about hundreds to thousands nucleotides in length and its characteristic hairpin stem-loop structure is recognized by the microprocessor complex composed of the RNase type III enzyme Drosha and the double stranded RNA-binding protein DGCR8 (DiGeorge syndrome critical region 8). Drosha excises the hairpin structures, resulting in the

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generation of a precursor-miRNA (pre-miRNA) composed of the characteristic 22-bp stem, a loop, and a 2-nucleotide overhang at the 3’end. This pre-miRNA is then exported to the cyto-plasm by exportin-5 in a Ran-GTP dependent manner and is further cleaved by the RNase III enzyme Dicer into a double-stranded miRNA-miRNA* duplex. After the separation of this duplex by a helicase, the mature strand (miRNA) is taken up into the Argonaute (Ago)-containing miRNA-induced silencing complex (RISC), whereas the other strand (miRNA*) is degraded. The mature miRNA within miRISC serves as a guide for recognizing target mRNAs by partial base-pairing, which leads to a block of the translation of the mRNA target and/or its degradation (Figure 5) (Berezikov, 2011; Esteller, 2011).

Figure 5: Biogenesis of miRNAs. miRNAs are initially transcribed as primary-miRNA (pri-miRNA) either as single units or as a cluster of several miRNAs by RNA polymerase II. Then the pri-miRNA is further processed by the microprocessor complex Drosha-DGCR8 into a precursor miRNA (pre-miRNA), which is then exported to the cytosol by exportin-5 in a Ran-GTP dependant manner. In the cytosol the pre-miRNA is further cleaved by Dicer into a double-stranded miRNA-miRNA* duplex structure. After unwinding the mature miRNA strand it is loaded into the Argonaute (Ago)-containing

RNA-induced silencing complex (RISC). Within this complex the miRNA interacts with the 3’

untrans-lated region (3’UTR) of target mRNA via complementary base-pairing. Depending in the degree of complementarity this initiates either mRNA degradation or translational repression (Berezikov, 2011; Esteller, 2011). ORF (open reading frame). The figure is reproduced from (He and Hannon, 2004).

1.8.2 miRNA targeting

miRNA target recognition involves base-pairing between the miRNA and the mRNA strand. It is hypothesized that depending on the degree of complementarity this results either in trans-lational repression or mRNA cleavage and degradation. In animals, mRNA targeting is main-ly mediated by the 5’end of the miRNA strand. Mutational profiles have revealed that

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cially the nucleotides 2 to 8 contribute to the sequence-specific binding of the 3’ untranslated region (3’ UTR) of the target mRNA (Bartel, 2009). This conserved region is also referred to as seed region and as this sequence is relatively short, a miRNA is able to target multiple mRNAs, thereby regulating entire gene networks. Furthermore, many miRNAs share the same seed sequence and are grouped into families and members of one seed family have a largely overlapping target spectrum (Bartel, 2009). Based on sequence information, 30% of all human miRNAs can be grouped into such seed families (Ambros et al., 2003). But seed matching alone is not an absolute prerequisite for efficient mRNA targeting as other nucleo-tides of the miRNA are known to be involved, too. Furthermore, these additional interactions can also compensate for a mismatch within the seed region and are schematically presented in Figure 6 (Grimson et al., 2007; Bartel, 2009). Another important aspect of stable binding is the local context of the mRNA target itself. Parameters such as the relative proximity of the miRNA binding site to the 3’UTR end or also the A and U content influence the efficiency of gene regulation. Finally, multiple binding of miRNAs in close proximity within the 3’UTR seems to enhance the degree of downregulation in a synergistic way (Bartel, 2009). This is either mediated by binding of identical or different miRNAs.

Figure 6: miRNA-mRNA interaction. Schematical presentation of the different interaction sites be-tween miRNA and mRNA contributing to efficient targeting. Perfect base-pairing of the seed region (red), comprising nucleotide 2-8, predominately contributes to efficient mRNA targeting. The presence of an adenosine at the opposite position of base 1 of the miRNA is thought to be recognized by RISC and improves the degree of silencing. Consecutive base pairing of additional 3-4 nucleotides at posi-tion 13-16 of the miRNA enhances the target efficiency and may compensate for a mismatch in the seed region.

