Prostate and breast cancer are among the most commonly diagnosed cancers and leading causes of cancer-related deaths in men and women worldwide. It is therefore evident that enhanced understanding of tumorigenesis is required to improve diagnostic tools, improve prognostics and identify novel therapeutic targets. The goal of this dissertation was to elucidate the role of primary cilia in prostate and breast cancer. Little is known about the role primary cilia may play in these cancers. Primary cilia are microtubule-based organelles which aid in sensing the extracellular environment and participate in signal transduction. Important developmental signaling pathways, such as Hedgehog (Hh) and Wnt signaling pathways, involve cilia. These pathways have also been implicated in prostate and breast cancer. In this work, we demonstrate that cilia are lost through prostate cancer progression. The few remaining cilia on prostate cancers appeared to be dysfunctional, as assessed by quantifying cilia lengths, an indirect measure of functionality. We also investigated a link between the observed cilia loss and canonical Wnt signaling in prostate cancers. Primary cilia have been determined to have a suppressive role in Wnt signaling, therefore we predicted loss of cilia to correlate with increased Wnt signaling. A link between cilia loss or shortened cilia and activated Wnt signaling was suggested in a subset of prostate cancers. Our lab has established that cilia are similarly lost in breast cancer. These data suggested the hypothesis that cilia may act as tumor suppressor organelles in the prostate and breast. To test this hypothesis, we knocked down cilia in an oncogenic mammary mouse model and assessed changes in tumor growth and characteristics. We observed enhanced tumor growth with cilia loss. The data supports the hypothesis that primary cilia may be playing a tumor suppressor role in the prostate and breast, and provides promising avenues for identifying novel therapeutic approaches for cancer patients.

La barrière hémato-encéphalique (BHE) protège le système nerveux central (SNC) en contrôlant le passage des substances sanguines et des cellules immunitaires. La BHE est formée de cellules endothéliales liées ensemble par des jonctions serrées et ses fonctions sont maintenues par des astrocytes, celles ci sécrétant un nombre de facteurs essentiels. Une analyse protéomique de radeaux lipidiques de cellules endothéliales de la BHE humaine a identifié la présence de la voie de signalisation Hedgehog (Hh), une voie souvent liées à des processus de développement embryologique ainsi qu’au niveau des tissus adultes. Suite à nos expériences, j’ai déterminé que les astrocytes produisent et secrètent le ligand Sonic Hh (Shh) et que les cellules endothéliales humaines en cultures primaires expriment le récepteur Patched (Ptch)-1, le co-récepteur Smoothened (Smo) et le facteur de transcription Gli-1. De plus, l’activation de la voie Hh augmente l’étanchéité des cellules endothéliales de la BHE in vitro. Le blocage de l’activation de la voie Hh en utilisant l’antagoniste cyclopamine ainsi qu’en utilisant des souris Shh déficientes (-/-) diminue l’expression des protéines de jonctions serrées, claudin-5, occcludin, et ZO-1. La voie de signalisation s’est aussi montrée comme étant immunomodulatoire, puisque l’activation de la voie dans les cellules endothéliales de la BHE diminue l’expression de surface des molécules d’adhésion ICAM-1 et VCAM-1, ainsi que la sécrétion des chimiokines pro-inflammatoires IL-8/CXCL8 et MCP-1/CCL2, créant une diminution de la migration des lymphocytes CD4+ à travers une monocouche de cellules endothéliales de la BHE. Des traitements avec des cytokines pro-inflammatoires TNF-α and IFN-γ in vitro, augmente la production de Shh par les astrocytes ainsi que l’expression de surface de Ptch-1 et de Smo. Dans des lésions actives de la sclérose en plaques (SEP), où la BHE est plus perméable, les astrocytes hypertrophiques augmentent leur expression de Shh. Par contre, les cellules endothéliales de la BHE n’augmentent pas leur expression de Ptch-1 ou Smo, suggérant une dysfonction dans la voie de signalisation Hh. Ces résultats montrent que la voie de signalisation Hh promeut les propriétés de la BHE, et qu’un environnement d’inflammation pourrait potentiellement dérégler la BHE en affectant la voie de signalisation Hh des cellules endothéliales. / The blood-brain barrier (BBB), composed of tightly bound endothelial cells (ECs), regulates the entry of blood-borne molecules and immune cells into the CNS. Recent studies indicate that the Hedgehog (Hh) signaling pathway in adult tissues plays an important role in vascular proliferation, differentiation and tissue repair. Using a lipid membrane raft-based proteomic approach, I have identified the Hedgehog (Hh) pathway as a signaling cascade involved in preserving and upkeeping BBB functions. My study shows that human astrocytes express and secrete Sonic Hh (Shh) and conversely, that human BBB-ECs bear the Hh receptor Patched-1 (Ptch-1), the signal transducer Smoothened (Smo) as well as transcription factors of the Gli family. Furthermore, activation of the Hh pathway in BBB-ECs restricts the passage of soluble tracers in vitro. By blocking the Hh signaling in vitro and by using Shh knock-out (-/-) embryonic mice, I demonstrate a reduced expression of TJ molecules claudin-5, occludin and ZO-1. Hh activation also decreases the surface expression of cell adhesion molecules ICAM-1 and VCAM-1, and decreases BBB-ECs secretion of pro-inflammatory chemokines IL-8/CXCL8 and monocytes chemoattractant protein 1 MCP-1/CCL2, resulting in a reduction of migrating CD4+ lymphocytes across human BBB-EC monolayers. In vitro treatment with inflammatory cytokines TNF-α and IFN-γ, upregulates the production of astrocytic Shh and the BBB-EC surface expression of Ptch-1 and Smo. In active Multiple Sclerosis (MS) lesions, in which the BBB is disrupted, Shh expression is drastically upregulated in hypertrophic astrocytes, while Ptch-1 and Smo expression is down-regulated or left unchanged, suggesting that a deregulation in the Hh signaling pathway may prevent the barrier stabilizing properties of Hh. Our data demonstrate an anti-inflammatory and BBB-promoting effect of astrocyte-secreted Hh and suggest that a pro-inflammatory environment disrupt the BBB by impacting, at least in part, on Hh signaling in brain ECs.

