Heat Shock Proteins, Hsp90 Inhibitors, and Protein Degradation

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Heat Shock Proteins, Hsp90 Inhibitors, and
Protein Degradation

• By Vince Centioni
• Paper A high affinity conformation of Hsp90
  confers tumour selectivity on Hsp90 inhibitors

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I. BackgroundA. Protein Degradation

• Protein molecules are continuously synthesized
  and degraded in all living organisms. The
  concentration of individual cellular proteins is
  determined by a balance between the rates of
  synthesis and degradation, which in turn are
  controlled by a series of regulated biochemical
  mechanisms.
• The only way that cells can reduce the steady
  state level of a particular protein is by
  degradation. Thus, complex and highly-regulated
  mechanisms have been evolved to accomplish this
  degradation.

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A. Protein Degradation cont.

• General principles Peptide bond Planar amide
  bond between a-carboxyl group a-amino group of
  2 adjacent amino acids.
• Proteolysis Biochemical degradation of protein
  through hydrolysis of peptide bonds.

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A. General Principle Diagram
Peptide Water Acid Amine
                                         
                                    
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A. Protein Degradation cont. (Intracellular
Proteolytic Systems)

• Lysosomal and Non-lysosomal
• Lysosomal Steps Uptake (Autophagy) into
  lysosome Secretory vesicles Cytoplasm
  Organelles, and enzymatic degradation .
• Non-lysosomal Steps Tagging of protein to be
  degraded (by ubiquitnation). Recognition of
  proteolytic system exposure of peptide sequence
  or distinction of unfolded protein segments
• Example Proteasome

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Ubiquitin-Proteasome Degradation

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A. Protein Degradation cont.

• Cells which are subject to stress such as
  starvation, heat-shock, chemical insult or
  mutation respond by increasing the rates of
  degradation.
• Selective degradation of particular proteins may
  occur in response to internal and external
  signals.

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B. Heat Shock Proteins

• Heat Shock proteins Family of proteins found in
  all cells that are expressed in response to cold,
  heat and other environmental stresses.
• Some serve to stabilize proteins in abnormal
  configurations, and play a role in folding and
  unfolding of proteins, acting as molecular
  chaperones.
• There are four major subclasses Hsp90, Hsp70,
  Hsp60, and small Hsps

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B. Heat Shock Proteins cont.

• Heat shock proteins are induced when a cell
  undergoes various types of environmental stresses
  like heat, cold, and oxygen deprivation.
• Heat shock proteins are also present in perfectly
  normal conditions where they act as chaperones
  making sure that the cells proteins are in the
  right shape and in the right place at the right
  time.
• HSPs also help shuttle proteins from one
  compartment to another inside the cell, and
  transport old proteins to garbage disposals
  inside the cell.

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B. Heat Shock Proteins cont.

• Heat shock proteins and the immune system under
  normal conditions HSPs are found outside the
  cell. But if a cancerous or infected cell has
  become so sick that it dies and its membrane
  bursts, all of cells contents spill out,
  including Hsps that are bound to peptides.
• These extracellular Hsps send a very strong
  danger r signal to the immune system,
  instructing it to destroy the other diseased
  cells.

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C. Hsp-danger signal to the immune system

• A sick cell dies and ruptures, spilling the
  Hsp-peptide complexes.
• These extracellular complexes of HSPs and
  peptides are detected by circulating immune
  system cells called APCs (antigen-presenting
  cells).
• The Hsp complexes bind the CD91 receptor on the
  APC cell surface. The APC can then take in the
  Hsp complexes, and then they travel to the lymph
  nodes.

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C. Hsp-danger signal cont.

• In the lymph nodes, the APCs take the peptides
  that were associated with HSPs and re-represent
  them on the cell surface.
• Specialized immune cells called T cells see
  these peptides and are then programmed to seek
  out the cells bearing these specific, abnormal
  peptides.
• Because every person and every cancer is
  different, the unique repertoire of antigenic
  peptides represents that individual specific
  cancers fingerprint.

