骨肉瘤是最常見的原發性骨腫瘤之一。青少年和年輕成年人屬于患上骨肉瘤的高危群組。利用現今的治療方法，非轉移性骨肉瘤病患者的長期存活率達百份之七十。不過，轉移性和復發骨肉瘤病人的預後並不理想，其五年存活率低於百份之二十。骨肉瘤的高死亡率帶出進一步瞭解骨肉瘤成因的重要性。雖然多個與骨肉瘤病發機理有關的遺傳風險因素已被發現，例如TP53基因和Rb基因的功能喪失性突變均會使其攜帶者傾向於出現骨肉瘤。但是這些結果仍未能找出專一性較強的骨肉瘤遺傳標記。因此有需要進一步研究骨肉瘤的主要病發機理。其中Hedgehog信號通路對於出生前後階段的骨絡生長和發育均有重要的調控作用。再者，之前在Ptch1{U+1D9C}/{U+1D9C}; HOC-Cre小鼠模型所做的研究也表明，於造骨細胞上調Hedgehog信號通路會導致長骨局部增生。另外，多種癌症的病發亦已知與Hedgehog信號通路失調有密切關係。根據這些發現，本研究項目假設Hedgehog信號通路失調是導致骨肉瘤病發的一個遺傳風險因素。一種新穎的小鼠模型 Ptch1{U+1D9C}/⁺; p53⁺/⁻; HOC-Cre被構建並採用於證明此假設。在這小鼠模型，Hedgehog信號通路只有在造骨細胞部分地上調。另外，p53的雜合敲除有助增加骨肉瘤的病發機率。實驗結果表明七至十二個月週歲的Ptch1{U+1D9C}/⁺; p53⁺/⁻; HOC-Cre小鼠出現類此骨肉瘤的症狀。原帶類骨肉瘤組織含有大量類骨質和多核細胞。由原帶類骨肉瘤組織所衍生的細胞系Kios-5有癌細胞在體和離體的特性。及後又發現由上調Hedgehog信號通路所導致的類骨肉瘤與Hippo信號通路有聯動關係。這個結果與一種由Hedgehog信號通路上調所導致的成神經管細胞瘤的結果互相吻合。故此，本研究也想探討Hippo信號通路和骨肉瘤的病變之間的關聯。實驗結果指出由Hedgehog信號通路上調而導致的骨肉瘤亦連帶上調Hippo信號通路的轉錄活度。與此同時，兩個Hippo信號通路的主要轉化啟動因子(Yap1和Taz) 還有Hippo信號通路的下游目標基因（Ctgf和Cyr61）的表達在原帶類骨肉瘤組織和類骨肉瘤衍生的細胞系均有上調。這印證了越來越多的研究結果表示Hippo信號通路在癌症的病變過程起關鍵作用。再者，類骨肉瘤細胞系的致腫瘤性也依賴於Yap1。更重要是，在組織陣列裡百份之八十七的人體骨肉瘤樣本表達YAP蛋白。這結果表明Hippo信號通路在骨肉瘤有臨床方面的一定重要性。另外，Kios-5活化一個多潛性標記基因Nanog的表達。這顯示本研究的骨肉瘤模型帶有類似幹細胞的特質。Kios-5還能夠被誘導分化成有骨質特性和脂肪特性的細胞。這些跡象顯示Kios-5的可塑性比較高。及後的一組實驗進一步地識別出潛在的分秘物質控制著Hedgehog與Hippo信號通路之間的相互作用。總結，我們的結果表明在雜合敲除p53基因的情況下於造骨細胞上調Hedgehog信號通路會引發骨肉瘤。當中亦上調Hippo信號通路的轉錄活度。而這兩條信號通路之間透過潛在的分秘物質而產生相互作用。 / Osteosarcoma (OS) is one of the most common primary bone tumors. Adolescence and young adults are the high risk groups of OS development. Under the current treatment, the overall survival rate for patients with non-metastatic OS approaches 75-80%. However, the 5-year survival rate for patients with metastatic and recurrent approached 60%. Though OS was treatable, the high incidence of OS metastasis disease and severe sequelae accompanying medical intervention necessitate better understanding of the etiology of OS. Multiple genetic risk factors, such as loss-of-function mutations in TP53 or RB1, predispose individual to OS development. However, these findings did not identify specific genetic markers for OS and thus, major mechanisms underlying OS pathogenesis require further investigation. In particular, Hedgehog (Hh) signaling has been well implicated in regulating bone growth and development at both embryonic and postnatal stages. Previous studies using Ptch1c/c; HOC-Cre mutant mice have shown that upregulation of Hh signaling in mature osteoblasts leads to focal bone overgrowth in the long bones. Dysregulated Hh signaling has also been implicated in a wide range of cancers. Based on these facts, the current studies hypothesized that dysregulation of Hh signaling in bones is a genetic risk factor that predispose to osteosarcoma development. A novel mouse model, Ptch1{U+1D9C}/⁺ ; p53⁺/⁻; HOC-Cre was generated to test this hypothesis in vivo. The mutants have partial upregulation of Hh signaling specifically in mature osteoblasts in a p53⁺/⁻ background to enhance the incident rate of OS. The results demonstrated that the mutant mice developed OS-like phenotypes starting from 7-12 months of age. The primary OS-like tumor tissues possessed massive osteoid with the presence of multinucleated cells. The tumor-derived cell line Kios-5 revealed cancer properties both in vitro and in vivo. The Hh signaling-induced OS in the mutant mice was shown to crosstalk with Hippo signaling pathway, which was previously demonstrated to be involved in a subset of medulloblastoma that is caused by Hedgehog signaling upregulation. Consistent with emerging evidence that Hippo pathway has critical roles in cancer development, results from the current studies suggested that OS initiated by upregulated Hedgehog signaling in mature osteoblasts led to upregulated transcriptional activity of Hippo signaling. The mutant mice showed upregulated expression of the main