Hedgehog is a lipid-linked morphogen that is carried on lipoprotein particles and that regulates both patterning and proliferation in a wide variety of vertebrate and invertebrate tissues. Hyperactivity of Hedgehog signaling causes numerous forms of cancer. Hedgehog acts by binding to its receptor Patched, relieving the suppression of Smoothened and initiating Smoothened signaling. The mechanism by which Patched represses Smoothened has been unclear, but correlates with reduced Smoothened levels on the basolateral membrane. The structural homology of Patched with the Niemann-Pick-Type C1 protein and bacterial transmembrane transporters suggests that Patched might regulate lipid trafficking to repress Smoothened. However, no endogenous lipid regulators of Smoothened have yet been identified, nor has it ever been shown that Patched actually controls lipid trafficking. This work shows that, in Drosophila melanogaster, the Sterol-Sensing Domain of Patched regulates Smoothened trafficking from Patched-positive endosomes. Furthermore, it demonstrates that Patched recruits internalized lipoproteins to Patched-positive endosomes. Thereby, Patched regulates the efflux of specific lipoprotein-derived lipids from this compartment via its Sterol-Sensing Domain and utilizes these lipids to destabilize Smoothened on the basolateral membrane. We propose that Patched normally promotes Smoothened degradation and subsequently downregulates its activity by changing the lipid composition of endosomes through which Smoothened passes. For this purpose, Patched utilizes a specific lipid – possibly a modified sterol or sphingolipid – derived from lipoproteins. Further, we suggest that the presence of Hedgehog on lipoprotein particles inhibits utilization of their lipids by Patched.

The presented doctoral thesis is focused on the role of the Hedgehog (HH) signaling pathway in cancer pathogenesis. HH signaling pathway is an evolutionarily conserved signaling pathway that plays an essential role in embryonic development. Its activity is strictly limited to stem and progenitor cells for example in brain, lung, skin or prostate. HH pathway also plays a key role in tissue homeostasis and regeneration. Aberrantly activated HH pathway is essential in cancer progression. The aim of the presented thesis was to elucidate new details about the HH signaling pathway. We identified a new target gene of the HH pathway - the anti-apoptotic protein survivin. Survivin is considered to be an important tumor marker associated with a poor prognosis of patients. We showed that the inhibitor of HH pathway effectors GLI1 and GLI2 GANT61 reduced the survivin level in cancer cells. Subsequently, we used GANT61 and the inhibitor of the anti-apoptotic BCL2 protein family obatoclax to inhibit melanoma cells growth. We showed that the combination of these inhibitors was very effective in the eradication of melanoma cells in vitro. We also proved that GANT61 triggers the process of apoptosis in melanoma cells. We found out that the HH signaling pathway is canonically activated in many cell lines of various...

The liver is the central metabolic hub in organisms and a complex, intertwining regulatory network guarantees efficient liver processes. The morphogenic Hedgehog pathway was recently shown to play a role in regulating the underlying genetic program. Transgenic mouse models with hepatocyte-specific inactivation of Hedgehog signaling showed alterations in insulin-like growth factor homeostasis and in energy metabolism associated with increased lipid accumulation in the liver. In this thesis, it was possible to connect the observed infertility of female knockout mice with an unexpected activation of sex steroid synthesis in the liver. Associated with increased steroidogenic gene expression exclusively in hepatocytes, the plasma testosterone level was significantly elevated, which led to androgenization and an anovulatory phenotype. With these characteristics, the mouse model mimicked the human polycystic ovarian syndrome and suggested an influence of liver and hepatic Hedgehog signaling on reproduction under disease conditions. Further, murine liver metabolism was challenged with starvation starting at different times of day. The transcriptomic results were analyzed with a self-organizing map approach, allowing an intuitive interpretation of data and a thus far unknown diurnally different response of hepatic regulatory mechanisms due to starvation was revealed. In contrast to the manifoldly published and observed switch from energy-consuming to energy-providing processes due to starvation started in the morning, evening starvation led to a novel hepatic expression signature with decreased gluconeogenic gene expression and increased levels of lipid and steroid metabolism-related genes. These differences can be explained by the equally diurnally regulated expression of the corresponding regulatory transcription factors and hormones. Additionally, lipidome analysis confirmed the diurnal differences after starvation. Thus, this study emphasized the immense impact of circadian regulation on liver metabolism and suggests high accuracy when starvation is the focus of research to avoid varying results.:BIBLIOGRAPHISCHE DARSTELLUNG ................................................................................ II LIST OF ABBREVIATIONS .................................................................................................. III TABLE OF CONTENTS ....................................................................................................... IV SUMMARY ............................................................................................................................ 1 ZUSAMMENFASSUNG ......................................................................................................... 5 INTRODUCTION ................................................................................................................... 9 Liver architecture and metabolism ..................................................................................... 9 Diverse possibilities of liver metabolism regulation .......................................................... 10 Connection of Hedgehog signaling to hepatic metabolism ............................................... 10 Impact of feeding schemes on hepatic metabolism .......................................................... 13 Aims of the thesis ............................................................................................................ 14 References ...................................................................................................................... 15 CHAPTER 1 ........................................................................................................................ 18 CHAPTER 2 ........................................................................................................................ 39 PERSPECTIVE ................................................................................................................... 64 CURRICULUM VITAE ........................................................................................................... V PUBLICATIONS AND PRESENTATIONS ............................................................................ VI Publications ...................................................................................................................... VI Oral presentations ............................................................................................................ VI Poster presentations ........................................................................................................ VII AUTHOR CONTRIBUTION STATEMENT .......................................................................... VIII SELBSTSTÄNDIGKEITSERKLÄRUNG .............................................................................. XII DANKSAGUNG .................................................................................................................. XIII