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D. Heat Shock Protein 90

• Cellular chaperone protein required for the
  activation of several eukaryotic protein kinases,
  including the cyclin-dependant kinase (CDK4).
• Geldanamycin, and inhibitor of the
  protein-refolding activity of Hsp90, has been
  shown to have antitumor activities.
• Hsp90 is the most abundant heat shock protein
  under normal conditions.

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D. Hsp90 cont.

• The protein exists as two major isoforms,
  Hsp90-alpha and Hsp90-beta.
• The proteins activity has been shown to be ATP
  dependant with a unique pocket located in the
  N-terminal region.
• This pocket is the site where ATP and ADP binding
  activity take place. Once ATP binds , a
  structural amendment by Hsp90 induces a
  conformational change from an open position to a
  closed position.

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D. Hsp90 cont.

• Hsp90s contributions in signal transduction,
  protein folding, and protein degradation.
• Hsp90 is a phosphoprotein containing two or
  three covalently bound phosphate molecules per
  monomer, and the phosphorylation is thought to
  enhance its function. The monomer of the Hsp90
  consists of a conserved 25-kaDa N-terminal domain
  and a 55-kaDa C-terminal domain linked by a
  35-kaDa charged linker region.
• Together the C-terminal domain and the linker
  region helps in the dimerization of the protein

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D. Hsp90 cont.

• Hsp90 exhibits ATPase activity which is essential
  for its chaperone function.
• Hsp90 binds to an array of client proteins, where
  its co-chaperone specificity varies and depends
  on the actual client protein.
• There is a growing list of Hsp90 client proteins
  and most of them include molecules involved in
  signal transduction.

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D. Hsp90 cont.

• Hsp90 forms several discrete sub-complexes, each
  containing different set of co-chaperones that
  function at different steps during the folding
  process of the client protein.
• Unlike other chaperones, Hsp90 contains two
  independent chaperone sites that differ in their
  substrate specificity, probably working in the
  form of a switch between Hsp90 and Hsp70 client
  protein interactions.

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D. Hsp90 cont. (Multi-chaperone complex)

• The best understood molecular association of
  Hsp90-multi-chaperone complexes was in
  conjunction with the maturation of steroid
  receptors.
• The folding process of steroid receptors and
  their translocation to the cell nucleus requires
  Hsp90.
• Steroid receptors also require molecular
  chaperones for their ligand binding,
  transcriptional activation and repression after
  stimulus.

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D. Hsp90 cont.

• Though there are reports that some Hsp90
  co-chaperones can work independently of Hsp90,
  the full competence of these co-chaperones
  requires Hsp90.
• Co-chaperones also help Hsp90-client protein
  binding interactions between Hsp70 client
  proteins, and docking of cytoskeletal proteins.

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Hsp90 Diagram
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III. Introduction A. Hsp90 and Cancer
Cells

• Hsp90 has been implicated in the survival of
  cancer cells.
• Hsp90 regulates the function and stability of
  many key signal proteins that help cancer cells
  to escape the inherent toxicity of their own
  environment, to evade chemotherapy, and to
  protect themselves from the results of their own
  genetic instability.

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A. Hsp90 and Cancer Cells cont.

• Hsp90 plays a key role in the conformational
  maturation of oncogenic signaling proteins,
  including HER-2/ErbB2, Akt, Raf-t, Bcr-Abl and
  mutated p53.
• Tumor Hsp90 is present entirely in
  multi-chaperone complexes with high ATPase
  activity.
• Tumor cells overexpress Hsp90 client proteins,
  suggesting that a greater amount of Hsp90 in
  tumor cells might be engaged in active
  chaperoning and present in multichaperone
  complexes that could modulate the binding
  affinity of ligands to Hsp90.

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A. Hsp90 and Cancer Cells cont.

• Hsp90 regulates many signaling pathways in cancer
  cells.
• Recent discoveries in cell biology have
  demonstrated that many of the key signaling
  molecules that are deregulated in human cancers
  require the action of Hsp90 chaperone family in
  order to maintain their function.(Neckers and Lee
  2003)

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Signaling Molecules Influenced by
Hsp90
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A. Hsp90 and Cancer Cells cont.

