Members of the insulin-like growth factor (IGF) family are mitogenic growth factors which have been shown to play critical roles in both normal growth and development, and tumour biology. The IGF system is complex and the biological effects of the IGFs are determined by diverse interactions between many molecules, including interactions with the extracellular matrix (ECM). Recent observations have demonstrated that IGFs can associate with the ECM protein vitronectin (VN) and this interaction can modulate IGF-stimulated biological functions. It has been demonstrated previously that IGF-II can bind directly to VN, while IGF-I associates with VN indirectly via the involvement of IGF-binding proteins (IGFBPs) -2, -3, -4 and -5. As the IGF system plays important roles in both normal breast development and in the transformation and progression of breast cancer, this study aimed to describe the effects of substrate-bound IGF-I:IGFBP:VN complexes on breast cell functions and to dissect the mechanisms underlying these responses. The studies reported in this thesis demonstrate that substrate-bound IGF-I:IGFBP:VN complexes, containing IGFBP-3 and IGFBP-5, are potent stimulators of proliferation and migration in the "normal", non-tumourigenic MCF-10A breast epithelial and MCF-7 breast carcinoma cell lines. Interestingly, substrate-bound IGF-I:IGFBP:VN complexes were less effective in increasing the migration of the metastatic MDA-MB-231 breast cancer cell line. This, however, is due to these cells expressing the αvβ3 integrin which can support a highly migratory phenotype independent of IGF-I-stimulation. Taken together this suggests a particularly important role for these complexes in stimulating a highly migratory phenotype in pre-invasive or poorly metastatic breast cells. Studies using IGF-I analogues were also undertaken to establish if there was a requirement for ternary complex formation and the type-1-IGF receptor (IGF-1R) in the enhanced migration responses observed. These studies determined IGF-I:IGFBP:VN-stimulated migration to be dependent upon both heterotrimeric IGF-I:IGFBP:VN complex formation and activation of the IGF-1R. Furthermore, the enhanced cellular migration was abolished upon incubation of MCF-7 and MCF-10A cells with function blocking antibodies directed at VN-binding integrins and the IGF-IR. In addition, analysis of the signal transduction pathways underlying the enhanced cell migration revealed that the complexes stimulate a transient activation of the ERK/MAPK signaling pathway, while simultaneously producing a sustained activation of the PI3-K/AKT pathway. Optimal intracellular signaling required activation of both the IGF-1R and VN-binding integrins, as antibody mediated inhibition of either receptor led to substantial decreases in both ERK/MAPK and PI3-K/AKT pathway activation. Furthermore, experiments using pharmacological inhibitors of these pathways determined a pivotal role for PI3-K/AKT activation in substrate-bound IGF-I:IGFBP:VN-stimulated cell migration. In order to confirm an important role for the PI3-K/AKT pathway in these responses, wild-type and activated-AKT was transiently overexpressed in MCF-10A cells. Overexpression of both wild-type and activated-AKT further enhanced cellular migration in response to substrate-bound IGF-I:IGFBP:VN complexes. However, these responses still required co-activation of the IGF-1R and VN-binding integrins. In an attempt to obtain a global view of the possible molecular mechanisms underpinning IGF-I:IGFBP:VN-stimulated cell migration, oligonucleotide microarrays were used to screen for candidate genes important for the observed migratory responses. The microarray studies identified 165 genes which were differentially expressed in cells migrating in response to substrate-bound IGF-I:IGFBP:VN complexes. Gene ontology and functional analysis revealed many of these genes to be significantly associated with biological functions relevant to cancer transformation and progression, including cell growth and proliferation, cell death and cellular movement. In regard to cell migration, a number of the genes identified have previously reported roles in cellular movement, migration and metastasis, which may provide future targets to augment IGF-I:IGFBP:VN-stimulated cell migration. Taken together, the studies reported throughout this thesis have provided the first mechanistic insights into the action of IGF-I:IGFBP:VN complexes and add further evidence to support the involvement of VN-binding integrins and their co-operativity with the IGF-IR in the promotion of tumour cell migration. Importantly, identifying the molecular mechanisms by which IGF:VN complexes enhance breast cell function will lead to not only a better understanding of this critical interaction, but also aid in developing diagnostic tests and therapeutics directed at treating breast cancer.

