多發性骨髓瘤(Multiple myeloma) 為骨髓內漿細胞異常增生的惡性腫瘤，到目前為止仍然難以治癒。其發生發展是一個複雜的多步驟事件，涉及腫瘤細胞中遺傳和表觀遺傳的改變，以及骨髓微環境的支持。現已確定骨髓瘤細胞和骨髓微環境之間的相互作用對於骨髓瘤的病理發生，以及骨髓瘤細胞的生長，遷移和抗藥性起著關鍵作用。血小根因子四(Platelet factor 4, or PF4) 是一種抗血管生成的趨化因子。它不僅在體外抑制血管內皮細胞增殖和遷移，而且在體內抑制腫瘤的生長。此前，我們發現PF4 基因在多發性骨髓瘤中等位缺失以及DNA 高度甲基化，因而導致其在骨髓瘤病人及細胞系中的表達缺失或降低。在本研究中，我們利用體內和體外實驗鑒定了PF4 對骨髓瘤細胞以及血管生成的作用，並闡明了其作用機制。 / 首先，我們在體外鑒定了PF4 在骨髓瘤細胞中的功能。我們發現PF4 抑制骨髓瘤細胞系以及從病人骨髓中分離出來的骨髓瘤細胞的生長，以及促進其凋亡。其促凋亡活性與caspase-3 和PARP 的激活有關。我們也檢測了PF4 在骨髓瘤中對血管生成的作用。我們首先分離了病人骨髓中的內皮細胞。結果顯示PF4抑制骨髓瘤內皮細胞的生長和管狀物的形成。這些結果證明PF4 在骨髓瘤中可能是一個抑癌因子。 / 接下來我們進一步檢測了PF4 在體內的抑癌功能。在第一種模型中，骨髓瘤細胞被皮下移植到重症聯合兔疫缺陷型(NOD-SCID) 小鼠中。尾靜脈注射200ngPF4 明顯的抑制了腫瘤的生長，並延長了小鼠的成活率。第二種小鼠模型稱為兔鼠融合模型(SCID-rab model) 。在這一模型中，大白兔的腿骨先被皮下移植到(NOD-SCID) 小鼠中，再將骨髓瘤細胞注射入已植入的大白兔腿骨的骨腔中。兩周後，小鼠被尾靜脈注射入20 或200ng PF4 。結果顯示200ng PF4 顯著抑制了腫瘤的生長。通過兔疫組化分析大白兔腿骨切片，我們進一步證明了PF4 在腫瘤細胞中的增瘟，凋亡以及血管生成的作用。我們的發現因此證實了PF4 是骨髓瘤中的一個抑制因子。 / 為了鑒定PF4 在骨髓瘤中的作用機制，我們用Protein/DNA 微陣列(Protein/DNA array) 分析了PF4 參與的信號通路。結果顯示PF4 調節了若干個轉錄因子，其中包括STAT3 。凝膠遷移(EMSA) 和螢光素酪報告基因(luciferase reporter assay )檢測進一步證實PF4 抑制了STAT3 的DNA 結合能力以及轉錄活性。因此PF4 可能通過抑制STAT3 信號通路而抑制骨髓瘤的生長。我們進一步發現PF4 能抑制組成性的以及自介素6 (IL-6) 誘導的STAT3的激活。我們發現PF4 下調了STAT3 下游的靶基因，包括Mc1-1， Survivin 以及血管內皮細胞生長因子(VEGF)。而過表達組成性激活的STAT3 能逆轉PF4 所誘導的細胞凋亡。在兔鼠敵合模型中，通過兔疫組化分析大白兔腿骨切片，我們發現PF4 能抑制STAT3 的入核。SOCS3 是STAT3 其中的一個抑制因子，我們發現PF4 能誘導SOCS3 的表達。而干擾掉SOCS3 能使PF4 喪失其抑制STAT3 激活的能力。這些結果表明PF4 可能通過誘導SOCS3 的表達，從而抑制STAT3 信號通路，引起骨髓瘤的生長抑制以及抗血管生成。 / 總而言之，本研究表明PF4 是骨髓髓中一個重要調節因子。在體外和體內，PF4 通過抑制STAT3 信號通路，從而抑制腫瘤細胞的生長，促進凋亡以及抑制血管生成。本文為PF4 的臨床研究，作為一種新的治療骨髓瘤藥物，提高骨髓瘤病人的治療效果提供基礎。 / Multiple myeloma (MM) is an incurable hematological malignancy characterized by accumulation of clonal plasma cells in bone marrow (BM). The development and progression of MM is a complex multistep tumorigenic event involving both genetic and epigenetic changes in the tumor cell as well as the support by the BM microenvironment. It has been well established that the physical interaction of MM cells with the BM milieu are crucial for MM pathogenesis, MM cell growth, survival, migration and drug resistance. Platelet factor 4 (PF4), a potent antiangiogenic chemokine, not only inhibits endothelial cell proliferation and migration in vitro but also solid tumor growth in vivo. Our group previously demonstrated loss of PF4 expression in patient MM samples and MM cell lines due to concurrent allelic loss and DNA hypermethylation. In this study, we characterized the effects of PF4 on MM cells and angiogenesis in the BM milieu both in vitro and in vivo and elucidated the mechanism of PF4 effects on MM. / To characterize the effects of PF4 on MM cells in vitro, assays on cell growth, cell cycle arrest and apoptosis were performed and we found that PF4 inhibited growth and induced apoptosis in both MM cell lines and MM cells from patients. The proapoptotic activity of PF4 is associated with activation of caspase-3 and poly (ADP) ribose polymerase (PARP). We also investigated the effects of PF4 on angiogenesis in MM using endothelial cells isolated from patient's BM aspirates (MMECs). Our results