1.8.3 Nomenclature of miRNAs

Classification of the mature miRNA follows a common nomenclature which was stated by Ambros et al. in 2003, exemplified here for hsa-miR-121 (Ambros et al., 2003). The first three letters of a miRNA refer to the organism, “hsa-“ for homo sapiens followed by the numbering of the miRNA, which is simply sequential corresponding to its discovery. The mature miRNA is designated as miR-121, while the gene or the primary stem-loop transcript is designated as miR-121. Identical mature sequences originated from different genomic loci are marked by an additional number, for example hsa-miR-121-1 and hsa-miR-121-2, while closely relat-ed mature sequences are denotrelat-ed by a letter, for example hsa-miR-121a and hsa-miR-121b. If two mature miRNAs are produced from the stem loop structure, this is indicated by a suffix; miR-121-5p (from the 5’ arm) and miR-121-3p (from the 3’ arm). Furthermore, when the rela-tive abundance clearly indicates which is the predominantly expressed miRNA, the mature

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sequences are assigned miR-121 (the predominant product) and miR-121* (from the oppo-site arm of the precursor). let-7 and lin-4 are obvious exceptions to the numbering scheme, and these names are retained for historical reasons (Ambros et al., 2003).

1.8.4 miRNA target prediction

Because of the high complexity of miRNA biogenesis, miRNA-target interactions as well as the immense amount of data received from high through-put sequencing screenings, bioinformatic approaches are pivotal for precise miRNA identification and target prediction. Most of the databases providing sequence information and target predictions are web-based tools and algorithms. The largest web-accessible database is the miRBase which lists all discovered miRNAs and provides additional information including their secondary structure and their precursor sequences. So far, numerous computational algorithms have been de-signed to predict miRNA-mRNA interactions based on the biological features of miRNAs, e.g. Watson-Crick base pairing, hairpin structure, conservation between different species or RNA conformation. An overview of commonly used target prediction algorithms is given in Table 1. Table 1: List of commonly used target prediction algorithms.

Prediction algorithm Parameters contributing to the final score

miRanda  complementarity to 3’UTR

 binding energy of the duplex structure  evolutionary conservation of the target site

 position within the 3’UTR

TargetScan  seed match

 3’ complementarity

 local AU content

 position contribution: proximity to 3’UTR ends

 conservation

PicTar  complementarity to 3’UTR

 binding energy of the duplex structure  conservation

DIANA-microT  complementarity to 3’UTR

 binding energy of the duplex structure

PITA  target site accessibility defined by secondary structure of the 3’UTR

 binding energy of the duplex structure

Rna22  pattern recognition and folding energy

Nevertheless, these prediction algorithms still yield many false positive targets and further-more, since the prediction algorithms differ in certain parameters, they do not always yield identical target lists. Therefore, the comparison of several target lists may be useful to narrow down the predicted list in order to obtain higher confidence targets. This is provided, for ex-ample, by the miRecords online database, which integrates the predicted targets of 11 miRNA target prediction tools (Farazi et al., 2013). Recently, certain databases, including miRecords, have integrated experimental data from validated targets and miRNA-mRNA pro-filing studies. But as global experimental data are very limited and many miRNAs have only been investigated in context-specific manner, the provided miRNA-mRNA interaction infor-mation covers only a small number of miRNAs and mRNAs. For example, miRecords cur-rently lists 644 miRNAs and 1901 target genes in 9 animal species. [April 27, 2013, miRecords]. Nevertheless, the increasing experimental knowledge largely contributes to the

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refinement of theoretical target predictions and helps to improve their reliability as most com-putational algorithms are based on machine-learning approaches.

