GU ONCOLOGY CENTER OF EXCELLENCE

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TEAM 1
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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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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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Synopsis of Specific Aims MT1-MMP (Michael
Cher)

• To assess the contribution of MT1-MMP to tumor
  growth, bone remodeling, and angiogenesis
• Overexpression approach with both wild type and
  mutant forms of MT1-MMP
• MT1-MMP inhibition approach using antisense
  technology.

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Tumor-derived MT1-MMP Enzymatic Activty Enhances
Growth of Tumor in Bone and Osteolytic Phenotype

• LNCaP cells transfected with
• E240A catalytically dead mutant MT1MMP
• WT wild type MT1MMP
• Neo vector alone

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Human Prostate Cancer Bone Biopsies
A
B
Anti-MT1MMP Immunostain
No primary antibody control
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MT1MMP Down-regulation in DU145 cells via siRNA
vector
Injection of media (no tumor cells)
Lytic response with control (scrambled) siRNA
transfection
Lytic/Blastic responses with downregulation of
MT1MMP
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Synopsis of Specific AimsTGF-beta (Theresa
Guise)

• Identify factors in PC3 cells regulated by
  TGF-beta (gene arrays)
• Test regulation of candidate factors by TGF-beta
  in CaP 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
  athymic mice and reliably metastasize to bone

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Adrenomedullin Therapeutic Target in Bone
Metastases

• Phenylacetic acid derivative
• Km 8nM

16311
16311
16311
Martinez et al, Endocrinology 145(8)3858-65, 2004
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Adrenomedullin Antagonist 16311 Inhibited New
Bone Formation In Calvarial Organ Culture
New bone area
-C
C
AM
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_
_
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Synopsis of Specific Aimson IL 17 Receptor
Like Molecule (Hari Reddi)

• Determine the role of IL-17 receptor-like
  molecule (IL-17RL) in normal prostate and CaP
  by
• identification of physiological ligand(s) by
  immunoprecipitation with specific antibodies and
  affinity chromatography using recombinant IL-17
  RL
• overexpression by stable transfection in CaP
  metastases to bone

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IL-17RL expression is significantly higher in
androgen-independent tumors than in
androgen-dependent tumors in CWR22 model IL-17RL
expression in CWR22 tumors, but not mouse stroma,
is induced by androgen deprivation
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IL-17RL promoter activities are regulated by
androgen-dependent and androgen-independent
pathways in LNCaP cells
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Synopsis of Specific AimsBSP and Endoglin (Ken
Koeneman)

• 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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Overexpress 2.3 kb. Endoglin Using C4-2B Cells
Transfected with pcDNA/neo-Endoglin
C4-2B-Endo
C4-2B
H2O
1kb
TSU
650 bp
GAPDH
Endoglin
Primer Endoglin E,F(380bp fragment
amplified) GAPDH G3P0, G3P2 (486bp fragment
amplified) 2ug total RNA, 59oC, 37 cycle, 2 gel
ncdi access nm-000118 nucleotide 341-2766. This
clone was sequenced and confirmed to be correct.
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Overexpress BSP in C4-2B Cells Followed by
Intra-femur Injection
Ratio of week 10 /week 6 post injection PSA
values of increase
C42B only vs. C42B-BSP p0.151 C42B only vs.
c42B-pcDNA p0.166 c42B-pcDNA vs. C42B-BSP
p0.022
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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.
• Overexpress PSA and prostin individually in CaP
  cell lines
• Express inactive forms (mutate pro-piece sequence
  or catalytic site) as control
• Inhibit development of the osteoblastic response
  in intra-tibia osseous models with
• anti-PSA antibodies
• PSA anti-sense

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Enhanced Inhibition of LuCaP 35V Tumor Growth By
Combining Docetaxel With An Anti-IGF-1R Antibody
Treatment stopped at 4 weeks
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Models General Topics

• Leland Establish novel transgenic animal
  models of CaP bone and visceral 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 Aims Novel Transgenic
Models(Leland Chung)

• To confirm the specificity of a human sPSA
  promoter in vivo.
• To improve our ability of monitoring normal
  prostate growth and development in live animals.
• To determine the effects of growth factors and
  cytokines in prostate cancer growth and
  metastasis.
• To create a mouse model of prostate cancer
  metastasis that can be imaged and used to monitor
  drug response in live animals.

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Demineralized Bone Matrix Supports Growth of
C4-2b-Luc Cells Implanted Subcutaneously
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Synopsis of Specific AimsAndrogen Ablation
Models(Theresa Guise)

• 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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Expansion of SCID-Hu Model System (Michael Cher)

• Transfected 3 different CaP cell lines with
  luciferase gene.
• Also transfected LNCaP and PC-3 with Green
  Fluorescent Protein (GFP)
• Evaluated in vitro various imaging modalities for
  in vivo imaging of SCID-Hu and intra-tibia
  minimal tumor burden.
• Based on in vitro data, spectrofluoremetry
  selected for initial in vivo imaging studies with
  GFP-LNCaP cells in bone.

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Synopsis of Specific Aims New Xenograft
Models(Bob Vessella)

• 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 of any xenografts
  derived from bone metastasis including the
  ability to cause an osteoblastic response upon
  growth in bone.

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Progress New Xenograft Generation

• Hypothesis is that the highly vascularized bed
  beneath the renal capsule will enhance the
  establishment of prostate cancer xenografts
  acquired from bone metastases at rapid autopsy.
• To date mice have been implanted beneath the
  renal capsule with tissue bits from seven rapid
  autopsies.
• Animals being monitored regularly for evidence of
  PSA production but currently none show growth.
• Technically challenging due to hard, sharp edged
  implants that may consist more of bone than
  tumor.
• However, success achieved with some subcutaneous
  implants from bone metastases.

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Bone Metastasis Team Progress Report Summary
After 20 Months

• Established human CaP cell lines that
  over-express MT1-MMP and demonstrated
  down-regulation MT1-MMP with siRNA leading to
  decreased osteolysis in vivo.
• Determined downstream targets of TGF-beta
  signaling in PC-3 identified adrenomedullin as a
  factor involved in CaP bone metastasis
  established osteoblastic LuCaP 23.1 tumors from
  Vessella lab studying hypoxia as target.
• Determined that IL 17 RL is a novel
  anti-apoptotic gene overexpressed in androgen
  independent prostate cancer siRNA can induce
  apoptosis promoter regulated by androgen
  dependent and androgen independent pathways.
• Generated CaP cell lines overproducing BSP and
  endoglin completed first series of in vivo
  intra-osseous studies -- histomorphometric
  analyses ongoing.

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Bone Metastasis Team Progress Report Summary
After 20 Months

• Succeeded in generating a highly expressing,
  inducible PSA and Prostin cell line in vivo
  studies ready to commence also conducted both
  PSA antisense and anti-PSA antibody studies in
  vivo but no drop in serum PSA anti-IGF-1R in
  vivo studies very promising.
• Generated and characterized a luciferase tagged
  mouse model with luciferase gene driven by super
  prostate specific antigen (sPSA) promoter mated
  with TRAMP to achieve highly innovative model
  reported on quantum dot nanotechnology for
  imaging CaP demonstrated bone matrix and BMP7
  enhanced sub-cutaneous CaP growth.
• Formulating 2 ideas for potential clinical trials
  with Team 3.