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Extracellular Hsp90alpha and Hsp70 Increase Activation of MMP-2 in Breast Cancer Migration and Invasion.

机译：细胞外Hsp90alpha和Hsp70增加了乳腺癌迁移和侵袭中MMP-2的激活。

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摘要

The goal of this thesis was to gain a better understanding of extracellular Heat Shock Protein 90alpha (Hsp90alpha) and its role in breast cancer migration and invasion through the activation of Matrix Metalloproteinase-2 (MMP-2). This work, which was begun by Brenda Eustace, a previous graduate student in the Jay Lab, initially identified Hsp90alpha outside of fibrosarcoma cells and demonstrated that Hsp90alpha functions to increase the invasiveness of cancer cells by influencing MMP-2 activation. I expanded upon this work by elucidating an export mechanism of Hsp90alpha from breast cancer cells, investigating how Hsp90alpha affects MMP-2 activation, and testing the ability of a cell-impermeable Hsp90alpha inhibitor to reduce breast cancer migration and invasion, both in vitro and in vivo.;In chapter 2 of this thesis, I address the mode of export of Hsp90alpha. Hsp90alpha was found to have two different isoforms, one of which contained an alternative start site and putative signal sequence, indicating that it could be exported through the canonical signal sequence pathway. However, we demonstrated that Hsp90alpha is not exported through the canonical signal sequence pathway or in an isoform specific manner. Hsp90alpha is instead exported from breast cancer cells via exosomes.;Chapter 3 explores the role of extracellular Hsp90alpha in the activation of MMP-2 and in breast cancer cell migration and invasion. I demonstrated that Hsp90alpha interacts with MMP-2, along with the co-chaperones Hsp70, Hop, Hsp40, and p23, both in vitro and in cancer cell conditioned media. This was the first time that all four of these co-chaperones were demonstrated to be present together outside of cancer cells. I showed that Hsp90alpha, in conjunction with these co-chaperones, was capable of assisting in the activation of MMP-2 in vitro. Also, when Hsp70 was inhibited, the activation of exogenously added MMP-2 in conditioned media was reduced, indicating the importance of Hsp70 in MMP-2 activation. I used wound healing and invasion assays to demonstrate that inhibition of Hsp90alpha or Hsp70 significantly reduced the ability of breast cancer cells to migrate or invade.;In the appendix, I address the role of Hsp90a and MMP-2 activation in breast cancer cell invasion and metastasis. In order to specifically target extracellular Hsp90alpha, I tested an Hsp90alpha function-inhibiting antibody for its ability to reduce cancer cell invasion. I tested the antibody in an in vitro invasion assay, where I observed a 40% reduction in invasion. In addition, I tested this antibody for its ability to inhibit metastasis in an in vivo breast-to-bone metastasis model, for which the data was inconclusive. The appendix also includes a paper that contains part of my work described in chapter 2 and contributions from Jessica McCready, Ph.D.;This thesis demonstrates that Hsp90alpha is exported from breast cancer cells via exosomes and describes one function of extracellular Hsp90alpha. In addition, this dissertation describes a novel mechanism for MMP-2 activation that is independent of MT1-MMP, the enzyme traditionally associated with MMP-2 activation. I also began testing an Hsp90a function-inhibiting antibody for its ability to specifically inhibit extracellular Hsp90alpha and reduce cancer cell migration and invasion. I demonstrated that the inhibitor is capable of causing a significant reduction in cancer cell invasion in vitro and warrants further study both in in vitro and in vivo models.
机译：本文的目的是通过激活基质金属蛋白酶2（MMP-2）更好地了解细胞外热休克蛋白90alpha（Hsp90alpha）及其在乳腺癌迁移和侵袭中的作用。这项工作由杰伊实验室前研究生Brenda Eustace开始，最初在纤维肉瘤细胞外部鉴定了Hsp90alpha，并证明Hsp90alpha通过影响MMP-2活化来增加癌细胞的侵袭性。我通过阐明乳腺癌细胞中Hsp90alpha的输出机制，研究Hsp90alpha如何影响MMP-2活化以及测试不渗透细胞的Hsp90alpha抑制剂在体外和体内减少乳腺癌迁移和侵袭的能力来扩展这项工作。在本文的第二章中，我讨论了Hsp90alpha的输出方式。发现Hsp90alpha具有两种不同的同种型，其中一种包含一个替代的起始位点和推定的信号序列，表明它可以通过规范的信号序列途径输出。但是，我们证明了Hsp90alpha不会通过规范信号序列途径或同工型特异性方式输出。相反，Hsp90alpha是通过外泌体从乳腺癌细胞输出的。第三章探讨了细胞外Hsp90alpha在激活MMP-2以及在乳腺癌细胞迁移和侵袭中的作用。我证明了Hsp90alpha在体外和癌细胞条件培养基中都与MMP-2以及伴侣伴侣Hsp70，Hop，Hsp40和p23相互作用。这是首次证明所有这四个伴侣分子都在癌细胞外部并存。我表明，Hsp90alpha与这些伴侣伴侣一起能够在体外辅助MMP-2的活化。同样，当Hsp70被抑制时，条件培养基中外源添加的MMP-2的激活减少，这表明Hsp70在MMP-2激活中的重要性。我使用伤口愈合和侵袭试验证明对Hsp90alpha或Hsp70的抑制作用显着降低了乳腺癌细胞迁移或侵袭的能力。在附录中，我探讨了Hsp90a和MMP-2激活在乳腺癌细胞侵袭和侵袭中的作用。转移。为了特异性靶向细胞外Hsp90alpha，我测试了Hsp90alpha功能抑制抗体减少癌细胞侵袭的能力。我在体外侵袭试验中测试了抗体，观察到侵袭减少了40％。另外，我在体内乳腺癌至骨转移模型中测试了该抗体抑制转移的能力，该数据尚无定论。附录还包括一篇论文，其中包含我在第2章中描述的部分工作以及Jessica McCready博士的文稿；本论文证明了Hsp90alpha是通过外泌体从乳腺癌细胞输出的，并描述了细胞外Hsp90alpha的一种功能。此外，本文描述了一种新型的MMP-2激活机制，该机制独立于传统上与MMP-2激活相关的酶MT1-MMP。我还开始测试Hsp90a功能抑制抗体的特异性抑制细胞外Hsp90alpha并减少癌细胞迁移和侵袭的能力。我证明了该抑制剂能够在体外显着减少癌细胞的侵袭，并且有必要在体外和体内模型中进行进一步的研究。

著录项

  • 作者

    Sims, Jessica D.;

  • 作者单位

    Sackler School of Graduate Biomedical Sciences (Tufts University).;

  • 授予单位 Sackler School of Graduate Biomedical Sciences (Tufts University).;
  • 学科 Biology Cell.;Biology Physiology.;Health Sciences Oncology.
  • 学位 Ph.D.
  • 年度 2011
  • 页码 179 p.
  • 总页数 179
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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