L’Ostéoarthrose (OA) est une maladie articulaire entrainant une dégénérescence du cartilage et une ossification de l’os sous-chondral. Elle touche un Canadien sur 10 et pourtant l’origine de cette pathologie est encore inconnue. Dans le cadre de ce projet, la contribution de deux facteurs de transcription, NFAT1 et PITX1, dans la régulation transcriptionnelle du promoteur d’IHH a été examiné compte tenu de l’implication potentielle de la voie hedgehog (Hh) et de ces facteurs dans la pathogenèse de l’OA. La voie de signalisation Hh régule la croissance et la différenciation des chondrocytes. Indian hedgehog (IHH), l’un des trois membres de la famille Hh, contrôle leur prolifération et leur différenciation. / Osteoarthritis (OA) is the most common joint disorder and is characterized by cartilage degradation and endochondral ossification. One in every ten Canadians is affected, yet its aetiopathogenesis remains unknown. In this present study, two new regulators of the IHH promoter, NFAT1 and PITX1, were studied. The downregulation of IHH expression by these factors could contribute to the OA pathogenesis. The Hedgehog (Hh) signaling pathway regulates chondrocyte growth and differentiation in the growth plate. Indian hedgehog (IHH), one of its members, stimulates chondrocyte proliferation and osteoblast differentiation. IHH is essential in skeletogenesis, osteoblastogenesis and cartilage growth.

7-Dehydrocholesterol (7-DHC) is the substrate of 7-dehydrocholesterol reductase (DHCR7) in the cholesterol synthesis pathway. Keratinocytes in human skin possess the enzymes necessary for cholesterol synthesis but are also responsible for vitamin D3 synthesis from 7-DHC by exposure to UVB irradiation. It has been well established that DHCR7 is regulated by the SREBP pathway in the regulation of cholesterol synthesis, but little is known about the regulation of DHCR7 by the vitamin D pathway. In this study, the regulation of DHCR7 activity by vitamin D was explored. Treatment of adult human epidermal keratinocyte (HEKa) cells with vitamin D3 resulted in a rapid decrease in DHCR7 activity which was not due to changes in the amount of enzyme present. This suppression of activity was observed only in HEKa cells, a primary cell line cultured from normal human skin, and not in an immortalized skin cell line (HaCaT cells) nor in two liver-derived hepatoma cell lines. Because vitamin D3 treatment of HEKa cells did not change the content of lanosterol nor 7-DHC, these results suggest that vitamin D3 rapidly down-regulates the entire cholesterolgenesis pathway, presumably at a very early step in the pathway. 25-Hydroxyvitamin D3, the first metabolite and circulating form of vitamin D3, had a lesser effect on DHCR7 activity, while 1,25-dihydroxyvitamin D3, the activated form of the vitamin, had no effect on DHCR7, indicating that the vitamin D receptor is not involved. The decrease in DHCR7 activity was due neither to the dephosphorylation of the enzyme, an established mechanism of inactivation, nor to direct inhibition by vitamin D3. Vitamin D3 markedly inhibited proliferation and induced differentiation of HEKa cells, suggesting a possible role for hedgehog signaling in the decrease in DHCR7 activity.

