GU ONCOLOGY CENTER OF EXCELLENCE

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TEAM 1
2
Investigators

• Michael Cher and Rafael Fridman Wayne State
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• Leland Chung and Haiyen Zhao Emory
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• Theresa Guise and John Chirgwin U Virginia
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• Kenneth Koeneman U Texas Southwestern
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• Hari Reddi -- UC Davis
•  Robert Vessella and Eva Corey U Washington

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The Enigma of Prostate CancerBone Metastases

• Very high propensity for metastasis to bone
• In contrast to other tumors metastatic to bone
  (e.g. breast cancer), prostate cancer causes new
  bone formation (osteoblastic response) rather
  than bone degradation (osteolytic response)
  mechanisms are unknown and not well studied
• In many autopsy series, including our own, gt50
  of patients expire with clinically insignificant
  non-osseous metastases begging the question --
  What is the cause of death?

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Inquiries Related to Prostate Cancer Bone
Metastases

• When does seeding of the bone marrow begin?
• How is the seeding process executed?
• What are the cellular interactions and factors
  that appear to favorably promote CaP growth in
  the bone?
• What cellular interactions and factors are
  involved in perturbing normal bone remodeling
  that leads to the classical osteoblastic
  response?
• What soluble factors play a role in inducing
  cachexia?
• How do we best model these events?
• What are the basic characteristics of bone
  metastases in a patient and how do they compare
  between patients?

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Project 1 Factors   Project 2 Models
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FACTORS Overarching Question What are the
important prostate cancer-derived factors and
bone-derived factors that facilitate the growth
of prostate cancer in bone, perturb normal bone
remodeling, and contribute to the morbidity
associated with bone metastases?
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Vicious Cycle
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Vicious Cycle
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Factors - General Topics
Michael Production by prostate cancer cells of
Matrix Metalloproteinase 14 (MT1-MMP)
supports their ability to form metastatic
deposits in bone Theresa Identify and test
factors (e.g. IGFBPs and TIMPs) regulated by
TGF-beta Hari Gain insight into the role of a
novel cytokine receptor (IL-17 receptor
like molecule IL-17 RL) as a potential
regulator of osteoclastogenesis
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Factors - General Topics (cont.)
Ken Explore biology and potential targeting
of bonesialoprotein (BSP) and endoglin, a
transmembrane TGF-beta binding protein Bob
Determine whether the prostate associated
serine proteases PSA and prostin participate in
the osteoblastic response  
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Synopsis of Specific Aims MT1-MMP

• To assess the contribution of MT1-MMP to tumor
  growth, bone remodeling, and ngiogenesis, we
  will use (1) an overexpression approach with
  both wild type and mutant forms of MT1-MMP, and
  (2) an MT1-MMP inhibition approach using
  antisense technology.

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Synopsis of Specific AimsTGF-beta

• Identify factors in prostate cancer PC3 cells
  regulated by TGF-beta, using analysis of gene
  arrays
• Test regulation of candidate factors by TGF-beta
  in prostate cells in vitro. Possible candidates
  include IGFBPs and tissue inhibitors of
  metalloproteinases, TIMPs
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• Validate physiological importance of identified
  factors using an animal model of bone metastasis
  in which genetically manipulated PC3 prostate
  cells are inoculated into the circulation of nude
  mice and reliably metastasize to bone

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Synopsis of Specific AimsIL-17 RL and IL-17

• To determine the role of IL-17 receptor-like
  molecule (IL-17RL) in normal prostate and
  prostate cancer by identification of the
  physiological ligand by immunoprecipitation with
  specific antibodies and affinity chromatography
  using recombinant IL-17 RL.
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• To investigate the role of IL-17 and IL17-RL by
  overexpression and stable transfection in
  prostate cancer metastases to bone.

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Synopsis of Specific AimsBSP and Endoglin

• Conduct an extensive IHC analysis of BSP and
  endoglin expression in human CaP cell lines and
  xenograft models and confirm observations in
  clinical CaP specimens
• Test the in vivo role of BSP and endoglin in the
  C4-2B subline transfected with either a BSP or
  endoglin plasmid expression vector. Determine
• differences in growth rate
• whether osteolytic, osteoblastic or mixed
  response using SCID-hu and intratibia injection
  models
• angiogenesis (e.g. microvessel density)

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Discussion

• BSP/LNSXII
• Packaging cell line
• Transfection
• Titration
• 3rd shot..continuing..

