大腸癌是全世界最常見的癌症之一，亦是一個癌症死亡率的首要原因。大腸癌患者約50%在病程中會出現轉移病灶。近十年來，雖然多種被批准用於臨床治療的新化療藥顯著提高了大腸癌的治療效果，但是轉移性大腸癌病人的預後仍然很差。隨著各種分子生物技術的進步，新的治療標靶可能在大腸癌細胞株中被發現，並得以在病人標本中驗證。 / 在本研究中，我們採用即時定量多聚酶鏈反應(qPCR)陣列分析，比較大腸癌細胞株和正常大腸細胞株基因表達譜，試圖識別潛在的新的治療標靶。結果提示，與正常大腸細胞株 CCD-18Co 比較，Jagged 2 (JAG2) 和 Frizzled-3 (FZD3)基因 在大腸癌細胞株 SW480 和 SW620 中表達升高。病人大腸癌組織的免疫組織化學染色 (IS) 檢查進一步證實了上述結果，大腸癌組織較其癌旁正常組織表達3.1倍JAG2和6.6倍FZD3蛋白。因此， 我們假設JAG2和FZD3在大腸癌的發生中起重要作用。 / 為了檢驗該假設的真偽，我們運用RNA 干擾的方法進行功能缺失研究。通過該方法，大腸癌細胞株中JAG2 信使RNA和蛋白均能夠被下調，但是FZD3蛋白卻沒有顯示降低。為了弄清JAG2基因的功能，我們進行了單層細胞劃痕傷口癒合試驗和Matrigel 侵襲試驗。結果提示，JAG2 基因下調顯著抑制大腸癌細胞遷移和侵襲的能力。 / 為了調查參與上述功能的機制，我們利用腫瘤轉移相關基因的qPCR陣列分析，試圖檢測出JAG2基因敲除後上調或下調表達的轉移相關基因。結果顯示組織蛋白酶K (CTSK)，一種溶酶體半胱氨酸蛋白酶，在JAG2基因沉默的大腸癌細胞株中表達下調。為了闡明CTSK 活性在大腸癌細胞株侵襲能力中起到的作用，我們採用CTSK抑制劑處理大腸癌細胞株HCT116和DLD-1，發現這兩種細胞株的侵襲能力分別下降了36%和59%。總之, 這些發現表明CTSK可能是JAG2的下游靶基因，活性CTSK可能參與了JAG2介導的大腸癌細胞株侵襲能力。 / 以前的研究表明p38 MAPK通路參與癌細胞遷和侵襲能力的調控。通過Western blot方法，磷酸化的p38和磷酸化的STAT3被發現在JAG2基因沉默的大腸癌細胞中表達降低。p38抑制劑處理的 HCT116和DLD-1細胞降低了侵襲能力下降，同時遷移能力也由於p38抑制劑的處理而降低，支持p38可調控癌細胞遷移和侵襲能力的事實。 / 總之，我們的結果顯示JAG2高表達通過啟動CTSK和p38 MAPK通路，可能促進大腸癌轉移。因此，JAG2可能成為轉移性大腸癌治療的潛在標靶。 / Colorectal cancer (CRC) is one of the most frequent cancers worldwide and is a leading cause of cancer mortality. Around 50% of patients with CRC will experience metastases. Although significant progress has been made in CRC treatment within the last decade with the approval of multiple new chemotherapeutic agents, the prognosis for patients with metastatic CRC remains poor. With the advancement of molecular techniques, novel therapeutic targets are able to be discovered in CRC cell lines and validated in patient samples. / Therefore in this project, I aim to identify potential novel therapeutic targets by comparing the gene expression profile of colon cancer cell lines and a normal colon cell line using quantitative polymerase chain reaction (qPCR) arrays. Results showed that Jagged 2 (JAG2) and Frizzled-3 (FZD3) were up-regulated in the CRC cell lines SW480 and SW620 as compared to the normal colon cell line CCD-18Co. Those results were further validated by immunohistochemical staining (IS), which detected up-regulated JAG2 (3.1-fold) and FZD3 (6.6-fold) proteins expression in CRC tissues as compared to adjacent normal tissues. Thus I hypothesized that JAG2 and FZD3 may play an important role in CRC carcinogenesis. / In order to study the roles of FZD3 and JAG2 in CRC, loss-of-function studies by RNA interference (RNAi) were carried out. While the expression of FZD3 protein failed to be down-regulated by RNAi, JAG2 expression was successfully knocked down in CRC cell lines at both the mRNA and protein levels. Functional analyses using the monolayer scratch wound-healing assay and Matrigel invasion assay showed that JAG2 knockdown significantly inhibited migration and invasion in CRC cell lines. / To investigate the mechanisms involved, a tumour metastasis qPCR array was used to examine the changes in the expression level of metastasis-related genes after JAG2 gene knockdown. Results showed that the expression of Cathepsin K (CTSK), a lysosomal cystein protease, was found to be down-regulated in CRC cell lines following JAG2 silencing. To demonstrate the importance of CTSK activity in CRC cell invasion, HCT116 and DLD-1 CRC cell lines