Die extrazelluläre Mikroumgebung spielt eine grundlegende Rolle bei der Entwicklung von Metastasen und hat einen großen Einfluss auf die Wahl der Migrationsstrategien, die von Karzinomzellen während der Invasion angewandt werden. In-vitro Anordnungen sind hilfreiche Instrumente für die Untersuchung von Zellmigration und -invasion, da sie grundlegende Merkmale von In-vivo-Geweben reproduzieren können. Ziel dieser Forschungsarbeit ist es, die Fähigkeit von mesenchymalen und epithelialen Brusttumorzellen zu untersuchen, sich zu verflüssigen und durch enge und starre Mikrostrukturen zu navigieren. Wir verwendeten eine mikrofluidische Vorrichtung mit trichterförmigen Mikroverengungen und verglichen das Verhalten von fünf verschiedenen menschlichen Brustkrebszelllinien in der Mikrovorrichtung bei Stimulation durch Chemoattraktoren. Wir fanden heraus, dass grundsätzlich verschiedene Zelllinien das gleiche invasive Potenzial haben, da normale Epithelzellen in der Lage waren, durch die stark komprimierenden Trichter zu wandern, ähnlich wie die invasiveren mesenchymalen Zellen. Wir fanden auch heraus, dass die Migration der normalen Epithelzellen auch ohne einen chemo-attraktiven Stimulus stattfindet. Wir konzentrierten unsere Beobachtungen auf die Rolle des Aktin- und Intermediärfilament-Zytoskeletts während der eingeschränkten Migration und zeigten, dass das Aktin-Zytoskelett eine starke und langanhaltende Reorganisation erfährt, damit die Zellen durch die engen Verengungen kriechen können. Wir sahen keinen Hinweis darauf, dass das Keratin- und Vimentin-Zwischenfilament- Zytoskelett während der Invasion in die Mikroverengungen eine aktive mechanische Rolle spielte. Insbesondere die Expression des Vimentin-Zwischenfilamentproteins korrelierte in unserem Versuchsaufbau nicht mit der Invasionsfähigkeit einzelner Zellen. Unter diesen Voraussetzungen wurden die passiven (Elastizität und Viskosität) und aktiven (Kontraktilität) viskoelastischen Eigenschaften der Zellen weiter untersucht und quantifiziert. Wir fanden keinen signifikanten Unterschied in der passiven viskoelastischen Reaktion der Zellen, nachdem sie oszillierenden Druckkräften mittels AFM-Sondierung ausgesetzt waren, was darauf hindeutet, dass Elastizität und Viskosität nicht zur Unterscheidung zwischen invasiven und nicht-invasiven Zellen verwendet werden können. Es wurde kein Hinweis darauf gefunden, dass die Kompressionsversteifung die Invasion durch die Mikroverengungen entweder behindert oder fördert. Schließlich haben wir bei der Betrachtung aktiver viskoelastischer Parameter die kontraktile Reaktion unserer Zelllinien verglichen, wenn sie mit dem mikrofluidischen optischen Strecker Laser-Streckkräften ausgesetzt wurden. Hier fanden wir eine klare Korrelation zwischen den Zelllinien, die ein invasives Verhalten in den Mikroverengungen zeigten, und denjenigen, die eine aktive (substratunabhängige) kontraktile Reaktion in der optischen Streckvorrichtung zeigten. Wir kommen zu dem Schluss, dass ein entscheidender Faktor für eine erfolgreiche Migration durch hohe räumliche Enge die Fähigkeit der Zellen ist, aktiv Aktin-Stressfasern zu erzeugen und abzubauen, was sich in der Fähigkeit manifestiert, von einer substratabhängigen und stressfaserbasierten Kontraktilität zu einer substratunabhängigen kortikalen Kontraktilität zu wechseln.

No description available.