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Synopsis of Specific AimsPSA and Prostin

• Determine whether PSA and/or prostin contribute
  to the osteoblastic response as a consequence of
  bone metastasis.
• a.) Overexpress PSA and prostin individually
  in C4-2 and CL-1 cell lines test in
  intra- tibia model for induction of the
  osteoblastic response
• b.) Express inactive forms (mutate pro-piece
  sequence or catalytic site) as control

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Synopsis of Specific AimsPSA and Prostin
c.) Attempt to inhibit development of the
osteoblastic response in intra-tibia osseous
models with anti-PSA antibodies (intact or
fragments) d.) Attempt to inhibit development
of the osteoblastic response in intra-tibia
osseous models by systemic adenoviral delivery
of anti-PSA ribozymes.
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Models General Topics

• Leland Establish novel transgenic animal models
  of CaP bone and viseral metastases
• Theresa Study the effects of androgen ablation
  in CaP bone metastases models
• Michael Expand the utility of the SCID-hu model
• Bob Develop new xenografts derived from bone
  metastases using the highly vascularized sub
  capsular region of the kidney as the site of
  initial implantation.

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Synopsis of Specific AimsNovel Transgenic Models

• Leland Establish these novel models of CaP bone
  and visceral metastasis to (a) enable
  monitoring of CaP cells in the host animals
  using a non-invasive imaging technique and (b)
  enhance the metastatic potential of CaP
  trafficking to bone by first generating and then
  mating the founder Luc-T mouse co-expressing the
  luciferase enzyme and large-T antigen in the
  prostate with mouse strains that overexpress
  chemoattractant factors for CaP metastasis to
  the skeleton.

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Biology of Tumor-Stroma InteractionLeland Chung
and Haiyen Zhau

• Progress Report
• Established a new mouse model of human prostate
  cancer bone metastasis based on the derivatives
  of a previously characterized ARCaP cell line
  (Zhau, et al. PNAS, 1996).
• Developed a novel luciferase-based imaging
  technique to identify normal prostate epithelial
  cells and chimeric prostate tumors in transgenic
  mouse models (Hsieh, et al. 2003).

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Biology of Tumor-Stroma InteractionLeland Chung
and Haiyen Zhau

• Progress Report (Continue)
• Tested the concept and identified genes that are
  differentially expressed in the reactive human
  prostate and bone stromal cells when exposed to
  tumor epithelium in culture (Sung, et al. 2003).
• Established novel 3-D culture models to study
  interactions between prostate cancer and prostate
  and bone stromal cells (Jin, et al. In Progress)

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Synopsis of Specific AimsAndrogen Ablation Models

• Theresa Using intra-cardiac injection of CaP
  cell lines, (a) test the effects of androgen
  ablation by orchiectomy on the development and
  progression of CaP bone metastasis and (b)
  determine if inhibition of the increased
  resorption induced by androgen ablation will
  reduce the development of progression of the
  CaP metastases to bone.

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Synopsis of Specific Aims SCID-Hu Model
Michael Further develop the SCID-human model of
bone metastases (a) additional cell lines
tested in model, (b) include intracardiac
injection, (c) manipulate the soil to
over-express genes of interest
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Synopsis of Specific Aims New Xenograft Models

• Bob From our rapid autopsy program, acquire
  bone metastases for implantation into the
  highly vascularized sub capsular region of the
  kidney in efforts to increase the take rate.
  Initiate characterization studies including the
  ability of derived xenografts to promote an
  osteoblastic response upon growth in bone.

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Synergy Among Team Members

• Share our current bone metastases models with
  other team members and greatly expand their
  utility
• Immediately inform team members and share as
  appropriate any of the new models being developed
• Engage in frequent intellectual exchanges
  regarding results from the Factors Project and
  continually seek opportunities for interaction.

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Synergy With Other Teams

• The fact that some of our team members sit on
  other teams will facilitate interactions, e.g.
  Theresa on the Signaling team
• Current and new models of CaP bone metastases
  will be made available to all consortium members
• Tissue resources, such as the bone metastases
  from the rapid autopsy program are available
  through the Core to all consortium members
• Ultimate goal is to derive insight on novel
  treatment strategies for CaP bone metastases and
  interact with Team 3 on translation to clinical
  trials.