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

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.