The goal of this thesis was to gain a better understanding of extracellular Heat Shock Protein 90α (Hsp90α) 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 Hsp90α outside of fibrosarcoma cells and demonstrated that Hsp90α functions to increase the invasiveness of cancer cells by influencing MMP-2 activation. I expanded upon this work by elucidating an export mechanism of Hsp90α from breast cancer cells, investigating how Hsp90α affects MMP-2 activation, and testing the ability of a cell-impermeable Hsp90α 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 Hsp90α. Hsp90α 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 Hsp90α is not exported through the canonical signal sequence pathway or in an isoform specific manner. Hsp90α is instead exported from breast cancer cells via exosomes.
Chapter 3 explores the role of extracellular Hsp90α in the activation of MMP-2 and in breast cancer cell migration and invasion. I demonstrated that Hsp90α 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 Hsp90α, 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 Hsp90α or Hsp70 significantly reduced the ability of breast cancer cells to migrate or invade.
In the appendix, I address the role of Hsp90á and MMP-2 activation in breast cancer cell invasion and metastasis. In order to specifically target extracellular Hsp90α, I tested an Hsp90α 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 Hsp90α is exported from breast cancer cells via exosomes and describes one function of extracellular Hsp90α. 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 Hsp90á function-inhibiting antibody for its ability to specifically inhibit extracellular Hsp90α 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.