• Cancer cells have numerous abnormalities that
  make them more dependent upon growth and survival
  which, are, in turn dependent upon Hsp90.
• Hsp90 works with other chaperone proteins and
  forms a Hsp90 multichaperone complex that
  maintains tumor progression, by stabilizing and
  interacting with a growing list of various
  kinases.
• Hsp90 chaperone complexes control protein folding
  and influence protein degradation.

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A. Hsp90 multichaperone pathway and
Ubiquitin-mediated degradation pathway
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A. Hsp90 and Cancer Cells cont.

• In tumors these signaling proteins are
  deregulated resulting in uncontrolled cell growth
  and survival.
• Certain Hsp90 inhibitors are designed to
  eliminate these deregulated signal transduction
  molecules from the tumor cell leading to death of
  the tumor.

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B. Hsp90 Inhibitors

• Certain Hsp90 inhibitors can bind into the ATP
  binding site of Hsp90 altering the function of
  the Hsp90 multichaperone complex.
• These inhibitors convert the Hsp90 complex from a
  catalyst for protein folding into one that
  induces protein degradation.
• The result is the degradation of a specific set
  of cancer signaling molecules, leading to cell
  cycle arrest and tumor cell death.

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B. Hsp90 inhibitors cont.

• Currently used Hsp inhibitors include
  geldanaymicn, herbimycin A and 17-AAG.
• In 1994, certain ansamycins were found to bind
  to Hsp90 and to cause the degradation of client
  proteins including Src kinases.
• Further efforts to develop anticancer drugs were
  made using geldanamycin analogs, and 17AAG was
  chosen as the best candidate for clinical trials.
  

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B. Hsp90 Inhibitors cont.

• Geldanaymicn is a natural Hsp90 inhibitor, that
  essentially causes the complete destruction of
  the receptor tyrosine kinase HER2/neu, a key
  driver of breast cancer growth.
• Geldanaymicn initially thought to be due to
  specific tyrosine kinase inhibition, later
  studies revealed that the antitumor potential
  relies on the depletion of oncogenic protein
  kinases via the proteasome.

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C. Geldanaymicn Structure
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C. Hsp90 inhibitors cont.

• Subsequent immunoprecipitation and X-ray
  crystallographic studies reveled that GA directly
  binds to Hsp90, and inhibits the formation of
  Hsp90 multichaperone complexes resulting in the
  ubiquitin-mediated degradation of Hsp90 client
  proteins.
• This represented the first generation of drugs
  that specifically targeted Hsp90.

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C. GA cont.

• GA binds to the N-terminal domain of Hsp90 and
  competes with ATP binding.
• The geldanamycin-Hsp90 crystal structure also
  shows that the binding inhibits substrate protein
  binding.
• GA also binds to Grp94, the Hsp90 analogue in the
  ER

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C. GA bound to Hsp90
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D. Advantages of Hsp90 inhibitors

• Preclinical trials emphasize the important role
  of Hsp90 inhibitors in clinical applications.
• Combination therapies, applying low doses of
  these drugs together with convention
  chemotherapeutic agents, seem to be an effective
  way to target various cancers.
• For example, in the case of Bcr/Abl-expressing
  leukemias, a low dose GA is sufficient to
  sensitize these cells to apoptosis.

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D. Advantages of Hsp90 Inhibitors

• Among the hallmarks of cancer, up regulation of
  growth signals and evasion of apoptosis are the
  most important.
• As most growth regulatory signals depend on Hsp90
  for their function stability, Hsp90 is an ideal
  molecule to intervene in complex oncogenic
  pathways.
• Hence, most drugs are targeting Hsp90, which is
  more beneficial than the selective oncogene
  pathway inhibitors.

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E. 17-Allylamino, 17-Demethoxygeldanamycin
(17-AAG)

• An analog of GA.
• There is a correlation between down-regulation of
  Hsp90s client proteins and growth inhibition
  caused by 17-AAG.
• 17-AAG has a higher affinity to bind to Hsp90
  multichaperone complexes than Hsp90 in normal
  cells. There is only speculation of why this is
  true.