Glioblastoma multiforme (GBM) is the most aggressive type of primary brain tumor. Even after patients undergo maximum and safe surgical resection followed by adjuvant chemotherapy and radiation therapy, residual GBM cells form secondary tumors which lead to poor survival times and prognoses for patients. This tumor recurrence can be attributed to the inherent GBM heterogeneity that makes it difficult to eradicate the therapy-resistant and tumorigenic subpopulation of GBM cells with stem cell-like properties, referred to as glioma stem cells (GSCs). Additionally, the migratory nature of GBM/GSCs enable them to invade into the healthy brain parenchyma beyond the resection cavity to generate new tumors. In an effort to address these challenges of GBM recurrence, this research aimed to develop a biomaterials-based approach to attract, capture, and eradicate GBM cells and GSCs with chemical and physical stimuli. Specifically, it is proposed that after surgical removal of the primary GBM tumor mass, an injectable hydrogel can be dispensed into the resection cavity for crosslinking in situ. A combination of chemical and physical cues can then induce the migration of the residual GBM/GSCs into the injectable hydrogel to localize and concentrate the malignant cells prior to non-invasively abating them. In order to develop this proposed treatment, this dissertation focused on 1) characterizing and optimizing the thiol-Michael addition injectable hydrogel, 2) attracting and entrapping GBM/GSCs into the hydrogel with CXCL12-mediated chemotaxis, and 3) assessing the feasibility of utilizing histotripsy to mechanically and non-invasively ablate cells entrapped in the hydrogel. The results revealed that hydrogel formulations comprising 0.175 M NaHCO3(aq) and 50 wt% water content were the most optimal for physical, chemical, and biological compatibility with the GBM microenvironment on the basis of their swelling characteristics, sufficiently crosslinked polymer networks, degradation rates, viscoelastic properties, and interactions with normal human astrocytes. Loading the hydrogel with 5 µg/mL of CXCL12 was optimal for the slow, sustained release of the chemokine payload. A dual layer hydrogel platform demonstrated in vitro that the resulting chemotactic gradient induced the invasion of GBM cells and GSCs from the extracellular matrix and into the synthetic hydrogel with ameboid migration and myosin IIA activation. This injectable hydrogel also demonstrated direct therapeutic benefits by passively eradicating entrapped GBM cells through matrix diffusion limitations as well as decreasing the GBM malignancy and GSC stemness upon cancer cell-hydrogel interactions. Research findings revealed the hydrogels can be synthesized under clinically relevant conditions mimicking GBM resection in vitro, and hydrogels were distinguishable with ultrasound imaging. Furthermore, the synthetic hydrogel was acoustically active to generate a stable cavitation bubble cloud with histotripsy treatment for ablation of entrapped red blood cells with well-defined, uniform lesion areas. Overall, the results from this research demonstrate this injectable hydrogel is a promising platform to attract and entrap malignant GBM/GSCs for subsequent eradication with chemical and physical stimuli. Further development of this platform, such as by integrating electric cues for electrotaxis-directed cell migration, may help to improve the cancer cell trapping capabilities and thereby mitigate GBM tumor recurrences in patients. / Doctor of Philosophy / Glioblastoma multiforme (GBM) is the deadliest type of primary brain cancer. Upon GBM diagnosis, patients first undergo surgery to remove the tumor from the brain. After waiting several weeks for the wound healing process due to surgery, patients are administered chemotherapy with drugs and radiation therapy to eradicate any remaining GBM cells. Even after undergoing these combinatorial treatments, the cancer returns and leads to median survival times of only 15 months in 90% of patients. Complete GBM eradication is difficult, since the cancer cells can migrate into healthy brain tissue beyond the original tumor site. Additionally, GBM is highly heterogenous and composed of different cell types that can resist chemotherapy and radiation therapy, which lead to secondary tumors and cancer relapse. To address these challenges, this dissertation aimed to develop a polymer-based material (specifically a hydrogel) that can attract, entrap, and localize the GBM cells into the material to subsequently eradicate them with chemical and physical signals. This hydrogel platform would have important clinical implications, as it can potentially be dispensed into the empty cavity after surgical removal of the tumor in the brain. The hydrogel can then be harnessed to attract residual GBM cells for directed migration into the hydrogel to concentrate and localize the cancer cells for their subsequent destruction with a non-invasive technology. In order to develop this proposed treatment, this dissertation investigated the following three aims: 1) to study and optimize the injectable hydrogel for chemical, physical, and biological compatibility with the GBM therapy; 2) to utilize chemical signals to attract and entrap the GBM cells into the hydrogel; and 3) to apply focused ultrasound with high amplitude, short duration negative pressure pulses to mechanically fractionate and destroy the cells entrapped in the hydrogel. The results revealed that the hydrogel comprising 0.175 M NaHCO3(aq) and 50 wt% water content was the most optimal formulation. CXCL12 chemokine proteins loaded into the hydrogel at 5 µg/mL released slowly from the hydrogel to generate a chemical gradient and thereby attract GBM cells to promote their invasion into the hydrogel matrix. The hydrogel was demonstrated to respond well to focused ultrasound treatment, which was capable of mechanically fractionating and destroying red blood cells in the hydrogel uniformly. Overall, the results from this research provide support that this hydrogel platform can attract, entrap, and eradicate GBM cells with chemical and physical stimuli. Hence, further improvement of this platform and implementation of this novel GBM treatment may in the future help minimize GBM cancer relapse in patients who undergo conventional therapies, thereby extending their survival times.