were treated with a CTSK inhibitor and its effect were assessed by the Matrigel invasion assay. Results showed that CTSK inhibition led to a 36% and 59% reduction in number of invaded cells in HCT116 and DLD-1 cell lines, respectively. Taken together, these findings show that CTSK may be a downstream target of JAG2 and that active CTSK may involve in JAG2 mediated invasion in CRC cell lines. / Previous works by others have shown that the p38 MAPK pathway is involved in the regulation of migration and invasive activity of cancer cell lines. Using Western blot analysis, the expression of phosphorylated p38 MAPK and phosphorylated STAT3 were found to be down-regulated following JAG2 depletion in CRC cell lines. In support of a role for p38 MAPK in the regulation of cancer cell migration and invasive capability, treatment with a p38 MAPK inhibitor was found to reduce the percentage of invasive cells and distance moved by migratory cells in HCT116 and DLD-1 cell lines. / In conclusion, my results show that JAG2 over-expression in CRC may promote cancer cell migration and invasion through activation of CTSK and the p38 MAPK pathway. Therefore, JAG2 may be a potential therapeutic target for treatment of metastatic CRC. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / He, Wan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 164-207). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Abstract in English --- p.i / Abstract in Chinese --- p.iv / Acknowledgements --- p.vi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Colorectal Cancer (CRC) --- p.1 / Chapter 1.1.1 --- Epidemiology and Incidence --- p.1 / Chapter 1.1.2 --- Histology --- p.2 / Chapter 1.1.3 --- Gender and Age --- p.4 / Chapter 1.1.4 --- Etiology of CRC --- p.4 / Chapter 1.1.4.1 --- Environment --- p.4 / Chapter 1.1.4.2 --- Hereditary Factors --- p.5 / Chapter 1.1.4.3 --- Dietary Factors --- p.6 / Chapter 1.1.4.4 --- Obesity --- p.6 / Chapter 1.1.4.5 --- Tobacco and alcoho --- p.7 / Chapter 1.1.4.6 --- Inflammatory bowel disease (IBC) --- p.7 / Chapter 1.1.5 --- Genetic Changes in CRC --- p.8 / Chapter 1.1.5.1 --- Chromosomal Aberration --- p.8 / Chapter 1.1.5.2 --- Tumor Suppressor Genes --- p.10 / Chapter 1.1.5.2.1 --- APC gene --- p.10 / Chapter 1.1.5.2.2 --- P53 gene --- p.11 / Chapter 1.1.5.2.3 --- SMAD4 gene --- p.11 / Chapter 1.1.5.3 --- Oncogenes --- p.12 / Chapter 1.1.5.3.1 --- Epidermal Growth Factor Receptor (EGFR) gene --- p.12 / Chapter 1.1.5.3.2 --- RAS gene and BRAF gene --- p.13 / Chapter 1.1.5.4 --- Proposed Two-hit Model for the Multistep Pathogenesis of CRC --- p.15 / Chapter 1.1.6 --- Clinical Presentation and Diagnosis --- p.16 / Chapter 1.1.7 --- Theatment --- p.16 / Chapter 1.1.7.1 --- Surgery --- p.16 / Chapter 1.1.7.2 --- Radiotherapy (RT) --- p.17 / Chapter 1.1.7.3 --- Concurrent Chemotherapy --- p.17 / Chapter 1.1.7.4 --- Target Therapy --- p.18 / Chapter 1.1.7.5 --- Colorectal Cancer Treatment by Stage --- p.19 / Chapter 1.1.7.6 --- Novel Strategies --- p.20 / Chapter 1.1.7.6.1 --- Epigenetic therapy --- p.20 / Chapter 1.1.7.6.2 --- Immunotherapy --- p.21 / Chapter 1.2 --- Pathways Involved in CRC Carcinogenesisand Progression --- p.22 / Chapter 1.2.1 --- Wnt Signaling Pathway --- p.22 / Chapter 1.2.2 --- Notch