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The Hedgehog (Hh) signalling pathway is one of the key signalling pathways orchestrating intricate organogenesis, including the development of neural tube, heart and skeletal muscle. Yet, insufficient mechanistic understanding of its diverse roles is available. Here, we show the molecular mechanisms regulating the neurogenic, cardiogenic and myogenic properties of Hh signalling, via effector protein Gli2, in embryonic and adult stem cells. In Chapter 2, we show that Gli2 induces neurogenesis, whereas a dominant-negative form of Gli2 delays neurogenesis in P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (ES) cell model. Furthermore, we demonstrate that Gli2 associates with Ascl1/Mash1 gene elements in differentiating P19 cells and activates the Ascl1/Mash1 promoter in vitro. Thus, Gli2 mediates neurogenesis in P19 cells at least in part by directly regulating Ascl1/Mash1 expression. In Chapter 3, we demonstrate that Gli2 and MEF2C bind each other’s regulatory elements and regulate each other’s expression while enhancing cardiomyogenesis in P19 cells. Furthermore, dominant-negative Gli2 and MEF2C proteins downregulate each other’s expression while imparing cardiomyogenesis. Lastly, we show that Gli2 and MEF2C form a protein complex, which synergistically activates cardiac muscle related promoters. In Chapter 4, we illustrate that Gli2 associates with MyoD gene elements while enhancing skeletal myogenesis in P19 cells and activates the MyoD promoter in vitro. Furthermore, inhibition of Hh signalling in muscle satellite cells and in proliferating myoblasts leads to reduction in MyoD and MEF2C expression. Finally, we demonstrate that endogenous Hh signalling is important for MyoD transcriptional activity and that Gli2, MEF2C and MyoD form a protein complex capable of inducing skeletal muscle-specific gene expression. Thus, Gli2, MEF2C and MyoD participate in a regulatory loop and form a protein complex capable of inducing skeletal muscle-specific gene expression. Our results provide a link between the regulation of tissue-restricted factors like Mash1, MEF2C and MyoD, and a general signal-regulated Gli2 transcription factor. We therefore provide novel mechanistic insights into the neurogenic, cardiogenic and myogenic properties of Gli2 in vitro, and offer novel plausible explanations for its in vivo functions. These results may also be important for the development of stem cell therapy strategies.

