WFYellow(F08) Research Proposal
Background: Heat shock proteins are molecular chaperons involved in maintaining the stability of certain proteins under stress induced conditions. Hsp90 is one particular type of heat shock protein that maintains a group of proteins called Hsp90 client proteins. Surprisingly, many of the client proteins, which include mutant p53, are involved in accelerating and sustaining breast cancer. It has been found that adding Hsp90 inhibitors increases the ubiquitination and degradation of these client proteins. Currently, an Hsp90 inhibitor, geldanamycin, is in phase II clinial trials.
Recently, it has been found that a pool of Hsp90 is exported out of the cell and bound to the cell membrane, either directly or through another membrane bound proteins. The functionality of such extracellular Hsp90 is unknown, but it is believed to promote cell metastasis.
Importance: Since geldanamycin affects both intracellular and extracellular Hsp90, one can expect that individuals who use this drug will suffer from negative side effects. Knocking extracellular Hsp90 not only be less harmful, since only cancerous cells express Hsp90 on their surface, but would also decrease cell metastasis. Doing so would still be very beneficial, since metastasis drastically reduces a patient's chances of surviving.
Idea(s): There are several directions that our project can take. We could:
- Identify compounds that decrease extracellular Hsp90 function (i.e. drugs that inhibit extracellular Hsp90, but not intracellular Hsp90)
- Identify proteins associated with extracellularlly-presented Hsp90 (in particular HER-2/ErbB2), which is known to be over-expressed in proliferating cancer cells - doing many identify other proteins needed for metastasis.
- Determine secretory pathway for extracellular Hsp90 - inhibiting this rare pathway would decrease extracellular Hsp90, and also eludicate a novel pathway that can be used to express other cytosolic proteins on the cell surface <- novel biotechnology technique?
- Identify the membrane targeting sequence of Hsp90 (if there is one), produce interfering RNA, and observe phenotypic changes
- High membrane-bound Hsp90 expression also correlates with the rounded morphology of proliferative cancer. One potential research idea is to study the induction of flat vs. rounded morphology pathways and observe whether or not return of flattened morphology reduces cell proliferation, metastasis, and tissue invasion.
Summaries of particular papers: Hsp90 has been found to interact with many client proteins inside cancerous cells. These include mutant versions of HER-2 receptor, Raf-1, CDK4 and p53. The inhibitor geldanamycin has been found to strongly bind to Hsp90 and decrease its activity, which in turn decreases the activity of these client proteins. Hsp90 has recently been found outside the cell, bound to the membrane. The authors suggest that Hsp90 interacts with the extracellular segment of HER-2, which in turn actives a protein called Erb-3, which, through a signaling pathway, leads to greater cell mobility. The authors propose that inhibiting extracellular Hsp90 can limit metastasis. 
The authors conducted a functional screen that found that Hsp90 alpha, but not Hsp90 beta, is expressed on the cell surface of fibrosarcoma and breast cancer cells. Hsap90 alpha is found to interact with MMP2, through co-immunoprecipitation. MMPs are involved in degrading the extracellular matrix, and their expression has been previously found to be increased in almost all cancer cells. The authors also showed that geldanamycin inhibits both extracellular and intracellular Hsp90. The authors suggest searching for drugs that inhibit only extracellular Hsp90, but not intracellular Hsp90, to reduce the toxicity of the drug. 
Links to papers:
- Cid C, Regidor I, Poveda PD, and Alcazar A. . pmid:18800240.
- Eustace BK, Sakurai T, Stewart JK, Yimlamai D, Unger C, Zehetmeier C, Lain B, Torella C, Henning SW, Beste G, Scroggins BT, Neckers L, Ilag LL, and Jay DG. . pmid:15146192.
- Sidera K, Gaitanou M, Stellas D, Matsas R, and Patsavoudi E. . pmid:18056992.
- Sidera K and Patsavoudi E. . pmid:18469526.
- Eustace BK and Jay DG. . pmid:15326368.
- Tsutsumi S and Neckers L. . pmid:17645779.
Methods (in brief - this would depend on what experiment we decide to do)
TAP (or other)-tagging