Extracellular Hsp90alpha and Hsp70 Increase Activation of MMP-2 in Breast Cancer Migration and Invasion.
Sims, Jessica.
2011
-
Abstract: The goal
of this thesis was to gain a better understanding of extracellular Heat Shock Protein
90&alpha (Hsp90&alpha) 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
Hsp90&alpha outside of fibrosarcoma cells ... read moreand demonstrated that Hsp90&alpha
functions to increase the invasiveness of cancer cells by influencing MMP-2 activation.
I expanded upon this work by elucidating an export mechanism of Hsp90&alpha from
breast cancer cells, investigating how Hsp90&alpha affects MMP-2 activation, and
testing the ability of a cell-impermeable Hsp90&alpha 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 Hsp90&alpha. Hsp90&alpha 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 Hsp90&alpha is not exported through the
canonical signal sequence pathway or in an isoform specific manner. Hsp90&alpha is
instead exported from breast cancer cells via exosomes. Chapter 3 explores the role of
extracellular Hsp90&alpha in the activation of MMP-2 and in breast cancer cell
migration and invasion. I demonstrated that Hsp90&alpha 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
Hsp90&alpha, 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 Hsp90&alpha or Hsp70 significantly reduced the ability of breast
cancer cells to migrate or invade. In the appendix, I address the role of
Hsp90&alpha and MMP-2 activation in breast cancer cell invasion and metastasis. In
order to specifically target extracellular Hsp90&alpha, I tested an Hsp90&alpha
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 Hsp90&alpha
is exported from breast cancer cells via exosomes and describes one function of
extracellular Hsp90&alpha. 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 Hsp90&alpha
function-inhibiting antibody for its ability to specifically inhibit extracellular
Hsp90&alpha 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.
Thesis (Ph.D.)--Tufts University, 2011.
Submitted to the Dept. of Cellular & Molecular Physiology.
Advisor: Daniel Jay.
Committee: Gary Sahagian, Michael Forgac, Charlotte Kuperwasser, and James Dice.
Keywords: Physiology, Oncology, and Cellular biology.read less - ID:
- 41687w218
- Component ID:
- tufts:20564
- To Cite:
- TARC Citation Guide EndNote