Abstract – The Role of Patched1 in Epidermal Homeostasis Hedgehog (Hh) signalling is a critical pathway involved in the development of many, if not all, organ systems. However the abnormal activation of Hh signalling in fully developed adult organs leads to cancer. Mutation of the Hh signal receptor, Patched1 (Ptc1), causes Naevoid Basal Cell Carcinoma Syndrome, which presents with developmental defects and cancer predisposition. The activation of Hh signalling is seen in a wide range of non-inherited cancer types also, including Medulloblastoma and Basal Cell Carcinoma (BCC) of the skin. BCC is the most common form of human cancer and over 90% of cases are linked to abnormally high Hh signalling. Hh signalling is known to regulate hair follicle morphogenesis during development and more recently has been linked to modulation of the embryonic epidermal stem cell compartment. However both the mechanisms behind this process and the mechanism behind its induction of BCC are still uncharacterised. The aim of this project was to determine the role of Ptc1 in the skin, particularly the adult stem cell compartment, and the role of Hh signalling in BCC formation. The deletion of Ptc1 specifically in the adult epidermis was enabled by the creation of a K14-Cre Recombinase induced Ptc1 Conditional (K14-Cre:Ptc1C/C) transgenic mouse line. Proliferation was increased throughout the epithelia and BCC-like lesions developed within 4 weeks of Ptc1 deletion. This indicates that Hh signalling plays a critical role in repressing cell turnover in the interfollicular epithelium (IFE) and bulge region in the adult despite being previously reported not to play a role in this area. Ptc1 deletion in the epithelia was also found to promote the IFE lineage over hair follicles and expand the expression of many proposed stem cell markers, including K15, Sox9 and p63. K14-Cre:Ptc1C/C transgenic mice also exhibited a severe growth defect, linked to low levels of Igf1 hormone in the serum. Igf1 binding protein alteration in the skin was determined to be the most likely cause and prompted the investigation of Igf axis signalling in Ptc1 deleted epidermis. Insulin-like growth factor binding protein 2 was found to localise to the bulge or stem cell region of the hair follicle, and was increased in K14-Cre:Ptc1C/C epidermis. Igfbp2 was coincident with a loss of PI3K/Akt signal translation. The majority of human BCC samples also expressed Igfbp2 at much higher levels than surrounding normal tissue indicating these results are relevant to the human BCC condition also. Interestingly Hh activation was also shown to increase p38 MAPK throughout the epidermis indicating it is a universal target of Hh signalling in the skin. In summary we have found that Hh signal activation in the epidermis promotes the bulge/stem cell and interfollicular lineages of the skin at the expense of hair follicles. Finally the modulation of PI3K/Akt signalling by Igfbp2 in the bulge is perhaps mediating the effect of Hh signalling via the promotion of the bulge lineage leading to the development of BCC.

Brain tumours comprise about 25% of all cancers in children. Medulloblastoma – which arise in the cerebellum – are the most common and severe malignant pediatric brain tumour and the leading cause of cancer-related deaths in children under the age of 9. Treatment of medulloblastoma remains conventional, with surgery followed by chemotherapy and radiation. These measures are successful in about 60-80% of cases but treatment results in severe side effects due to its toxicity to the central nervous system. Therefore it is of utmost importance to define the signalling pathways and genetic changes involved in the formation of medulloblastoma in order to allow for better diagnosis and treatments with higher efficiency and decreased toxicity. The cell of origin for medulloblastoma is thought to be the granule neuron progenitor, a cell type arising from cerebellar stem cells of the ventricular zone. After birth granule neuron progenitors differentiate into mature granule neurons which populate the majority of the cerebellum and are crucial for its cognitive functions and motor coordination. The Hedgehog signalling pathway plays an important role in medulloblastoma generation and murine models with activated Hedgehog signalling develop medulloblastoma at high frequencies. In addition, the Notch pathway has been implicated in the generation of medulloblastoma, and interaction between the two pathways has been suggested. Inhibitors of both Hedgehog and Notch are currently in clinical trials however knowledge of possible interactions between them could lead to more effective treatment strategies. The aim of this project was to investigate the interaction of Hedgehog and Notch signalling in normal brain development and medulloblastoma. Two mouse models allowed activation of Hedgehog and inactivation of Notch signalling in granule neuron progenitors and cerebellar ventricular zone stem cells. In granule neuron progenitors canonical Notch signalling is not required and the layering and cell types of RBP-Jlox/lox;Math1-Cre cerebella appear identical to control brains. In contrast, Notch inactivation in ventricular zone stem cells with GFAP-Cre resulted in increased differentiation of stem cells into progenitor cells accompanied by an overall developmental delay in neuronal differentiation. Medulloblastoma generated by Hedgehog activation (through inactivation of the negative Hedgehog regulator Ptc1) in both cell types cannot be blocked by Notch inactivation. Furthermore, medulloblastoma of Ptc1lox/lox;RBP-Jlox/lox;GFAP-Cre and those of Ptc1lox/lox;RBP-Jlox/lox;Math1-Cre mice are identical in incidence as well as histology to the tumours in which only Hedgehog signalling is activated. This implies that even though Notch signalling plays an important role in cerebellar stem cells it is not required for the initiation and development of Hedgehog induced medulloblastoma. Therefore it may be crucial to consider the Hedgehog status of patients in order to interpret clinical data of Notch pathway inhibitors and even more importantly these results suggest that determining the Hedgehog status might be crucial before treatment of medulloblastoma patients with Notch pathway inhibitors.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

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