Prostate Cancer Bone Metastasis: Biology and Targeting P'I' Leland W' K' Chung, Ph'D'

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Transformation
Tumor-Stroma Interaction
Invasion Migration
PI-88
Heparanase MMPs
EMT
E-cadherin
Osteomimicry
Aberrant ß2-microglobulin and TGF-ß signaling
Bone Tropism and Colonization
Initiation
ROS
Perlecan VEGF FGF-2
VEGFR1 MMP2,9
Hypoxia Hematomimicry
HIP
Micrometastasis Balance of Growth and Dormancy
Neovasculogenesis
Endoglin, Smad in Stroma
FGF-2 VEGF FGFR1,2 VEGFR2 PSA mTOR
Tumor Growth and Neoangiogenesis
Project 1 Biology and Targetingof Tumor-Stroma
InteractionP.I. Leland W. K. Chung, Ph.D.
BMP-2 Runx2 FGF-2 VEGF Perlecan Shh FGFR1,2,4
VEGFR2 PSA Bone Markers
Tumor growth and visceral organ metastasis are
accelerated by osteomimicry which also promotes
the expression of lethal invasive phenotype
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Project 1 Biology and Targetingof
Tumor-Stroma InteractionP.I. Leland W. K.
Chung, Ph.D.

• Specific Aim 1 To determine the molecular
  mechanisms governing osteomimicry and potential
  downstream therapeutic targets in human prostate
  cancer bone metastasis
• Specific Aim 2 To study molecular interactions
  between prostate cancer cells and bone
  marrow-derived primary stromal, and mesenchymal
  stem cells under 3D co-culture conditions to
  assess epithelial to mesenchymal transition
• Specific Aim3 To search for stroma-derived
  genes that may predict the lethality of human
  prostate cancer

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Specific Aim 1 To determine the molecular
mechanisms governing osteomimicry and potential
downstream therapeutic targets in human prostate
cancer bone metastasis

• Goals
• To determine b2M-mediated signaling in the
  regulation of integrin expression by prostate
  cancer cells
• To determine the effect of b2M-mediated signaling
  in the regulation of androgen receptor in
  prostate cancer cells
• To determine the molecular link between
  b2M-mediated signaling and growth and survival of
  prostate cancer cells

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a2b1 avb3 avb5

• BSP supports breast cancer cell adhesion,
  proliferation and migration through differential
  usage of avb3 and avb5 integrins. (J Cell Physiol
  176 482-94, 1998)

Huang, et. al 2007
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C4-2B (-) Scramble-siRNA
b2M-siRNA
a2b1
avb3
avb5
Huang, et. Al 2007
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Experiment 1

• To assess the effect of b2M on integrin
  expression and assess the altered ability of
  prostate cancer cells to attach to bone matrix
  proteins
• To determine if the effects of b2M on integrin
  expression may be mediated by androgen receptor
• To assess if isotype-specific integrin siRNA or
  anti- b2M antibody may exert additive or
  synergistic inhibition of prostate cancer growth
  and survival in mouse skeleton with standard
  chemotherapy

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Experiment 2

• To determine the minimal b2M mimetic peptides,
  based on b2M protein crystalline structure, to
  stimulate osteomimicry
• To assess b2M induced downstream signaling that
  mediates EMT in prostate cancer cells
• To assess the biological activity of peptide
  mimetics and drugs that can block b2M-mediated
  osteomimicry, tumor growth in mouse skeleton and
  EMT of prostate cancer cells

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The crystal structure of a monomeric human
b2M
8-stranded b-sheet
(Trinh C.H. et al., 2002, PNAS, 99 9771-9776)
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Experiment 3

• To determine the possible involvement of
  G-protein coupled receptor (GPCR) in mediating
  b2M downstream cell signaling of human prostate
  cancer cells
• To assess the effects of uncoupling of pKA/pCREB
  and GPCR signaling by blocking Gs and Gi on the
  growth and the expression of osteomimicry by
  prostate cancer cells

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Specific Aim 2 To study molecular interactions
between prostate cancer cells and marrow stromal
or mesenchymal stem cells under 3D co-culture
conditions

• Goals
• To evaluate if bone marrow stromal or mesenchymal
  stem cells can promote permanent transition of
  prostate cancer cells from their epithelial to
  mesenchymal phenotype
• To validate these transitions and investigate the
  molecular mechanisms by biochemical, molecular
  and behavioral analyses

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3D model Rotary Wall Vessel
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Bioreactor Apparatus
Powers et al. 2002
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Hep/Du Coculture
Day 2
Day 4
Red Du-145 Prostate cancer cells Green GFP
Hepatocytes
Yates, et al. 2006
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Day 6
Day 9
Yates, et al. 2006
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Bioreactor Tissue Morphology
Yates, et al. 2006
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ARCaP Androgen Refractory Cancer of the
Prostate