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21257号 / 医博第4375号 / 新制||医||1029(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 増永 慎一郎, 教授 妹尾 浩, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The Hedgehog (Hh) signaling pathway plays many important roles throughout embryonic development, including the regulation of tissue patterning, cell differentiation, proliferation, and apoptosis. The loss of SHH signaling in human development has been shown to cause holoprosencephaly. Conversely, inappropriately activated Shh signaling in adults has been implicated in many cancers. Furthermore, Shh has been found to be a key regulator of neural stem cells in the mammalian brain. To further study the roles of Hh, I have developed a transgenic zebrafish line as a tool to monitor tissues that respond to Hh signaling throughout the vertebrate life-cycle. A number of genes have been identified that are transcriptionally up-regulated by Hh signaling. Transcription of these genes is initiated through binding of activated Gli transcription factors to an identified Gli binding site (GBS) in the cis-regulatory region. This Gli binding site is largely conserved across vertebrate species. I have generated transgene constructs in which 12 GBSs have been placed upstream of a minimum promoter that drives GFP, RFP, or Kaede fluorescent proteins. These plasmid constructs are activated in embryonic regions known to be Hh responsive, such as the ventral CNS. Treatment with cyclopamine eliminates this expression, confirming that these transgenes accurately report an active Hh response. These transgenic lines will be extremely powerful tools for research into the mechanisms by which Hh signaling regulates adult cell types such as neural stem cells. These lines will also be important tools that will help understand how misregulation of Hh signaling can lead to cancer.

Hedgehog (Hh) proteins control animal development and tissue homeostasis. They activate gene expression by regulating processing, stability, and activation of Gli/Cubitus interruptus (Ci) transcription factors. Hh proteins are secreted and spread through tissue, despite becoming covalently linked to sterol during processing. Multiple mechanisms have been proposed to release Hh proteins in distinct forms; in Drosophila, lipoproteins facilitate long-range Hh mobilization but also contain lipids that repress the pathway. Here, we show that mammalian lipoproteins have conserved roles in Sonic Hedgehog (Shh) release and pathway repression. We demonstrate that lipoprotein-associated forms of Hh and Shh specifically block lipoprotein-mediated pathway inhibition. We also identify a second conserved release form that is not sterol-modified and can be released independently of lipoproteins (Hh-N*/Shh-N*). Lipoprotein-associated Hh/Shh and Hh-N*/Shh-N* have complementary and synergistic functions. In Drosophila wing imaginal discs, lipoprotein-associated Hh increases the amount of full-length Ci, but is insufficient for target gene activation. However, small amounts of non-sterol-modified Hh synergize with lipoprotein-associated Hh to fully activate the pathway and allow target gene expression. The existence of Hh secretion forms with distinct signaling activities suggests a novel mechanism for generating a diversity of Hh responses.

