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首页> 外文学位 >Determination of the Biological Significances of Platelet Factor 4 (PF4), a Tumor Suppressor Gene Encoding an Angiogenesis Inhibitor in Multiple Myeloma.
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Determination of the Biological Significances of Platelet Factor 4 (PF4), a Tumor Suppressor Gene Encoding an Angiogenesis Inhibitor in Multiple Myeloma.

机译：确定血小板因子4（PF4）的生物学意义，PF4是编码多发性骨髓瘤中血管生成抑制剂的肿瘤抑制基因。

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摘要

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 ceils 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 ofPF4 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 CD138 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-1, 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.
机译：多发性骨髓瘤（MM）是一种无法治愈的血液系统恶性肿瘤，其特征是克隆性浆细胞在骨髓（BM）中积累。 MM的发展和进展是一个复杂的多步骤致瘤事件，涉及肿瘤细胞的遗传和表观遗传变化以及BM微环境的支持。众所周知，MM细胞与BM环境的物理相互作用对于MM发病机理，MM细胞生长，存活，迁移和耐药性至关重要。血小板因子4（PF4）是一种有效的抗血管生成趋化因子，不仅在体外抑制内皮细胞的增殖和迁移，而且在体内也抑制实体瘤的生长。我们的研究小组先前证实，由于同时发生等位基因缺失和DNA甲基化过多，导致患者MM样本和MM细胞系中PF4表达的缺失。在这项研究中，我们表征了PF4对BM环境中MM细胞和血管生成的影响，并阐明了PF4对MM的作用机理。为了表征PF4在体外对MM细胞的影响，对细胞进行测定进行了生长，细胞周期停滞和凋亡的研究，我们发现PF4抑制了MM细胞系和患者MM细胞的生长并诱导了凋亡。 PF4的凋亡活性与caspase-3和聚（ADP）核糖聚合酶（PARP）的激活有关。我们还使用分离自患者BM抽吸物（MMEC）的内皮细胞研究了PF4对MM中血管生成的影响。我们的结果表明PF4抑制MMECs在基质胶上的生长和管形成具有剂量依赖性。;鉴于PF4体外抑制MM细胞生长和血管生成的能力，我们在体内评估了其抑癌功能。在人皮下基质胶异种移植小鼠模型中，尾静脉注射200ng PF4可显着降低MM肿瘤的生长并延长生存期。接下来，我们使用了SCID-rab小鼠模型，该模型在体内概括了人类BM环境。在该模型中，将MM细胞直接注射到兔骨中，然后将其皮下植入NOD-SCID小鼠中。注射后两周，SCID小鼠通过尾静脉注射用各种剂量的PF4（每次注射20或200ng，每周3次）或PBS治疗。 ELISA法检测hig（λ），与对照组相比，用200ng PF4处理的小鼠的肿瘤生长明显降低了58％，这通过兔骨切片CD138染色的免疫组织化学分析进一步证实。与体外结果一致，体内也可证明MM4诱导MM细胞凋亡并抑制血管生成，这在治疗后的兔和兔的兔骨切片上ki67，Casped caspase-3，CD31和VEGF染色中得到了证实。对照小鼠。因此，我们的发现证实了PF4是MM中的一种新型肿瘤抑制因子。然而，关于PF4如何抑制MM肿瘤发生的分子机制仍不清楚。为了鉴定参与MM的信号途径PF4，进行了蛋白质/ DNA阵列。我们发现PF4在U266​​细胞中调节了几种转录因子，包括STAT3。 EMSA和荧光素酶报告基因检测进一步证实了PF4抑制了STAT3 DNA的结合和转录活性。因此，PF4可能通过抑制MM细胞中的STAT3途径来介导其肿瘤抑制功能。我们进一步发现，PF4的预处理在人类MM细胞中以时间依赖的方式阻断了组成型和白介素6诱导的STAT3激活。 PF4还可以下调STAT3调控的基因产物，包括Mcl-1，Survivin和血管内皮生长因子（VEGF）。此外，从PF4诱导的凋亡中，组成型活性STAT3的表达得以抢救。在SCID-rab小鼠模型中，我们还发现PF4通过兔骨切片的免疫染色抑制STAT3核易位。进一步检查时，我们发现PF4诱导了STAT3抑制剂SOCS3之一的表达，并且通过小的干扰RNA使SOCS3的基因沉默消除了PF4抑制STAT3激活的能力，表明SOCS3在PF4的作用中起着关键作用。因此，我们的发现表明，通过诱导SOCS3表达，PF4消除了STAT3活性，从而诱导了肿瘤生长抑制和抗血管生成。；这些新颖的研究共同表明，PF4是MM肿瘤发生的重要调节剂。通过废除STAT3信号，它靶向细胞生长，诱导细胞凋亡，并在MM体内和体外抑制血管生成。这些科学观察为这种趋化因子的临床研究提供了框架，该趋化因子是治疗MM以改善MM患者预后的新型药物。

著录项

  • 作者

    Liang, Pei.;

  • 作者单位

    The Chinese University of Hong Kong (Hong Kong).;

  • 授予单位 The Chinese University of Hong Kong (Hong Kong).;
  • 学科 Oncology.;Molecular biology.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 182 p.
  • 总页数 182
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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