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E. 17-AAG Structure
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IV. Paper

• A high affinity conformation of Hsp90 confers
  tumor selectivity on Hsp90 inhibitors
• by Kamal, Thao, Sensintaffar, Zhang, Boehm,
  Fritz, and Burrows

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Important Discoveries

• Tumour Hsp90 is present entirely in
  multichaperone complexes with high ATPase
  activity, whereas Hsp90 from normal tissue is in
  a latent uncomplexed state. (Kamal 2003)
• In vitro reconstitution of chaperone complexes
  with Hsp90 resulted in increased binding affinity
  to 17-AAG, and increased ATPase activity. (Kamal
  2003)

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Important Discoveries cont.

• Tumour cells contain Hsp90 complexes in an
  activated, high-affinity conformation that
  promotes malignant progression, and that may
  represent a unique target for cancer therapeutics
• How do we prove if Hsp90 from tumour cells had a
  higher binding affinity to 17-AAG than that from
  normal cells?

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Figure 1 Hsp90 binding affinity to 17-AAG

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Figure 1 Results

• Hsp90 in tumour cells has a significantly higher
  binding affinity for 17-AAG than does Hsp90 from
  normal cells. (Kamal 2003)
• The binding affinity of 17-AAG to Hsp90 from the
  different cells directly correlates with the
  cytotoxic/cytostatic activity of 17-AAG in those
  cells (Fig. 1d)

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Further Questions

• Hsp90 interacts with many co-chaperone proteins
  that assemble in multi-chaperone complexes
• Tumour cells overexpress Hsp90 client proteins
• How to find out whether Hsp90 in tumour cells was
  present in multi-chaperone complexes?

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Figure 2 Levels of p23 and Hop associated with
Hsp90
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Figure 2a Results

• Co-immunoprecipitation in the normal and tumour
  cell lystates with antibodies to Hsp90 revealed
  that more Hsp90 in tumour cell was present in
  complexes with p23 and Hop compared to to normal
  cells (Kamal 2003)
• Control immunoprecipitaions without antibodies
  did not immunoprecipitate any Hsp90
• Immunoprecipitation with antibodies to both p23
  and Hop revealed that the entire tumour cell pool
  of Hsp90 was present in complexes unlike normal
  cells.

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Figure 2a Results cont.

• Immunoprecipitation with an antibody specific for
  the uncomplexed form of Hsp90 pulled down far
  more Hsp90 from normal cells than from tumour
  cells.
• All Hsp90 in tumour cells is in the form
  multi-chaperone complex that are actively engaged
  in chaperoning client oncoproteins, and that the
  heightened complex formation is not due to
  increased expression of Hsp90 or co-chaperones.

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Figure 2b Results

• Since the chaperone function of Hsp90 is
  dependent upon ATPase activity, they
  immunopreipitated Hsp90 from cell lysates and
  performed ATPase assays
• Tumour Hsp90 had markedly higher ATPase activity
  compared to Hsp90 from normal cells, and was
  inhibited by 17-AAG. (Kamal 2003)
• Control immunoprecipitation in the absence of
  Hsp90 antibody did not have any significant
  ATPase activity (data not shown). (Kamal 2003)

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Figure 2 Combined Results

• These results suggest that essentially all
  soluble Hsp90 in tumour cells is present in fully
  active multi-chaperone complexes, whereas Hsp90
  in normal cells is in an uncomplexed inactive
  form. (Kamal 2003)
• Data demonstrates that the Hsp90 from tumour
  cells had higher binding affinity to 17-AAG, and
  this correlates with presence of increased
  multi-chaperone complexes and increased ATPase
  activity.
• Could there by any further tests?