• AR and PSA positive
• readily culture in vitro
• grow in both intact and castrated hosts
• predictable rapid bone and soft tissue metastases

Xu, et al. 2006
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ARCaP Classic EMT Model
Xu, et al. 2006
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Tumor Cell-Bone Interaction Induces Rapid Bone
and Adrenal Metastases
Xu, et al. 2006
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Experiment 1

• To characterize bone marrow derived stromal cells
  and mesenchymal stem cells isolated from patients
• To utilize the well characterized marrow stromal
  or mesenchymal stem cells to co-culture with
  ARCaPE cells to observe if they can be promoted
  to undergo ARCaPM transition

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Experiment 2

• To evaluate if RANKL expression may be activated
  upon prostate cancer and bone stroma / marrow
  mesenchymal stem cell interaction
• To determine RANK-luc expression in human
  prostate cancer cells stably transfected with
  RANKL promoter luciferase reporter under 3-D
  co-culture conditions in a cell context and
  time-dependent manner

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Experiment 3

• To assess the behavior of ARCaPE cells subsequent
  to cellular interaction with marrow stromal or
  mesenchymal stem cells in vitro under 3D
  conditions
• The objectives of this study are 1) to assess
  the behavior of ARCaPE cells isolated from
  chimeric organoids subsequent to cellular
  interaction with marrow stromal/mesenchymal stem
  cells in vitro 2) to determine factors (growth
  factors, ROS, ECM) that may affect EMT of ARCaPE
  cells

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Specific Aim 3 To establish and validate stromal
genes that may predict lethality of human
prostate cancer

• Goals
• Because cancer-induced stromal gene expression
  changes are non-random and are accompanied by
  molecular, morphologic and behavioral alterations
  in cultured stromal cells, we will validate the
  expression of stromal genes in clinical specimens

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Lethal map-related genes

• BDNF
• Brain-derived neurotrophic factor is a
  prosurvival factor induced by cortical neurons
  that is necessary for survival of striatal
  neurons in the brain.
• Lu et al. (2001) showed that SDF1 and BDNF are
  chemoattractants for cerebellar granule cells.
• Induced hepatocellular carcinoma cell
  proliferation.
• CCL5
• Also called RANTES Regulated upon Activation,
  Normally T-Expressed, Secreted.
• Marker for CD8 T cell response to HIV infection.
• MCP-1?, MCP-1? and CCL5 are major factor for
  macrophage infiltration into cancer areas during
  neoplastic development.

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Lethal map-related genes (cont.)

• CxCL5 (Epithelial cell-derived neutrophil-activati
  ng peptide, ENA78)
• CCL5 is an inflammatory chemokine that is
  produced concomitantly with IL8 in response to
  stimulation with either IL1B or TNF?.
• CXCL16
• Chemotaxis assays found that CXCL16 induced a
  strong chemotactic response in activated CD8 T
  cells. In addition, CXCL16 induced calcium
  mobilization. Human and mouse cells expressing
  CXCR6 showed a strong chemotactic response to
  CXCL16 but not to other chemokines.
• ADAM10 is involved in constitutive cleavage of
  the transmembrane adhesion molecule CXCL16 to a
  soluble chemoattractant for activated T cells.
  Constitutive cleavage was markedly reduced in
  Adam10-deficient mouse fibroblasts and was
  restored by retransfection with ADAM10.

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BDNF
Sung, et al. 2007
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ELISA of prostate cancer patient sera sample in
Emory clinic for BDNF expression
Sung, et al. 2007
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CCL5
Sung, et al. 2007
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CXCL5
Sung, et al. 2007
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CXCL16
Sung, et al. 2007
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Opportunities for clinical translation

• Can the levels of expression of chemokines and
  chemokine receptors predict prostate cancer
  recurrence, distant dissemination and therapeutic
  resistance?
• Are the chemokine and receptor axes attractive
  therapeutic targets?

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Experiment 1

• To evaluate promising stromal genes that might be
  associated with prostate cancer progression
• To confirm the expression of stromal genes in
  clinical specimens
• To correlate stromal gene expression with the
  prostate cancer behaviors in patients

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Experiment 2

• To construct a lethal map that predicts prostate
  cancer progression
• Construction of relevant signaling component that
  may be intimately linked to prostate cancer
  progression
• Assess osteomimicry-related downstream target
  genes and convergent signaling pathway components
  as the lethal marker for prostate cancer
  progression
• Assess the relationship between EMT and stromal
  markers with prostate cancer progression

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Proposed Core Usage

• Pathology and laboratory services
• Cell culture and transgenic services
• Biostatistical microarray and bioinformatic
  support