Signaling --- p.23 / Chapter 1.2.3 --- Nuclear Factor-kappa B (NF-κB) Signaling Pathway --- p.23 / Chapter 1.2.4 --- Phosphatidylinositol 3-kinase (PI3K) Signaling Pathway --- p.24 / Chapter 1.2.5 --- Crosstalk Among WNT, NOTCH, NF-κB and PI3K Signaling Pathway in CRC --- p.24 / Chapter 1.3 --- Hypothesis and Objectives of this Study --- p.28 / Chapter Chapter 2 --- Identification of Differentially Expressed Genes between Colorectal Cancer Cell Lines and A Normal Colon Cell Line --- p.29 / Chapter 2.1 --- Background --- p.29 / Chapter 2.2 --- Materials and Methods --- p.33 / Chapter 2.2.1 --- Cell Lines --- p.33 / Chapter 2.2.2 --- Identification of Differetially Expressed Genes by qPCR Arrays --- p.33 / Chapter 2.2.2.1 --- Total RNA Extraction --- p.33 / Chapter 2.2.2.2 --- RNA Quality Contol --- p.34 / Chapter 2.2.2.3 --- Reverse Transcription (RT) --- p.34 / Chapter 2.2.2.4 --- PCR Arrays --- p.34 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- Differentially Expressed Genes in WNT Signaling Pathway --- p.36 / Chapter 2.3.2 --- Differentially Expressed Genes in Notch Signaling Pathway --- p.40 / Chapter 2.3.3 --- Differentially Expressed Genes in NF-κB Signaling Pathway --- p.43 / Chapter 2.3.4 --- Differentially Expressed Genes in PI3K-AKT Signaling Pathway --- p.46 / Chapter 2.3.5 --- Choice of over-expressed genes for further validation and characterization --- p.49 / Chapter 2.4 --- Discussions --- p.53 / Chapter 2.4.1 --- WNT Signaling Pathway --- p.53 / Chapter 2.4.2 --- NOTCH Signaling Pathway --- p.54 / Chapter 2.4.3 --- NF-κB Signaling Pathway --- p.55 / Chapter 2.4.4 --- PI3K-AKT Signaling Pathway --- p.56 / Chapter 2.4.5 --- Choice of over-expressed genes for further validation and characterization --- p.56 / Chapter Chapter 3 --- JAG2, FZD3 and NOTCH4 Expression in Colorectal Cancer Cell Lines and Colorectal Cancer Tissues --- p.59 / Chapter 3.1 --- Background --- p.59 / Chapter 3.1.1 --- JAG2 Ligand --- p.59 / Chapter 3.1.2 --- FZD3 Receptor --- p.61 / Chapter 3.1.3 --- NOTCH4 Receptor --- p.62 / Chapter 3.2 --- Materials and Methods --- p.64 / Chapter 3.2.1 --- CRC Cell Lines --- p.65 / Chapter 3.2.2 --- CRC Tissues --- p.65 / Chapter 3.2.3 --- Quantitative RT-PCR --- p.66 / Chapter 3.2.4 --- Detection of JAG2, FZD3 and NOTCH4 Protein Expression in CRC Tissues by Immunohistochemical Staining (IS) --- p.67 / Chapter 3.2.5 --- Western Blot Assays --- p.68 / Chapter 3.2.5.1 --- Protein extraction --- p.68 / Chapter 3.2.5.2 --- SDS-PAGE gel electrophroresis --- p.68 / Chapter 3.2.5.3 --- Protein blotting --- p.68 / Chapter 3.2.6 --- Detection of JAG2 and FZD3 Protein Expression in CRC and Normal Colon Cell Lines by Western Blotting --- p.69 / Chapter 3.2.7 --- Statistical Analysis --- p.70 / Chapter 3.3 --- Results --- p.71 / Chapter 3.3.1 --- JAG2 and FZD3 but not NOTCH4 mRNA were Over -expressed in CRC Cell Lines --- p.71 / Chapter 3.3.2 --- Over-expression of JAG2 and FZD3 Proteins in CRC Tissues --- p.72 / Chapter 3.3.3 --- FZD3 Over-expression Correlated with Tumour-Node Metastasis (TNM) stages --- p.76 / Chapter 3.3.4 --- JAG2 and FZD3 Protein Expression in Colorectal Cancer and Normal Cell Lines --- p.77 / Chapter 3.4 --- Discussions --- p.78 / Chapter Chapter 4 --- Functional Analyses of