1.8.5 miRNAs and their biological function

The fact that about 75% of the human genome is transcribed into RNA, whereas only 3% is transcribed into protein-coding genes, indicates that the number of non-coding RNAs such as miRNAs is potentially much higher than that of protein-coding genes (Djebali et al., 2012). Furthermore, Bernstein et al. reported in 2003 that the presence of miRNAs is of vital im-portance as the disruption of miRNA biogenesis by inhibiting Dicer function in mice causes embryo death before gastrulation (Bernstein et al., 2003). Meanwhile, miRNAs are implicated in the regulation of almost all fundamental biological cellular processes such as embryogen-esis, organogenembryogen-esis, development, proliferation and apoptosis. For example, the miR-290 cluster is strongly associated with self-renewal and pluripotency of embryonal stem cells by suppressing proteins that inhibit Oct4 expression, while miR-21 was reported to counteract miR-290 activity as it directly targets Oct4. Subsequently, levels of these miRNAs are in-versely correlated in embryonic stem cells. (Singh et al., 2008; Sinkkonen et al., 2008). miR-1 and miR-miR-133 have been shown to possess distinct roles in modulating skeletal muscle pro-liferation and differentiation. miR-1 promotes myogenesis by targeting histone deacetylase 4 (HDAC4), a transcriptional repressor of muscle gene expression, while miR-133 enhances myoblast proliferation by repressing serum response factor (SRF) (Chen et al., 2006). Ex-pression of miRNAs is also reported to display regulatory functions in response to certain stimuli. For example, oxidative stress induced the expression of the miR-200 family which regulates epithelial-mesenchymal transition through inhibition of the E-cadherin transcrip-tional repressors ZEB1 and ZEB2. Thus, downmodulation of ZEB1 has a key role in ROS-induced apoptosis and senescence of endothelial cells (Magenta et al., 2011). Like other regulatory molecules, the expression level of each miRNA has to be controlled in a context-specific and spatiotemporal manner as the dysregulation of miRNA activity interferes with proper cell function and is at the root of many diseases.

1.8.6 miRNAs in cancer

Due to their pivotal role in various biological processes and their diverse target spectrum, miRNAs are strongly associated with the development and progression of various diseases. Especially in the context of cancer, they are obviously of major interest as according to Pubmed 40% of all publications dealing with miRNAs are connected to cancer. The first miRNAs involved in the development of cancer, miR-15 and miR-16, were reported by Calin et al. in 2002 and were found to be downregulated in B cell chronic lymphocytic leukemia (B-CLL) (Calin et al., 2002). Meanwhile, ablation of these miRNAs has been implicated in many other cancers as well and, additionally, their tumor-suppressive function was attributed to the downregulation of cell cycle proteins like Bcl2 and CDK6 (Cimmino et al., 2005; Klein et al., 2010).

Several expression profiling studies of different tumor samples have revealed that the ex-pression patterns of several miRNAs are profoundly altered in malignant cells and, moreover,

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their expression often correlates with developmental lineages, differential states and clinical prognoses of cancer. In a study by Enerly et al., microarray analyses of primary human breast tumors identified 26 miRNAs that separated almost perfectly the basal-like and lu-minal a samples (Enerly et al., 2011). Among them was the oncogenic miR-17-92 cluster, which was distinctively overexpressed in highly proliferative samples including the basal-like subtype. This cluster is frequently upregulated in a broad range of cancers and miRNA members of this cluster are effectors of the oncogene MYC and are known to target the tu-mor suppressors PTEN and BIM (Mu et al., 2009; Ling et al., 2013). In another study, altered expression of single miRNAs, like miR-21, miR-126, miR-199a, and miR-335, was closely associated with clinicopathologic features of breast cancer such as histological tumor grades and hormone receptor expression (Iorio et al., 2005; Wang et al., 2010). Based on these ob-servations, many research groups focused on the role and function of single miRNAs, em-phasizing their dual function acting as oncogenes and/or tumor suppressors as well.

One of the best characterized oncogenic miRNAs, a so called oncomir, is miR-21 which is frequently overexpressed in a variety of tumors (Volinia et al., 2010). miR-21 exerts its onco-genic function at multiple levels, for example, overexpression of miR-21 enhances KRAS-dependent lung carcinogenesis in mice by inhibiting negative regulators of the Ras/MEK/ERK pathway (Hatley et al., 2010) and vice versa, genetic deletion of miR-21 pro-tects against tumor formation. Furthermore, miR-21 mediates the downregulation of PTEN, which was reported to contribute to acquired resistance against trastuzumab treatment in HER2 overexpressing tumors (Gong et al., 2011). Underscoring the enormous potential of gene regulation, it was shown that miR-221, which is frequently overexpressed in various types of cancer, affects multiple oncogenic pathways by modulating the mRNA level of ap-proximately 600 genes (Lupini et al., 2013). Among these targeted genes were prominent proteins, such as PTEN and PUMA, involved in apoptosis and proliferation (Sarkar et al., 2013). Interestingly, a study by Fornari, et al. revealed that miR-221 can stimulate its own expression in a transcription factor feedback mechanism, where the miRNA-mediated downregulation of Mdm2 activates p53 which in turn stimulates the expression of miR-221 (Fornari et al., 2014). Beside these oncogenic functions, there are also various miRNAs with inherent tumor-suppressive activity. A prominent example is the let-7 family which is often downregulated in advanced stage and high grade tumors. (Dangi-Garimella et al., 2009). Members of the let-7 family target the oncogenes KRAS and MYC, resulting in reduced proliferation and metastasis of cancer cells (Sampson et al., 2007; Dangi-Garimella et al., 2009; Ling et al., 2013). miR-31 on the other hand mainly exerts its tumor-suppressive function in later stages of cancer progression by affecting metastasis. Overexpression of this miRNA in mice causes the regression of metastasis without affecting the growth of the prima-ry tumor via the suppression of prometastatic target genes including integrin α5 (ITGA5), radixin (RDX), and RhoA (Valastyan et al., 2010, 2011).