Single cell migration in artificial collagen gels as an in vitro model system in the context of cancer are studied. Cell and matrix mechanical properties are determined using atomic force microscopy and an advanced analysis method. Matrix pore-size is studied using a novel approach and analysis method. A novel, minimally invasive approach to determine the amount of displacement of the cell microenvironment due to force generation of single cells during migration in artificial 3D collagen gels is introduced. An automated analysis and user friendly software to analyze high-throughput cell invasion is introduced. These methods are used to study cell migration and mechanical properties of the breast cancer cell lines MDA-MB-231 and MCF-7 and the influence of cell nuclear elasticity is investigated. Using mouse embryonic fibroblasts, the role of focal adhesion kinase (FAK) during cell migration is studied using FAK deficient knock-out cell lines FAK-/- and control FAK+/+ as well as kinase-dead mutants FAKR454/R454 and control FAKWT/WT.:Abstract i Acknowledgements iii 1 Introduction 1 2 Background 5 2.1 Cancer — An ever-changing Disease 5 2.1.1 Carcinogenesis and Neoplasm 6 2.1.2 Hallmarks of Cancer 7 2.1.3 Metastasis— The malignant Progression of Cancer 7 2.1.4 Metastatic Cascade 9 2.2 The Cell— Where it begins 10 2.2.1 Actomyosin Complex 12 2.2.1.1 Actin Monomer 12 2.2.1.2 Polymerization 12 2.2.1.3 Structures 14 2.2.1.4 Actin Cortex 15 2.2.1.5 Filopodia 16 2.2.1.6 Lamellipodium 16 2.2.1.7 Invadopodium 17 2.2.1.8 Stress Fibers 17 2.2.1.9 Actin in Cancer and Metastasis 17 2.2.1.10 Myosin and Actin 18 2.2.2 Focal Adhesions 19 2.2.3 Microtubules 20 2.2.4 Intermediate Filaments 21 2.2.5 Cellular Stiffness 22 2.2.6 Nuclear Deformability 23 2.3 The Extracellular Matrix— Where it happens 24 2.3.1 Components and Structure 25 2.3.2 Collagen as a Model System 26 2.3.2.1 Collagen I Fibril Formation 27 2.3.2.2 The Rat/Bovine-Collagen-Mix Model System 28 2.4 Single Cell Migration— Why it spreads 29 3 Materials and Methods 31 3.1 Cell Culture 31 3.1.1 Cancer Cells 31 3.1.2 Mouse fibroblasts 32 3.1.3 Pharmacological treatment 34 3.2 Collagen matrices 34 3.3 Cell Elasticity 36 3.3.1 Atomic Force Microscopy 36 3.3.2 Preparation 37 3.3.3 Data Aquisition 38 3.3.4 Data Analysis 38 3.4 Matrix Stiffness 40 3.4.1 Preparation 40 3.4.2 Data Aquisition 41 3.4.3 Data Analysis 41 3.5 Invasion Assay 42 3.5.1 Preparation 42 3.5.2 Data aquisition 44 3.5.3 Data Analysis 44 3.6 Matrix Topology 48 3.6.1 Preparation 49 3.6.2 Data Acquisition 50 3.6.3 Data Analysis 51 3.6.3.1 Binarization 51 3.6.3.2 Pore-Size 53 3.6.3.3 Fiber Thickness 54 3.7 Fiber Displacement 55 3.7.1 Preparation 56 3.7.2 Data Aquisition 56 3.7.3 Data analysis 57 3.7.3.1 Fiber Displacement 59 3.7.3.2 Cell Segmentation 60 3.7.3.3 Shell Analysis 61 3.8 A toolset to understand Single Cell Migration and what influences it 62 4 Results 65 4.1 Cell Elasticity 65 4.1.1 Example Force-Distance Curves 66 4.1.2 Single Cell Elasticity 67 4.2 Matrix Stiffness 69 4.3 Invasion 71 4.4 Matrix Topology 75 4.5 Influence of Cell Nucleus on Cell Migration 79 4.5.1 Cellular Elasticity 79 4.5.2 Invasion 81 4.6 Fiber Displacement 89 4.7 Effect of FAK on Cell Invasion and Fiber Displacement 93 4.7.1 FAK Knock-Out 93 4.7.2 Kinase-dead FAK Mutant 96 5 Discussion 103 References 107 / Die Einzelzellmigration in künstlichen Kollagennetzwerken als ein in vitro Modellsystem im Kontext von Krebs wurde studiert. Mechanische Eigenschaften von Zellen und der verwendeten Kollagennetzwerke wurden mithilfe der Atomic Force Microscopy (AFM) und weiterentwickelten Analysemethoden bestimmt. Die Porengröße der verwendeten Kollagennetzwerke wurde mit einer neuentwickelten Auswertemethode analysiert. Eine neuartige, minimal-invasive Methode zur Bestimmung der Verformung der