heat shock proteins...previous information from dr. repasky regarding hsps and stress responses,...
TRANSCRIPT
Heat Shock Proteins in Cancer Immunology:
Friends or Foes?
Michelle Messmer Trends in Tumor Immunology
9/17/14
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What did you think of the paper? How are topics for research selected?
Heat Shock Proteins
• Protein/peptide chaperones
• Up-regulated during times of stress
• Multiple families defined by: • Structure • Localization • Function
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Previous information from Dr. Repasky regarding HSPs and stress responses, ability to act as DAMPs, evolutionarily ancient roles in cellular biology When/why would proteins/peptides need chaperones? Basic biochemistry – AA side chains, effects on tertiary structure Give examples of cellular stress, pertinence to cancer – hypoxia, low glucose, altered pH What family of proteins is discussed in the paper? What is their localization? Primarily intracellular, HSP70/HSC70 cytoplasmic, Grp78 in the ER. Upregulated: hyperthermia, oxidative stress, and changes in pH. Client proteins: steroid hormone receptors, Raf kinase, eIF2α-kinase, CyclinB1/Cdk1 and heat shock transcription factors–1 (HSF-1), c-Myc and pRb.
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HSC70 and the other HSPs utilize the power of ATP hydrolysis to assist in protein folding– the binding partners that associate with the HSP determine trafficking and client protein specificity. HSPs also important in regulating protein expression, recycling, and degradation – protect clients and bring them in association with quaternary subunits, determine exit from ER to golgi, target misfolded proteins to the proteasome. Constitutive vs. inducible forms can have different partners- thus different, specialized functions Inhibitors of HSPs largely focus on blocking ATP binding domain (17-DAAG, others), catalytic domain, can also block binding partners. What would be the effect of these inhibitors? Selective death of cells experiencing stress – but still non-specific – similar to other chemotherapeutics – off target effects – additional problems with immune responses as will be discussed. Many of the details of HSP function have been worked out in yeast and bacterial systems; their mammalian counterparts have comparable functions, but with additional complexity
HSP70
HSP90
Grp78
Grp94 Calreticulin
HSP110
Grp170
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Classic image of MHC processing and presentation of intracellular and extracellular antigens. What’s missing from this picture? Peptides can not freely exist inside or outside the cell HSP70, HSP90, HSP110 primarily cytosolic Grp94, grp78, calreticulin, grp170 in the ER
Roles for HSPs in Immunity
• Extracellular roles: • Identification as tumor
antigens in the 1980s • Depletion of HSPs
abrogates cell lysate immunogenicity
• Receptor specific uptake
• Targeting of HSP chaperoned antigens to peptide processing pathway
• DAMP signaling
• Intracellular roles • Peptide processing relay
line • Transport from
endosome to proteasome to MHC
• Immunologically relevant clients
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Extracellular roles: Identification as tumor antigens in the 1980s Depletion of HSPs abrogated cell lysate immunogenicity: HSPs were redundant – depletion of only one fails to abrogate, but adding back only one can rescue Responses are peptide specific: MHC independent, different size requirements Receptor specific uptake: receptor identity still questioned, however, highly efficient processing Evolutionarily ancient: identified in xenopus, targeting of HSP chaperoned antigens to peptide processing pathway: retained in early endosomes, transported to cytosol DAMP signaling: some controversy- contamination with LPS, differential cytokines dependent on HSP, can differentially impact outcomes, upregulation of co-stimulatory molecules Intracellular roles Peptide processing relay line:Transport from proteasome to MHC: HSP90 interaction with endosome, interaction with proteasome and TAP in cytosol, hand off within the endoplasmic reticulum from TAP to grp94 to calreticulin to MHC class I Immunologically relevant clients: TLRs, Immunoglobulin
• Two ‘opposing’ roles for heat shock proteins:
• Tumor Promoter
• Immune Stimulator
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What were the major functions of HSP70 under investigation in this paper? Why is it important to distinguish between these roles?