In the context of breast cancer, miRNAs influencing the ErbB signaling pathway are of spe-cial interest due to its major contribution to tumorigenesis and therapy resistance. One of the first published miRNAs regulating the ErbB pathway at the receptor level was miR-7, which targets EGFR and its downstream signaling molecules including the MAPK effector protein

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Raf1 (Webster et al., 2009). Furthermore, miR-125a and miR-125b* were shown to target ErbB2 and ErbB3, which was accompanied by the suppression of the downstream Akt-PI3K signaling pathway. In line with this, the expression levels of these miRNAs have been found to be decreased in various cancer tissues when compared to normal tissues (Scott et al., 2007). A common mechanism for sustained ErbB receptor signaling is the downregulation of the lipid phosphatase PTEN and several miRNAs have been described to exert their onco-genic function partly by targeting PTEN like 21, the 17-92 cluster, 26a or miR-214, respectively. In contrast, miR-199a mediates its oncogenic effect by reducing the pro-tein level of Necl-2 which is a negative effector of ErbB3-signaling. Necl-2 promotes the dephosphorylation of the receptor via PTPN13 resulting in enhanced HRG-induced ErbB2-ErbB3 signaling (Kawano et al., 2009; Minami et al., 2013).

This complex interaction network of protein-coding genes and miRNAs emphasizes the high capacity of miRNAs for prognostic and therapeutic purposes and has already led to the de-velopment of miRNA-based therapies with some of them being tested in clinical trials. But nevertheless, the knowledge about the detailed regulation of miRNAs and their target net-work has barely skimmed the surface and needs to be further investigated.

1.8.7 Aim of the thesis

microRNAs are small non-coding RNAs and their discovery unveiled a novel mechanism of regulating gene expression at the post-transcriptional level. In their function as master regu-lators of the genome, they are essential for the regulation of a multitude of biological pro-cesses but they are also implicated in the development of various diseases including breast cancer. Their capacity to simultaneously target multiple genes and pathways represents a powerful tool for diagnostic and therapeutic purposes. Nevertheless, the clinical use of miRNAs requires the investigation of miRNAs in a context specific manner as their function is often tissue-specific and, furthermore, a profound knowledge about the molecular targets of each miRNA.

The objective of the first part of this thesis was to investigate the extent to which miRNAs modulate the ErbB receptor signaling pathway. In breast cancer, the ErbB2-ErbB3 receptor dimer has attracted growing attention because upregulation of signaling through this receptor dimer plays an important role in the resistance to targeted therapies. This can be explained by the very efficient coupling of ErbB3 to the PI3K/Akt survival pathway. Therefore, I per-formed a genome-wide miRNA screen in the breast cancer cell line MCF7 based on heregulin-induced Akt phosphorylation as a read-out. Using a bioinformatical approach, I aimed to identify the molecular targets within the ErbB/PI3K/Akt signaling network followed by the validation of these predictions using biochemical and cellular assays. The second part of this thesis focuses on the screen hit miR-149, which is indicated to have a tumor-suppressive function in a broad range of tumors including the basal-like breast cancer sub-type. Applying a genome wide pathway analysis, I aimed to identify the signaling modules responsible for this tumor-suppressive phenotype, the contribution of which was investigated in the basal like breast cancer cell line MDA MB 231 using a broad range of different biologi-cal assays.