Mikroumgebung von Zellen während der Migration verursacht durch Kräftegenerierung der Zelle wird beschrieben. Die Analyse des Invasions-Assays wurde automatisiert und eine nutzerfreundliche Software entwickelt, mit der große Datenmengen ausgewertet werden können. Diese Methoden wurden verwendet, um mechanische Eigenschaften und Migration der humanen Brustkrebszellinien MDA-MB-231 und MCF-7 zu studieren. Die Rolle der focal adhesion kinase (FAK) wurde mithilfe von embryonalen Maus-Fibroblasten studiert. Sowohl eine FAK knock-out Zellinie FAK-/- und Kontrolle FAK+/+, als auch eine kinase-dead Mutante FAKR454/R454 und Kontrolle FAKWT/WT wurden hinsichtlich ihrer Invasion und Verformung der Mikroumgebung analysiert.:Abstract i Acknowledgements iii 1 Introduction 1 2 Background 5 2.1 Cancer — An ever-changing Disease 5 2.1.1 Carcinogenesis and Neoplasm 6 2.1.2 Hallmarks of Cancer 7 2.1.3 Metastasis— The malignant Progression of Cancer 7 2.1.4 Metastatic Cascade 9 2.2 The Cell— Where it begins 10 2.2.1 Actomyosin Complex 12 2.2.1.1 Actin Monomer 12 2.2.1.2 Polymerization 12 2.2.1.3 Structures 14 2.2.1.4 Actin Cortex 15 2.2.1.5 Filopodia 16 2.2.1.6 Lamellipodium 16 2.2.1.7 Invadopodium 17 2.2.1.8 Stress Fibers 17 2.2.1.9 Actin in Cancer and Metastasis 17 2.2.1.10 Myosin and Actin 18 2.2.2 Focal Adhesions 19 2.2.3 Microtubules 20 2.2.4 Intermediate Filaments 21 2.2.5 Cellular Stiffness 22 2.2.6 Nuclear Deformability 23 2.3 The Extracellular Matrix— Where it happens 24 2.3.1 Components and Structure 25 2.3.2 Collagen as a Model System 26 2.3.2.1 Collagen I Fibril Formation 27 2.3.2.2 The Rat/Bovine-Collagen-Mix Model System 28 2.4 Single Cell Migration— Why it spreads 29 3 Materials and Methods 31 3.1 Cell Culture 31 3.1.1 Cancer Cells 31 3.1.2 Mouse fibroblasts 32 3.1.3 Pharmacological treatment 34 3.2 Collagen matrices 34 3.3 Cell Elasticity 36 3.3.1 Atomic Force Microscopy 36 3.3.2 Preparation 37 3.3.3 Data Aquisition 38 3.3.4 Data Analysis 38 3.4 Matrix Stiffness 40 3.4.1 Preparation 40 3.4.2 Data Aquisition 41 3.4.3 Data Analysis 41 3.5 Invasion Assay 42 3.5.1 Preparation 42 3.5.2 Data aquisition 44 3.5.3 Data Analysis 44 3.6 Matrix Topology 48 3.6.1 Preparation 49 3.6.2 Data Acquisition 50 3.6.3 Data Analysis 51 3.6.3.1 Binarization 51 3.6.3.2 Pore-Size 53 3.6.3.3 Fiber Thickness 54 3.7 Fiber Displacement 55 3.7.1 Preparation 56 3.7.2 Data Aquisition 56 3.7.3 Data analysis 57 3.7.3.1 Fiber Displacement 59 3.7.3.2 Cell Segmentation 60 3.7.3.3 Shell Analysis 61 3.8 A toolset to understand Single Cell Migration and what influences it 62 4 Results 65 4.1 Cell Elasticity 65 4.1.1 Example Force-Distance Curves 66 4.1.2 Single Cell Elasticity 67 4.2 Matrix Stiffness 69 4.3 Invasion 71 4.4 Matrix Topology 75 4.5 Influence of Cell Nucleus on Cell Migration 79 4.5.1 Cellular Elasticity 79 4.5.2 Invasion 81 4.6 Fiber Displacement 89 4.7 Effect of FAK on Cell Invasion and Fiber Displacement 93 4.7.1 FAK Knock-Out 93 4.7.2 Kinase-dead FAK Mutant 96 5 Discussion 103 References 107

During the epithelial-to-mesenchymal transition, the intracellular cytoskeleton undergoes severe reorganization which allows epithelial cells to transition into a motile mesenchymal phenotype. Among the different cytoskeletal elements, the intermediate filaments keratin (in epithelial cells) and vimentin (in mesenchymal cells) have been demonstrated to be useful and reliable histological markers. In this study, we assess the potential invasiveness of six human breast carcinoma cell lines and two mouse fibroblasts cells lines through single cell migration assays in confinement. We find that the keratin and vimentin networks behave mechanically the same when cells crawl through narrow channels and that vimentin protein expression does not strongly correlate to single cells invasiveness. Instead, we find that what determines successful migration through confining spaces is the ability of cells to mechanically switch from a substrate-dependent stress fibers based contractility to a substrate-independent cortical contractility, which is not linked to their tumor phenotype.