JAG2 and FZD3 in CRC Cell Lines by RNA Interference --- p.81 / Chapter 4.1 --- Background --- p.81 / Chapter 4.2 --- Materials and Methods --- p.84 / Chapter 4.2.1 --- Transfection of siRNA into CRC Cell Lines --- p.84 / Chapter 4.2.2 --- Cell Proliferation Assay --- p.85 / Chapter 4.2.3 --- Monolayer Scratch Wound Healing Assay --- p.85 / Chapter 4.2.4 --- Matrigel Invasion Assay --- p.86 / Chapter 4.2.5 --- Statistical Analysis --- p.87 / Chapter 4.3 --- Results --- p.88 / Chapter 4.3.1 --- Knockdown of JAG2 and FZD3 Expression by RNA Interference --- p.88 / Chapter 4.3.2 --- Effect of JAG2 Knockdown on Migration of CRC Cell Lines --- p.91 / Chapter 4.3.3 --- JAG2 Knockdown by siRNA 2 Transfection Reduced Migratory Capability of HCT116, DLD-1and HT29 cell lines --- p.94 / Chapter 4.3.4 --- JAG2 Knockdown Impaired the Invasiveness of HCT116 and DLD-1 Cell Lines --- p.97 / Chapter 4.3.5 --- Decreased Migratory and Invasive Capabilities Induced by JAG2 Knockdown was not Due to Reduced Cell Proliferation --- p.100 / Chapter 4.4 --- Discussions --- p.102 / Chapter Chapter 5 --- NOTCH Pathway Inactivation by JAG2 Silencing Reduces Oncogenic Properties of HT29 but not HCT116 andDLD-1 CRC Cell Lines --- p.106 / Chapter 5.1 --- Background --- p.106 / Chapter 5.2 --- Materials and Methods --- p.109 / Chapter 5.2.1 --- CRC Cell lines --- p.109 / Chapter 5.2.2 --- Pharmacological Inhibition of NOTCH signaling by DAPT --- p.109 / Chapter 5.2.3 --- Combination of DAPT Treatment and JAG2 Silencing by siRNA --- p.109 / Chapter 5.2.4 --- Western Blotting --- p.109 / Chapter 5.2.5 --- Cell Proliferation Assay (MTS Assay) --- p.110 / Chapter 5.2.6 --- Monolayer Scratch Wound Healing Assay --- p.110 / Chapter 5.2.7 --- Matrigel Invasion Assay --- p.111 / Chapter 5.2.8 --- Statistical Analysis --- p.111 / Chapter 5.3 --- Results --- p.112 / Chapter 5.3.1 --- JAG2 Silencing Down-regulates Notch Pathway Signaling in CRC Cell Lines --- p.112 / Chapter 5.3.2 --- Inhibition of NOTCH Signaling by DAPT Treatment in CRC Cell Lines --- p.112 / Chapter 5.3.3 --- NOTCH Inhibition Does not Significantly Affect Cell Proliferation in CRC Cell Lines --- p.114 / Chapter 5.3.4 --- Suppression of NOTCH Signaling by DAPT Inhibits Migration in HT29 but not in HCT116 and DLD-1 CRC Cell Lines --- p.115 / Chapter 5.3.5 --- Suppression of NOTCH Signaling by DAPT does not Significantly Affect Invasiveness of HCT116 and DLD-1 CRC Cell Lines --- p.117 / Chapter 5.4 --- Discussions --- p.118 / Chapter Chapter 6 --- JAG2 Knockdown Inhibits Invasion in CRC Cell Lines through Inactivation of Cathepsin K --- p.121 / Chapter 6.1 --- Background --- p.121 / Chapter 6.2 --- Materials and Methods --- p.123 / Chapter 6.2.1 --- Human Tumour Metastasis RT2 Profiler[superscript TM] PCR Array --- p.123 / Chapter 6.2.2 --- Measurement of CTSK Gene expression level by Quantitative Real-Time PCR --- p.123 / Chapter 6.2.3 --- Immunohistochemical Staining (IS) of CTSK in CRC Tissues --- p.124 / Chapter 6.2.4 --- Pharmacological Inhibitior of CTSK in CRC Cell Lines --- p.124 / Chapter 6.2.5 --- Inhibition of CTSK in CRC Cell Lines for Migration Study --- p.124 / Chapter 6.2.6 --- Inhibition of CTSK in CRC Cell Lines for Invasion Study --- p.125 / Chapter 6.2.7 --- Western