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Further Questions and Tests

• How to examine whether reconstitution of purified
  Hsp90 with co-chaperones would result in
  increased binding affinity and ATPase activity?
• In vitro reconstitution, they used five proteins
  that have been shown to be required for the in
  vitro chaperoning activity of Hsp90. (Hsp90, 70,
  40, Hop, and p23)

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Figure 3 In vitro reconstitution of purified
Hsp90 with co-chaperones
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Figure 3a Results

• Of all four co-chaperones to purified Hsp90
  increased the apparent affinity of 17-AAG from
  600 nM for Hsp90 alone to 12 nM in the presence
  of the added proteins, where as partial complexes
  did not. (Fig3a)
• Therefore, Hsp90 when reconstituted with
  co-chaperones has nanomolar binding activity, in
  concordance with that observed in tumour cell
  lysates. (Kamal 2003)

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Figure 3b Results

• Hsp90 reconstituted with the indicated
  co-chaperones has increased ATPase activity,
  which can be inhibited by 10 micromolar 17-AAG
• The ATPase activity of Hsp90 was also
  significantly enhanced by all four co-chaperones
  and was inhibited by the addition of 17-AAG.
  (Kamal 2003)
• Hsp70 had some minimal ATPase activity, but was
  not inhibited by 17-AAG

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Figure 3 Combined Results

• These results suggest that Hsp90 present in
  multi-chaperone complexes not only had a higher
  binding affinity for 17-AAG but was also more
  biochemically active. (Kamal 2003).

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Further Experiments

• How to determine if these observations in vitro
  also apply to mice and to clinical cancer?
• They examined the binding affinity of 17-AAG to
  Hsp90 in normal and malignant mouse and human
  tissue samples (Fig4a,b)

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Figure 4 Hsp90 from clinical tumour samples
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Figure 4a,b Results

• The apparent binding affinity of 17-AAG to Hsp90
  from mouse tumours (3T3-src, B16, and CT26) was
  8-35 nM compared to 200-600 nM for the mouse
  normal tissues, even though Hsp90 was more
  abundantly expressed in several of the normal
  tissues (Fig4a)
• For the human tissues, using four samples of each
  tissue type Normal breast vs. Breast carcinoma
  and Normal colon vs. colon carcinomas (Fig4b)

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Figure 4c,d Results

• Co-immunoprecipitations and observed that there
  were increased amounts of p23 with Hsp90 from the
  human tumours compared to the normal tissues
  (Fig4c). (Kamal 2003)
• Conversely, there was significantly more
  uncomplexed Hsp90 co-immunoprecipitated with the
  Hsp90 antibody from the normal tissues compared
  to tumour tissues (Fig4c)

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Figure 4c,d Results cont.

• The Hsp90 activity in both clinical tumour
  samples was significantly higher relative to the
  normal tissue and was inhibited by 17-AAG,
  radicicol, and 0.5 M KCl (Fig.4d) (Kamal 2003).
• Resulting that the Hsp90 human clinical tumour
  tissues is in a high-affinity, multi-chaperone
  complex with increased ATPase activity
• The markedly higher affinity tumour Hsp90 in
  vivo explains the remarkable ability of
  ansamycins to accumulate progressively at tumour
  sites in animals. (Kamal 2003)

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Final Results

• The data showed that Hsp90 in tumour cells exists
  in a functionally distinct molecular form, and
  therefore clarify three fundamental aspects of
  the role of Hsp90 in tumour biology.

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Fundamental Aspects

• First, the nanomolar binding of 17-AAG to
  high-affinity tumour Hsp90 is now consistent with
  the nanomolar anti-tumour of this family of drugs
• Second, the markedly higher affinity of tumour
  Hsp90 in vivo explains the remarkable ability of
  ansamycins to accumulate progressively at tumour
  sites in animals.
• Third, the complete usage of tumour Hsp90 may
  provide a selection pressure leading to further
  upregulation of Hsp90 that is observed in many
  advanced tumours.

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Final Thoughts

• Dependence on the activated, high-affinity
  chaperone could make Hsp90 an Achilles heel of
  tumour cells, driving the selective accumulation
  and bioactivity of pharmacological Hsp90
  inhibitors, and making tumour Hsp90 a unique
  cancer target. (Kamal 2003)
• What makes Hsp90 better able to bind 17-AAG when
  the protein is part of the super-chaperone
  complex? Can only speculate

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References

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  2003.
• Lee and Neckers. Nature. V425. pg. 357-359.
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  281-290. (2001).
• Stebbins, C.E., Russo, A.A. Cell V89. pg.
  239- 248. 1997.
• Yano, M Tanakasa, S. Ansano. Gene Expression
  and Roles of Heat Shock Proteins in Human Breast
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