Models in this Paper
• HSP Nomenclature • HSP70 & HSC70 • HSP90AA1 & HSP90AB1
• Oncogenes
• E1A/Ras • Bcr-Abl
• Mice • HSP70.1.3-/-
• HSF-1-/-
• CD1-Foxn1nu
• Eµ-Myc
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HSP70 & HSC70: inducible vs. constitutive, HSP90AA1 & HSP90AB1: inducible vs. constitutive E1A/Ras vectors: E1A associates with p300/CBP histone acetyltransferases at c-fos promoters to form inactive chromatin structure, Ras constitutively activates the MAP/ERK kinase cascade, Bcr-Abl: interaction with HSP70 HSP70.1.3-/- mice: increased radiosensitivity and sensitivity for heat, fertile with no obvious phenotype HSF-1-/- mice: loss of Hsf1 function did not prolong tumor-free survival in p53(-/-) mice, but altered the spectrum of tumors that arose. Tumor development is rapid in p53(-/-) mice, which predominantly (about 70%) succumb to lymphomas. In contrast, hsf1(-/-)p53(-/-) mice rarely develop lymphomas (<8%), but succumb to other tumor types including testicular carcinomas and soft tissue sarcomas. Eliminating HSF1 also protects mice from tumors induced by mutations of the RAS oncogene CD1-Foxn1nu: The nu allele on chromosome 11 is an autosomal recessive mutation. Dysfunctional rudimentary thymus. Phenotypically hairless (sparse, intermittent hair growth possible). Athymia. No generation of cytotoxic effector cells. No graft versus host response Eµ-Myc: c-myc gene driven by the IgH enhancer (E mu-myc) develop lymphomas, 90% succumbing in the first 5 mo of life.
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HSP70 is neither required for in vitro transformation nor tumor growth in vivo. Selected to upregulate HSP70 to support growth but tumors can survive without HSP70, HSP70-/- increase HSP90 expression
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Neither BCR-ABL nor Eu myc requires HSP70 to induce lymphoma. HSP70 is not required for BCR-Abl, Eu-Myc oncogene expression – model: bone marrow induced lymphoma BCR-ABL – same survival +/- HSP70 Eu-Myc - better survival in HSP70-/- Maturity of B cell lymphoma
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Growth of WT and hsp70-/- tumors is dependent on the immune status of the host Worse survival for transformed MEFs injected subcutaneously- WT were rejected, KO grew Difference dependent on immune status Selection for HSP70 expression; hsp90, hsc70 to make up for it
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HSP70-/- tumors are associated with a significant reduction in immune cell infiltration Reduced immune infiltration – Mac 2 staining in a (WT) & b (nude), T cell staining in c
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HSP70-/- tumors are associated with a significant reduction in immune cell infiltration
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Tumor growth in vivo selects against HSP70 expression in an immune competent host Tumors selected for shRNA knockdown of HSP70, retention of RFP; b – nude, d – WT Mac2 on f & g, cd3 on h
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Tumor-derived hsp70 functions in a chemokine-like manner to facilitate intra-tumoral infiltration of immune cells Tumor growth depended on loss of HSP70 within the tumor, not the host Mac2 (b) and CD3 (c) staining D & E migration assay – how was it done? What does it actually show?
Conclusions Is HSP70 required for tumor growth? • Pubmed search:
HSP70 inhibitor: 1395 HSP90 inhibitor: 1912 Grp94 inhibitor: 116 Total: 3423
Is HSP70 required for immune responses? • Pubmed search:
HSP70 vaccine: 562 HSP90 vaccine: 93 Grp94 vaccine: 30 Total: 685
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1980 1990 2000 2010 2020
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HSP70 inhibitorHSP70 vaccine
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Is HSP70 required for tumor growth? Tumor specific, can selectively adapt. General form of therapeutic: small molecule inhibitors, good bioavailability, relatively cheap, but if targeted: toxic, off target effects Is HSP70 required for immune responses? Not required, but can stimulate. General form of therapeutic: purified protein from patient, or from cell lines, DC based vaccines, if targeted: relatively safe but may not be effective in immunocompromised states Consider combination therapies for both approaches – which best with immunotherapies?