Blotting --- p.125 / Chapter 6.2.8 --- Statistical Analysis --- p.125 / Chapter 6.3 --- Results --- p.126 / Chapter 6.3.1 --- Identification of Metastasis Related Genes Which were Down-regulated by JAG2 Knockdown in HCT116 Cells --- p.126 / Chapter 6.3.2 --- Validation of Down-regulation of CTSK Gene by JAG2 Knockdown in HCT116 Cell Line by qRT-PCR --- p.126 / Chapter 6.3.3 --- JAG2 Knockdown Reduced Expression of Active CTSK Protein in CRC Cell Lines --- p.128 / Chapter 6.3.4 --- CTSK Protein Expression in CRC Tissue Samples --- p.130 / Chapter 6.3.5 --- Pharmacological Inhibition of CTSK Suppressed Invasiveness of CRC Cell Lines --- p.131 / Chapter 6.3.6 --- Pharmacological Inhibition of CTSK did not Affect Migration of CRC Cell Lines --- p.132 / Chapter 6.4 --- Discussions --- p.133 / Chapter Chapter 7 --- Depletion of JAG2 Inhibits Migration and Invasion in CRC Cell Lines through Inactivation of p38 MAPK/HSP27 Pathway --- p.137 / Chapter 7.1 --- Background --- p.137 / Chapter 7.2 --- Materials and Methods --- p.140 / Chapter 7.2.1 --- Pharmocological Inhibition of p38 MAPK Phosphorylation CRC Cell Lines --- p.140 / Chapter 7.2.2 --- Inhibition of p38 MAPK Phosphorylation for Migration Study in CRC Cell Lines --- p.140 / Chapter 7.2.3 --- Inhibition of p38 MAPK Phosphorylation for Invasion Study in CRC Cell Lines --- p.140 / Chapter 7.2.4 --- Knockdown of STAT3 by RNA interference --- p.141 / Chapter 7.2.5 --- Knockdown of STAT3 for Migration Study in CRC Cell Lines --- p.141 / Chapter 7.2.6 --- Knockdown of STAT3 for Invasion Study in CRC Cell Lines --- p.141 / Chapter 7.2.7 --- Western Blotting --- p.141 / Chapter 7.2.8 --- Statistical Analysis --- p.142 / Chapter 7.3 --- Results --- p.143 / Chapter 7.3.1 --- JAG2 Knockdown Inhibits p38 MAPK / HSP27 Pathway in CRC Cell Lines --- p.143 / Chapter 7.3.2 --- Inhibition of p38 MAPK / HSP27 Signaling Pathway Down-regulated Invasive Capability of CRC Cell Line --- p.145 / Chapter 7.3.3 --- Inhibition of p38 MAPK / HSP27 Signaling Pathway Down-regulated Migration of CRC Cell lines --- p.147 / Chapter 7.3.4 --- JAG2 Knockdown Inactivated p38 MAPK / HSP27 Pathway Independently of NOTCH Pathway in CRC Cell Lines --- p.149 / Chapter 7.3.5 --- JAG2 Knockdown Inhibits STAT3 Activation in CRC Cell Lines --- p.151 / Chapter 7.3.6 --- STAT3 Silencing Reduced Invasive Capability in CRC Cell Lines --- p.152 / Chapter 7.3.7 --- STAT3 Silencing Reduced Migratory Capability in CRC Cell Lines --- p.154 / Chapter 7.3.8 --- Inhibition of p38 MAPK Activity Suppressed STAT3 Activation in HCT116 Cells --- p.156 / Chapter 7.4 --- Discussions --- p.157 / Chapter Chapter 8 --- Conclusions and Future Works --- p.161 / Chapter 8.1 --- Conclusions --- p.161 / Chapter 8.2 --- Future work --- p.163 / References --- p.164 / Chapter Appendix 1 --- List of Figures and Tables --- p.208 / Chapter Appendix 2 --- Abbrevations used in this thesis --- p.212
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328244 |
| Date | January 2013 |
| Contributors | He, Wan, Chinese University of Hong Kong Graduate School. Division of Medical 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 (xi, 217 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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