showed that PF4 suppressed MMECs growth and tube formation on matrigel in a dose-dependent manner. / Given the ability of PF4 to suppress MM cell growth and angiogenesis in vitro, we evaluated its tumor suppressive function in vivo. In human subcutaneously matrigel xenograft mouse model, tail vein injection of 200ng PF4 significantly reduced MM tumor growth and prolonged survival. We next used the SCID-rab mouse model which recapitulates the human BM milieu in vivo. In this model, MM cells were directly injected into the rabbit bone which was subcutaneously implanted into the NOD-SCID mice. Two weeks after injection, SCID mice were treated with various dose of PF4 (20 or 200ng per injection, three times per week) or PBS by tail vein injection. ELISA assay for hIg (lambda) showed that tumor growth in 200ng PF4-treated mice was markedly reduced by 58% compared with the control group, which was further confirmed by immunohistochemistry analysis of CD 138 staining on rabbit bone section. Consistent with the in vitro results, induction of apoptosis in MM cells and inhibition of angiogenesis by PF4 could also be demonstrated in vivo, as evidenced by the findings on ki67, Cleaved caspase-3, CD31 and VEGF staining on rabbit bone sections from treated versus control mice. Our findings thus confirmed that PF4 is a novel tumor suppressor in MM. / However, the molecular mechanism of how PF4 inhibits MM tumorigenesis is still unclear. To identify the signal pathway PF4 involved in MM, Protein/DNA array was performed. We found that PF4 regulated several transcription factors including STAT3 in U266 cells. EMSA and luciferase reporter assay further confirmed that PF4 suppressed STAT3 DNA binding and transcriptional activity. So it is possible that PF4 mediates its tumor suppressive function, through suppressing STAT3 pathway in MM cells. We further found that pre-treatment of PF4 blocked both constitutive and interleukin-6-induced STAT3 activation in a time-dependent manner in human MM cells. PF4 could also down-regulate the STAT3-regulated gene products including Mcl-I, Survivin and vascular endothelial growth factor (VEGF). Moreover, enforced expression of constitutively active STAT3 rescued cells from PF4-induced apoptosis. In SCID-rab mouse model, we also found that PF4 inhibited STAT3 nuclear translocation by immunostaining of rabbit bone sections. When examined further, we found that PF4 induced the expression of one of the STAT3 inhibitor SOCS3, and gene silencing of SOCS3 by small interfering RNA abolished the ability of PF4 to inhibit STAT3 activation, suggesting a critical role of SOCS3 in the action of PF4. Our findings therefore suggest that by inducing SOCS3 expression, PF4 abrogates STAT3 activity, thus induces tumor growth inhibition and anti-angiogenesis. / Together, these novel studies have shown that PF4 is an important regulator of MM tumorigenesis. By abrogating STAT3 signaling it targets cell growth, induces apoptosis, suppresses angiogenesis both in vitro and in vivo in MM. These scientific observations provide the framework for clinical studies of this chemokine, as a novel drug for treatment of MM to improve patient outcome in MM. / 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. / Liang, Pei. / "November 2011." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 139-161). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract in English --- p.I / Abstract in Chinese --- p.IV / List of Publications --- p.VI / Acknowledgements --- p.VII / List of abbreviations --- p.IX / List of Tables --- p.XII / List of Figures --- p.xm / Table of Contents --- p.XV / Chapter Chapter1 --- Introduction and Literature Review --- p.1 / Chapter 1.1 --- Multiple myeloma-General description --- p.1 / Chapter 1.1.1 --- Epidemiology of MM --- p.1 / Chapter 1.1.2 --- Stages of MM --- p.1 / Chapter 1.2 --- The bone marrow (BM) microenvironment in MM --- p.3 / Chapter 1.3 --- Signal pathways in MM cells --- p.5 / Chapter 1.3.1 --- JAK/STAT3 in cancers and MM --- p.5 / Chapter 1.3.1.1 --- IL-6 and its receptor --- p.7 / Chapter 1.3.1.2 --- Activation of downstream signals-The "on" signals --- p.9 / Chapter 1.3.1.3 --- Inactivation of downstream signaling --- p.11 / Chapter 1.3.1.3.1 --- Phosphatases --- p.12 / Chapter 1.3.1.3.2 --- SOCS family --- p.13 / Chapter 1.3.1.3.3 --- The PIAS family --- p.14 / Chapter 1.3.2. --- NF-κB pathway --- p.15 / Chapter 1.3.3 --- RAS-MAPK pathway --- p.17 / Chapter 1.3.4 --- Phosphatidyl inositol-3 kinase (PI3K)/AKT --- p.18 / Chapter 1.4 --- Angiogenesis in MM --- p.18 / Chapter 1.4.1 --- The process of angiogenesis --- p.18 / Chapter 1.4.2 --- Angiogenesis in caner --- p.20 / Chapter 1.4.3 --- Angiogenesis in MM --- p.22 / Chapter 1.5 --- Animal models in MM --- p.24 / Chapter 1.6 --- Treatment of MM --- p.27 / Chapter 1.6.1 --- Chemotherapy --- p.27 / Chapter 1.6.2 --- Autologous stem cell transplantation --- p.28 / Chapter 1.6.3 --- Biologically based therapies --- p.28 / Chapter 1.7 --- Platelet factor 4 (PF4) --- p.30 / Chapter 1.8 --- Structure of PF 4 --- p.30 / Chapter 1.9 --- Role of PF4 in physiological process --- p.32 / Chapter 1.9.1 --- Inhibition of megakaryocytopoiesis --- p.32 / Chapter 1.9.2 --- PF4 and coagulation --- p.33 / Chapter 1.10 --- Role of PF4 in pathological process --- p.34 / Chapter 1.10.1 --- PF4 and cancer --- p.34 / Chapter 1.10.2 --- PF4 is an angiogenic inhibitor --- p.35 / Chapter 1.11 --- Clinical applications of PF4 --- p.37 / Chapter 1.12 --- Summary and project aims --- p.37 / Chapter Chapter 2 --- Materials and Methods --- p.40 / Chapter 2.1 --- Reagents and antibodies --- p.40 / Chapter 2.2 --- MM Cell lines --- p.40 / Chapter 2.3 --- CD138⁺ primary MM cells --- p.41 / Chapter 2.4 --- CD31⁺ MM endothelial cells (MMECs) --- p.42 / Chapter 2.5 --- WST-1 assay --- p.43 / Chapter 2.6 --- Trypan blue exclusion --- p.43 / Chapter 2.7 --- Cell cycle analysis --- p.44 / Chapter 2.8 --- Apoptosis analysis --- p.44 / Chapter 2.9 --- In vitro tube formation assay --- p.45 / Chapter 2.10 --- SCID-rab mice model --- p.45 / Chapter 