transcriptional coactivators of the Hippo pathway, Yap1 and Taz as well as the downstream target genes of Hippo pathway, Ctgf and Cyr61. The tumorigenicity of OS-like tumor derived cells showed Yap1-dependence. Importantly, Hippo pathway has clinical relevance in OS pathogenesis as 78% of human OS samples in tissue array showed YAP expression. Besides, tumor cells derived from the OS model showed stem cells properties through upregulating the expression of the key pluripotent marker gene, Nanog. And the OS cells revealed higher lineage plasticity as they were inducible to undergo osteoblastic and adipogenic differentiation. Further, the results suggested potential secretory factor(s) was/were mediated the interaction between Hh and Hippo pathways were recognized. In conclusion, the findings indicated that upregulated Hedgehog signaling in the mature osteoblasts under the p53⁺/- context initiated osteosarcoma development, which also led to upregulated Hippo pathway transcriptional activity. The crosstalk between these two signaling pathways was mediated through some potential secretory factor(s). / Detailed summary in vernacular field only. / Chan, Lok Hei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 187-205). / Abstracts also in Chinese. / ABSTRACT OF THESIS ENTITLED --- p.iii / 中文摘要 --- p.vi / ACKNOWLEDGEMENTS --- p.viii / CONTENTS --- p.ix / LIST OF ABBREVIATIONS --- p.xii / LIST OF FIGURES --- p.xiv / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Introductory statement --- p.1 / Chapter 1.2 --- Introduction of osteosarcoma --- p.1 / Chapter 1.2.1 --- Clinical feature and treatment --- p.4 / Chapter 1.2.2 --- Genetic risk factors --- p.5 / Chapter 1.2.3 --- Invasion and metastasis of osteosarcoma --- p.8 / Chapter 1.3 --- Bone formation in mammals and its regulation by Hedgehog signaling --- p.10 / Chapter 1.3.1 --- Overview of bone formation in vertebrates --- p.10 / Chapter 1.3.2 --- Hedgehog signaling in bone formation --- p.19 / Chapter 1.4 --- Hedgehog signaling and cancer development --- p.27 / Chapter 1.4.1 --- Hedgehog signaling pathway --- p.27 / Chapter 1.4.2 --- Hedgehog signaling in cancer pathogenesis --- p.34 / Chapter 1.4.2.1 --- Mutation-driven, ligand-independent activation of Hedgehog in cancer development --- p.38 / Chapter 1.4.2.2 --- Hedgehog signaling in cancer stem cells --- p.42 / Chapter 1.4.2.3 --- Hedgehog-targeting therapy in cancers --- p.44 / Chapter 1.5 --- Emerging roles of Hippo signaling in organ development and cancer pathogenesis --- p.47 / Chapter 1.5.1 --- Hippo signaling pathway --- p.47 / Chapter 1.5.1.1 --- Sensing the environmental cues by the Hippo pathway --- p.47 / Chapter 1.5.1.2 --- Salvador-Warts-Hippo (SWH) tumor-suppressor kinase cascade --- p.51 / Chapter 1.5.1.3 --- Transcriptional regulation of Hippo pathway target genes --- p.52 / Chapter 1.5.2 --- Emerging roles of Hippo signaling in cancer pathogenesis --- p.54 / Chapter 1.5.3 --- Therapeutic potential through targeting the Hippo pathway --- p.56 / Chapter 1.6 --- Aim of studies --- p.57 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.58 / Chapter 2.1 --- Animal studies --- p.58 / Chapter 2.1.1 --- Genotyping --- p.58 / Chapter 2.1.1.1 --- DNA purification --- p.58 / Chapter 2.1.1.2 --- PCR amplification, gel electrophoresis and imaging --- p.59 / Chapter 2.2 --- Cell culture --- p.61 / Chapter 2.2.1 --- Isolation and culturing of primary osteosarcoma cells --- p.61 / Chapter 2.2.2 --- Primary culture of wild-type calvarial osteoblasts --- p.62 / Chapter 2.2.3 --- Culture of MC3T3-E1, Hela, SaOS-2, U2-OS and HEK-293 --- p.62 / Chapter 2.3 --- MTT proliferation assay --- p.63 / Chapter 2.4 --- Differentiation assay --- p.64 / Chapter 2.4.1 --- Alkaline phosphatase (ALP) staining --- p.64 / Chapter 2.4.2 --- Alizarin Red S staining --- p.65 / Chapter 2.4.3 --- Von Kossa staining --- p.65 / Chapter 2.4.4 --- Oil Red O staining --- p.65 / Chapter 2.5 --- Immunofluorescence staining --- p.66 / Chapter 2.6 --- Anchorage-independent (soft agar) assay --- p.67 / Chapter 2.7 --- Migration assay --- p.68 / Chapter 2.8 --- Transwell invasion assay --- p.69 / Chapter 2.9 --- In vitro treatment of Hedgehog agonists/antagonists --- p.70 / Chapter 2.10 --- RNA extraction and qRT-PCR --- p.71 / Chapter 2.10.1 --- RNA extraction --- p.71 / Chapter 2.10.2 --- Reverse-transcription --- p.72 / Chapter 2.10.3 --- Real-time PCR --- p.72 / Chapter 2.11 --- Nude mice injection --- p.75 / Chapter 2.11.1 --- Subcutaneous injection --- p.75 / Chapter 2.11.2 --- Intra-tibial inoculation --- p.75 / Chapter 2.12 --- Western blot --- p.76 / Chapter 2.12.1 --- Protein extraction --- p.76 / Chapter 2.12.2 --- Protein concentration determination --- p.77 / Chapter 2.12.3 --- Western blot analysis --- p.77 / Chapter 2.13 --- Dual-luciferase reporter gene assay --- p.79 / Chapter 2.13.1 --- Transfection of plasmids by electroporation --- p.79 / Chapter 2.13.2 --- Sample harvesting and dual-luciferase assay --- p.80 / Chapter 2.14 --- Lentivirus preparation and Transduction of Kios-5 --- p.80 / Chapter 2.14.1 --- Lentivirus preparation --- p.80 / Chapter 2.14.2 --- Transduction of Kios-5 --- p.81 / Chapter 2.15 --- Conditioned medium studies --- p.82 / Chapter 2.15.1 --- Conditioned medium preparation --- p.82 / Chapter 2.15.2 --- Neutralizing OPN antibody treated conditioned medium --- p.83 / Chapter 2.15.3 --- Conditioned medium treatment --- p.83 / Chapter 2.16 --- Recombinant OPN treatment --- p.84 / Chapter 2.17 --- Immunohistochemistry --- p.84 / Chapter 2.18 --- Statistical analysis --- p.85 / Chapter CHAPTER 3 --- RESULTS --- p.87 / Chapter 3.1 --- Introductory statement --- p.87 / Chapter 3.2 --- Design of the OS mouse model Ptch1{U+1D9C}/+; p53+/-; HOC-Cre --- p.87 / Chapter 3.3 --- Characterization of the osteosarcoma mouse model --- p.93 / Chapter 3.3.1 --- Development of osteosarcoma-like tumor from the mouse model --- p.93 / Chapter 3.3.2 --- Characterization of osteosarcoma-like tumor derived cell line --- p.94 / Chapter 3.3.2.1 --- Osteoblastic properties of Kios-5 --- p.94 / Chapter 3.3.2.2 --- Functional assays of Kios-5 --- p.101 / Chapter 3.3.2.3 --- In vivo cancer properties of Kios-5 --- p.106 / Chapter 3.3.2.4 --- Molecular characterizations of Kios-5 and its derived secondary tumors --- p.109 / Chapter 3.4 --- Interaction between Hedgehog signaling and Hippo pathway in osteosarcoma mouse model --- p.114 / Chapter 3.5 --- Secretory factor-mediated interaction between Hedgehog signaling and Hippo pathway --- p.133 / Chapter CHAPTER 4 --- DISCUSSION --- p.160 / Chapter 4.1 --- Introductory statement --- p.160 / Chapter 4.2 --- Mouse model of Hedgehog signaling-induced osteosarcoma --- p.160 / Chapter 4.3 --- Hedgehog signaling interacted with Hippo pathway in the osteosarcoma mouse model --- p.167 / Chapter 4.4 --- Autocrine/paracrine mechanism in Hedgehog signaling-induced osteosarcoma --- p.171 / Chapter 4.5 --- Future studies --- p.174 / APPENDIX --- p.177 / Chapter A-1 --- List of reagents and chemicals --- p.177 / Chapter A-2 --- Recipes of buffers --- p.181 / Chapter A-3 --- Data sheet of human osteosarcoma tissue array --- p.185 / REFERENCES --- p.187
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328567 |
| Date | January 2012 |
| Contributors | Chan, Lok Hei., Chinese University of Hong Kong Graduate School. Division of Biomedical Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xv, 205 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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