Micro (mi)RNAs have recently been found to play an important role in cancer biology. In order to further understand how miRNAs affect prostate tumour progression, we evaluated miRNA expression in two invasive prostate tumour lines, PC3 and DU145. We then focused our evaluation on a novel miRNA, miR-105, whose levels were significantly decreased in both tumour cell lines as compared to normal prostate epithelial cells. As miR-105 levels were reduced in prostate tumour cell lines, we restored its expression following transfection of cells with mimic constructs to over-express miR-105 in both cell lines, in order to determine its effect on various tumourigenic properties. Over-expression caused decreased tumour cell proliferation, anchorage-independent growth and invasion in vitro and inhibited tumour growth in vivo. We further identified CDK6 as a putative target of miR-105, which likely contributed to its inhibition of tumour cell growth. Our results suggest that miR-105 inhibits tumour cell proliferation and may be an interesting target to regulate tumour growth or potentially used as a biomarker to differentiate between less and more aggressive tumours in patients.

Micro (mi)RNAs have recently been found to play an important role in cancer biology. In order to further understand how miRNAs affect prostate tumour progression, we evaluated miRNA expression in two invasive prostate tumour lines, PC3 and DU145. We then focused our evaluation on a novel miRNA, miR-105, whose levels were significantly decreased in both tumour cell lines as compared to normal prostate epithelial cells. As miR-105 levels were reduced in prostate tumour cell lines, we restored its expression following transfection of cells with mimic constructs to over-express miR-105 in both cell lines, in order to determine its effect on various tumourigenic properties. Over-expression caused decreased tumour cell proliferation, anchorage-independent growth and invasion in vitro and inhibited tumour growth in vivo. We further identified CDK6 as a putative target of miR-105, which likely contributed to its inhibition of tumour cell growth. Our results suggest that miR-105 inhibits tumour cell proliferation and may be an interesting target to regulate tumour growth or potentially used as a biomarker to differentiate between less and more aggressive tumours in patients.

Micro (mi)RNAs have recently been found to play an important role in cancer biology. In order to further understand how miRNAs affect prostate tumour progression, we evaluated miRNA expression in two invasive prostate tumour lines, PC3 and DU145. We then focused our evaluation on a novel miRNA, miR-105, whose levels were significantly decreased in both tumour cell lines as compared to normal prostate epithelial cells. As miR-105 levels were reduced in prostate tumour cell lines, we restored its expression following transfection of cells with mimic constructs to over-express miR-105 in both cell lines, in order to determine its effect on various tumourigenic properties. Over-expression caused decreased tumour cell proliferation, anchorage-independent growth and invasion in vitro and inhibited tumour growth in vivo. We further identified CDK6 as a putative target of miR-105, which likely contributed to its inhibition of tumour cell growth. Our results suggest that miR-105 inhibits tumour cell proliferation and may be an interesting target to regulate tumour growth or potentially used as a biomarker to differentiate between less and more aggressive tumours in patients.