2.10.1 --- Construction of SCID-rab mice --- p.45 / Chapter 2.10.2 --- Establishment and monitoring of myeloma in SCID-rab mice --- p.46 / Chapter 2.10.3 --- Enzyme-linked immunosorbent assay (ELISA) --- p.46 / Chapter 2.10.4 --- PF4 treatment --- p.47 / Chapter 2.10.5 --- Immunohistochemistry --- p.48 / Chapter 2.11 --- Protein/DNA arrays --- p.49 / Chapter 2.12 --- Electrophoretic mobility shift assay (EMSA) --- p.50 / Chapter 2.13 --- Luciferase reporter assay --- p.52 / Chapter 2.14 --- Western blotting --- p.53 / Chapter 2.15 --- RNA extraction --- p.54 / Chapter 2.16 --- Real-time Polymerase Chain Reaction (Real-time PCR) --- p.54 / Chapter 2.17 --- Nuclear transfection --- p.55 / Chapter 2.18 --- Statistical analysis --- p.55 / Chapter Chapter3 --- The role of PF4 in MM: in vitro studies --- p.58 / Chapter 3.1 --- Results --- p.58 / Chapter 3.1.1 --- PF4 inhibited growth of human MM cell lines --- p.58 / Chapter 3.1.2 --- PF4 did not cause cell cycle arrest --- p.59 / Chapter 3.1.3 --- PF4 induced apoptosis of myeloma cell lines --- p.63 / Chapter 3.1.4 --- PF4 caused cell apoptosis in primary MM cells cultured in vitro --- p.64 / Chapter 3.1.5 --- PF4 suppressed MMECs growth --- p.69 / Chapter 3.1.6 --- PF4 suppressed MMECs tube formation --- p.69 / Chapter 3.2 --- Discussion --- p.73 / Chapter 3.2.1 --- Negative regulation of PF4 in MM cells growth in vitro --- p.73 / Chapter 3.2.2 --- PF4 induces apoptosis in MM cell lines and primary MM cells --- p.74 / Chapter 3.2.3 --- PF4 inhibits angiogenesis in MM in vitro --- p.76 / Chapter 3.3 --- Summary --- p.79 / Chapter Chapter4 --- The role ofPF4 in MM tumorigenesis: in vivo studies --- p.82 / Chapter 4.1 --- Results --- p.82 / Chapter 4.1.1 --- PF4 inhibited MM tumor growth and prolonged survival in subcutaneous matrigel xenograft model --- p.82 / Chapter 4.1.2 --- PF4 inhibited MM tumor growth and prolonged survival in SCID-rab mouse model --- p.85 / Chapter 4.1.3 --- PF4 reduced human MM cell proliferation, angiogenesis and induced apoptosis in SCID-rab mice --- p.88 / Chapter 4.2 --- Discussion --- p.91 / Chapter 4.2.1 --- PF4 inhibited human tumor growth in subcutaneous matrigel xenograft mouse model --- p.91 / Chapter 4.2.2 --- SCID-rab mouse model was successfully established and PF4 inhibited human MM turnor growth in this model --- p.92 / Chapter 4.2.3 --- PF4 inhibited human MM cell proliferation, angiogenesis and induced apoptosis in SCID-rab mice --- p.95 / Chapter 4.3 --- Summary --- p.96 / Chapter Chapter 5 --- The molecular mechanisms of PF4 in MM tumorigenesis --- p.98 / Chapter 5.1 --- Results --- p.98 / Chapter 5.1.1 --- ProteinlDNA array hybridization and Quantification of protein/DNA array spots --- p.98 / Chapter 5.1.2 --- PF4 suppressed DNA binding and transcriptional activity of STAT3 --- p.102 / Chapter 5.1.3 --- PF4 inhibited constitutive STAT3 phosphorylation in MM cells --- p.104 / Chapter 5.1.4 --- PF4 inhibited IL-6-induced STAT3 activation --- p.105 / Chapter 5.1.5 --- PF4 suppressed STAT3 regulated gene expression --- p.107 / Chapter 5.1.6 --- Enforced expression of constitutively active STAT3 rescued cells from PF4-induced apoptosis --- p.109 / Chapter 5.1.7 --- PF4 induced the expression of SOCS3 --- p.111 / Chapter 5.1.8 --- PF4-induced inhibition of STAT3 activation was reversed by gene silencing of SOCS3 --- p.111 / Chapter 5.1.9 --- PF4 inhibited nuclear accumulation of STAT3 and induced expression of SOCS3 in vivo --- p.114 / Chapter 5.2 --- Discussion --- p.115 / Chapter 5.2.1 --- PF4 regulated several TFs --- p.115 / Chapter 5.2.2 --- PF4 inhibited constitutive activation of STAT3 --- p.118 / Chapter 5.2.3 --- PF4 inhibited IL-6 induced activation of STAT3 --- p.120 / Chapter 5.2.4 --- PF4 suppressed STAT3 regulated gene expression --- p.121 / Chapter 5.2.5 --- PF4 induced the expression of SOCS3 --- p.124 / Chapter 5.3 --- Summary --- p.125 / Chapter Chapter 6 --- Conclusion and future studies --- p.128 / Chapter 6.1 --- Conclusion --- p.128 / Chapter 6.2 --- Future studies --- p.135 / Appendices --- p.137 / References list --- p.139

Kuok, Chiu Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 139-154). / Abstract and appendix also in Chinese. / Abstract --- p.i / Abstract (in Chinese) --- p.iv / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Tables --- p.xi / List of Figures --- p.xiv / List of Abbreviations --- p.xv / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- The Impact of Malignant Diseases and Multiple Myeloma --- p.4 / Chapter 1.3 --- Pharmacoeconomics --- p.6 / Chapter 1.4 --- Macao Healthcare System --- p.9 / Chapter 1.5 --- Study Hypothesis --- p.12 / Chapter 1.6 --- Study Objectives --- p.12 / Chapter 1.7 --- Perspective of the Study --- p.13 / Chapter Chapter 2 --- Literature Review / Chapter 2.1 --- Hematopoietic System --- p.14 / Chapter 2.1.1 --- Specific Blood Cell Lineages and Blood Cells --- p.15 / Chapter 2.1.2 --- Bone Marrow Microenvironment --- p.19 / Chapter 2.2 --- The Hematologic Malignancies --- p.20 / Chapter 2.2.1 --- Leukemia --- p.21 / Chapter 2.2.2 --- Lymphoma --- p.23 / Chapter 2.2.3 --- Plasma Cell Disorders --- p.24 / Chapter 2.3 --- Multiple Myeloma --- p.25 / Chapter 2.3.1 --- Epidemiology --- p.26 / Chapter 2.3.2 --- Pathology --- p.29 / Chapter 2.3.3 --- Clinical Presentation and Disease Complications --- p.31 / Chapter 2.3.4 --- Classification and Diagnostic Criteria --- p.35 / Chapter 2.3.5 --- Disease Staging and Prognosis --- p.42 / Chapter 2.3.6 --- Treatment --- p.45 / Chapter 2.3.6.1 --- Treatment Regimens and Strategies --- p.47 / Chapter 2.3.6.1.1 --- Standard Chemotherapy --- p.48 / Chapter 2.3.6.1.1.1 --- Melphalan-based Regimens --- p.51 / Chapter 2.3.6.1.1.2 --- VAD-based Regimens --- p.52 / Chapter 2.3.6.1.1.3 --- High-dose Glucocorticoid Regimens --- p.53 / Chapter 2.3.6.1.2 --- Treatment Strategies --- p.53 / Chapter 2.3.6.1.2.1 --- Initial Chemotherapy --- p.53 / Chapter 2.3.6.1.2.2 --- High-dose Chemotherapy --- p.55 / Chapter 2.3.6.1.2.3 --- Newer Therapeutic Agents for Multiple Myeloma --- p.58 / Chapter 2.3.6.1.2.4 --- Salvage Chemotherapy --- p.60 / Chapter 2.3.6.2 --- Treatment Responses --- p.63 / Chapter 2.3.6.3 --- Treatment for Disease Complications --- p.66 / Chapter Chapter 3 --- Methodology / Chapter 3.1 --- Study Design --- p.69 / Chapter 3.2 --- Patients Selection Criteria --- p.71 / Chapter 3.2.1 --- For Retrospective Cost Analysis --- p.71 / Chapter 3.2.2 --- For Health-related Quality of Life Measurement --- p.71 / Chapter 3.3 --- Patient Screening --- p.72 / Chapter 3.4 --- Data Collection --- p.72 / Chapter 3.5 --- Overview of Assessment Methods --- p.73 / Chapter 3.5.1 --- Outcomes --- p.73 / Chapter 3.5.2 --- Cost Analysis --- p.74 / Chapter 3.5.3 --- Cost Effectiveness Analysis --- p.74 / Chapter 3.5.4 --- Cost Utility Analysis --- p.75 / Chapter 3.5.5 --- Health-related Quality of Life Assessment --- p.75 / Chapter 3.6 --- Statistical Analysis --- p.76 / Chapter 3.7 --- Ethic approval --- p.77 / Chapter Chapter 4 --- Results / Chapter 4.1 --- Study Population --- p.78 / Chapter 4.1.1 --- Cost and Pharmacoeconomic Analysis --- p.78 / Chapter 4.1.2 --- Health-related Quality of Life Assessment --- p.79 / Chapter 4.2 --- Study Results --- p.81 / Chapter 4.2.1 --- Comparison of All Patients --- p.81 / Chapter 4.2.1.1 --- Differences in Treatment Protocols --- p.81 / Chapter 4.2.1.2 --- Differences in Treatment Responses --- p.82 / Chapter 4.2.1.3 --- Differences in Treatment Outcomes --- p.82 / Chapter 4.2.1.4 --- Differences in Treatment Costs --- p.84 / Chapter 4.2.2 --- Comparison for Patients Treated by Melphalan-based Regimens and VAD-based Regimens --- p.90 / Chapter 4.2.2.1 --- Differences in Treatment Responses --- p.90 / Chapter 4.2.2.2 --- Differences in Treatment Outcomes --- p.90 / Chapter 4.2.2.3 --- Differences in Treatment Costs --- p.93 / Chapter 4.2.3 --- Melphalan-based Regimens Versus VAD-based Regimens by Patients with Different DS Staging --- p.96 / Chapter 4.2.3.1 --- Patients in Stage 3-A MM --- p.96 / Chapter 4.2.3.2 --- Patients in Stage 3-B MM --- p.98 / Chapter 4.2.4 --- Melphalan-based Regimens versus VAD-based Regimens in Patients with Different IS Staging --- p.101 / Chapter 4.2.4.1 --- Patients in Stage I MM --- p.101 / Chapter 4.2.4.2 --- Patients in Stage II MM --- p.104 / Chapter 4.2.4.3 --- Patients in Stage III MM --- p.107 / Chapter 4.2.5 --- Comparison for Patients with and without Transplantation --- p.110 / Chapter 4.2.6 --- Cost-effectiveness Assessment --- p.117 / Chapter 4.2.7 --- Cost-utility Assessment --- p.118 / Chapter 4.2.8 --- Sensitivity Analysis --- p.119 / Chapter 4.2.9 --- Health-related Quality of Life Assessment --- p.120 / Chapter Chapter 5 --- Discussion and Conclusion / Chapter 5.1 --- Summary of Results --- p.123 / Chapter 5.2 --- Implication for Treatment --- p.126 / Chapter 5.3 --- Economic Evaluation --- p.129 / Chapter 5.4 --- Health-related Quality of Life --- p.132 / Chapter 5.5 --- Limitations of the Study --- p.134 / Chapter 5.6 --- Conclusion and Implications for Future Studies --- p.135 / Appendix --- p.137 / References --- p.139