Schweinfurthins are a family of natural products with significant anti-cancer activities. They were originally identified in the National Cancer Institute (NCI) human 60 cancer cell line screening. The growth inhibition profile of schweinfurthins is distinct from other clinically used anti-cancer agents, indicating that they have a novel mechanism of action or have a previously unrecognized protein target. Previous studies showed that schweinfurthins affect multiple cellular processes in cancer cells. For example, schweinfurthins can alter cytoskeleton organization, induce ER stress and apoptosis, and inhibit the mevalonate pathway. The mevalonate pathway is responsible for the production of isoprenoids and cholesterol, which have been shown to play regulatory roles in the Hedgehog (Hh) signaling pathway. In this study, we found that the Hh signaling pathway in NIH-3T3 and SF-295 cells was inhibited by schweinfurthins. The supplementation of mevalonate and cholesterol partially restored Hh signaling, indicating that schweinfurthins inhibit Hh signaling partially by down-regulating the products from the mevalonate pathway. Interestingly, schweinfurthins in combination with cyclopamine, an inhibitor of the Hh singaling pathway, synergistically decreased cell viability. In order to better understand the underlying mechanism of the anti-cancer action of schweinfurthins, we attempted to identify the protein target of schweifnurthins. Affinity chromatography was performed to pull down the protein target. We found that schweinfurhtins bound to the M2 isoform of pyruvate kinase (PKM2) and inhibit its pyruvate kinase activity. Knockdown of PKM2 by siRNA increased the sensitivity of SF-295 cells to schweinfurthins. The inhibition of PKM2 by schweinfurthins led to a reduction in the rate of glycolysis in cancer cells. Fructose 1,6-bisphosphate (FBP), an activator of PKM2, could alleviate schweinfurthin-mediated inhibition on PKM2 and glycolysis. Notably, FBP could also partially reverse the reduction of cell viability in the presence of schweinfurthins. Taken together, these studies revealed the mechanism by which schweinfurthins inhibit Hh signaling. In addition, we uncovered PKM2 as a schwienfurthin target and highlighted the importance of glycolysis suppression as a mechanism of the anti-cancer action of schweinfurthins.

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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...

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 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.

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...

Les genodermatoses sont des maladies génétiques rares à expression cutanée. Parmi elles, les dysplasies ectodermiques (DE) caractérisées par des anomalies du développement d’au moins deux structures ectodermiques (dents, ongles, glandes sudorales et poils), constituent un groupe hétérogène de genodermatoses de plus de 200 syndromes rares. Si la plupart de ces syndromes associent des anomalies des seuls dérivés ectodermiques, d'autres plus complexes, tels que le syndrome de Bazex-Dupré-Christol et l’Incontinentia Pigmenti, rassemblent en plus des manifestations disparates. Le syndrome de Bazex-Dupré-Christol (SBDC) associe une DE à la prédisposition aux carcinomes basocellulaires (CBCs) de survenue précoce. L’étude de 6 familles nous a permis d’identifier, chez 2 d’entre elles, une mutation tronquante dans le gène ACTRT1, codant la protéine Arp-T1. Dans l’épiderme, la protéine Arp-T1 est diminuée chez tous les patients atteints de SBDC, porteurs ou non de mutations dans le gène ACTRT1. Le séquençage à haut débit de la région candidate a permis d’identifier des mutations dans des régions transcrites, régulatrices du gène ACTRT1 chez les patients des 4 autres familles. Notant que la voie Hedgehog est dérégulée dans 70 % des CBCs, nous avons démontré qu’ACTRT1 est un nouvel inhibiteur de cette voie, en particulier par sa liaison au promoteur de GLI1 dont il inhibe l’expression. Enfin, ACTRT1 est un nouveau gène suppresseur de tumeur capable de réduire in vivo la progression tumorale de certaines lignées cancéreuses par la régulation de gènes impliqués dans la prolifération, la mort et la survie cellulaire, ou encore la migration. L’Incontinentia Pigmenti (IP) est une affection multisystémique caractérisée par une atteinte de la peau, des dents, des yeux et parfois du système nerveux central. Elle résulte de mutation dans le gène NEMO et l’abolition de l’activation de la voie NF-KB. L’étude d’une famille concernée par l’IP à permis d’identifier une nouvelle mutation d’épissage du gène NEMO aboutissant à l’expression d’une protéine tronquée. Cette protéine conserve l’intégralité des domaines fonctionnels de NEMO connus à ce jour. Sa caractérisation a révélé une perte d’interaction avec SHARPIN, composants du complexe LUBAC permettant l’ubiquitination linéaire. Il s’agit de la première mutation humaine de NEMO montrant l’importance de son ubiquitination linéaire dans l’activation de la voie NF-KB. Mes travaux de thèse ont ainsi mis en évidence de nouveaux mécanismes physiopathlogiques responsables de deux formes de dysplasie ectodermique. Ces mécanismes reflètent la complexité des voies moléculaires impliquées dans le développement de la peau et le maintien de son homéostasie durant la vie adulte. / Genodermatoses are rare genetic diseases with cutaneous expression. Among them, ectodermal dysplasia (ED) characterized by abnormal development of at least two ectodermal structures (teeth, nails, sweat glands and hair) constitute a heterogeneous group of genodermatoses of more than 200 rare syndromes. While most of these syndromes associate only abnormalities of the ectodermal derivatives, others more complex, such as Bazex-Dupré-Christol syndrome and Incontinentia Pigmenti, bring together disparate manifestations. Bazex-Dupré-Christol syndrome (BDCS) associates ED with predisposition to early basal cell carcinoma (BCCs). The study of 6 families allowed us to identify, in 2 of them, a truncated mutation in the ACTRT1 gene, encoding the Arp-T1 protein. In the epidermis, Arp-T1 protein is decreased in all patients with BDCS, carrying or not of mutations in ACTRT1 gene. High-throughput sequencing of the candidate region allowed to identify mutations in transcribed enhancer regions, regulating the ACTRT1 gene in patients of the remaining 4 families. Noting that the Hedgehog pathway is deregulated in more than 70% of BCCs, we have demonstrated that ACTRT1 is a novel inhibitor of this pathway, via its binding to GLI1 promoter and inhibiting its expression. Finally, ACTRT1 is a new tumor suppressor gene capable of reducing in vivo the tumor progression of certain cancer lines by the regulation of genes involved in proliferation, death and cell survival, or migration. Incontinentia Pigmenti (IP) is a multisystemic disorder characterized by involvement of skin, teeth, eyes and sometimes the central nervous system. It results from mutation in the NEMO gene and the abolition of activation of NF-KB pathway. The study of a family concerned with IP allowed to identify a new splicing mutation of NEMO gene leading to a truncated protein expression. This protein retains all the functional domains of NEMO known. Its characterization revealed a loss of interaction with SHARPIN, components of LUBAC complex allowing linear ubiquitination. This is the first human mutation of NEMO showing the importance of its linear ubiquitination in the activation of the NF-KB pathway. Thus, my thesis work revealed novel physiopathological mechanisms responsible for two forms of ectodermal dysplasia. These mechanisms reflect the complexity of the molecular pathways involved in the development of the skin and the maintenance of its homeostasis during adult life.

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.

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.