The objective of this study was to investigate primary cilia and their mechanotransduction role in lung adenocarcinoma tumor progression. The main focus investigated the effect of primary cilia on cell cycle progression, survival, adhesion and migration analysis of these cells and the role of sonic hedgehog signaling pathway in mechanotransduction. Human Non-Small Cell Lung Cancer (NSCLC) adenocarcinoma biopsies contain more primary cilia than non-tumor lung sections. To observe the effects of primary cilia presence in lung cancer cells in-vitro, formation of primary cilia is inhibited using small interfering RNA. A549 cells with intact primary cilia observe less cell cycle progression than cells deficient in primary cilia under static and cyclic stretch conditions. Primary cilia cause higher cell survival and adhesion. Increase in cell adhesion also increases the migration and wound closure rates in control samples compared to samples treated with inhibition of IFT88, thereby increasing the metastasis of these cells. Several downstream regulatory genes in sonic hedgehog signaling pathway observe significantly decreased gene expressions in primary cilia deficient cells, thus indicating inefficient mechanotransduction. Therefore, cancer cells need primary cilia to survive, adhere and migrate and continue tumor progression.

Hedgehog (Hh) proteins control animal development and tissue homeostasis. They activate gene expression by regulating processing, stability, and activation of Gli/Cubitus interruptus (Ci) transcription factors. Hh proteins are secreted and spread through tissue, despite becoming covalently linked to sterol during processing. Multiple mechanisms have been proposed to release Hh proteins in distinct forms; in Drosophila, lipoproteins facilitate long-range Hh mobilization but also contain lipids that repress the pathway. Here, we show that mammalian lipoproteins have conserved roles in Sonic Hedgehog (Shh) release and pathway repression. We demonstrate that lipoprotein-associated forms of Hh and Shh specifically block lipoprotein-mediated pathway inhibition. We also identify a second conserved release form that is not sterol-modified and can be released independently of lipoproteins (Hh-N*/Shh-N*). Lipoprotein-associated Hh/Shh and Hh-N*/Shh-N* have complementary and synergistic functions. In Drosophila wing imaginal discs, lipoprotein-associated Hh increases the amount of full-length Ci, but is insufficient for target gene activation. However, small amounts of non-sterol-modified Hh synergize with lipoprotein-associated Hh to fully activate the pathway and allow target gene expression. The existence of Hh secretion forms with distinct signaling activities suggests a novel mechanism for generating a diversity of Hh responses.

Non-small cell lung cancer belongs to most frequent malignant tumours at all worldwide. Despite significant progress in knowledge about etiopathogenesis and targeted anticancer therapy, basic scientific research in this particular field and development of more effective treatment remains challenging. In case of its inadequate activation, the Hedgehog signaling pathway is involved in non-small cell cancer development. P53 is well known tumour suppressor gene, that serves as anticancer barrier. Its activity is mostly determined by the transcriptional activation of many pro-apoptotic genes, one of which is SIVA-1. Recently, it has been surprisingly shown, that SIVA-1 has also pro-oncogenic properties in a mouse model of non-small cell lung cancer. The aim of this study was to clarify the importance of Hedgehog signaling pathway and protein SIVA-1 and their potential relationship in development and progression of human non-small cell lung cancer. In selected cell lines of human non-small cell lung cancer, expression of each single component of Hedgehog signalign pathway was detected. In the tissue samples of tumour obtained from 39 patients that underwent surgery for non-small cell lung cancer and selected cell lines of the same tumour, expression of SIVA-1 protein was revealed. These findings indicate...

The Hedgehog signaling pathway regulates many processes during embryogenesis and the homeostasis of adult organs. Recent data suggest that central metabolic processes and signaling cascades in the liver are controlled by the Hedgehog pathway and that changes in hepatic Hedgehog activity also affect peripheral tissues, such as the reproductive organs in females. Here, we show that hepatocyte-specific deletion of the Hedgehog pathway is associated with the dramatic expansion of adipose tissue in mice, the overall phenotype of which does not correspond to the classical outcome of insulin resistance-associated diabetes type 2 obesity. Rather, we show that alterations in the Hedgehog signaling pathway in the liver lead to a metabolic phenotype that is resembling metabolically healthy obesity. Mechanistically, we identified an indirect influence on the hepatic secretion of the fibroblast growth factor 21, which is regulated by a series of signaling cascades that are directly transcriptionally linked to the activity of the Hedgehog transcription factor GLI1. The results of this study impressively show that the metabolic balance of the entire organism is maintained via the activity of morphogenic signaling pathways, such as the Hedgehog cascade. Obviously, several pathways are orchestrated to facilitate liver metabolic status to peripheral organs, such as adipose tissue.