VEGF Signaling in Prostate Cancer

1
VEGF Signalingin Prostate Cancer
Daqing Wu, Ph.D. Emory University
2
VEGF isoforms and receptors
Ferrara et al., Nature Medicine, 2003
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VEGF paracrine signaling on endothelial cells
angiogenesis
Ferrara et al., Nature Medicine, 2003
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Pro-osteoblastic effect of VEGFin prostate
cancer bone metastasis
cancer cells (C4-2B)
BMPs (BMP-2,-4,6,-7)
VEGF
osteoblast precursor
osteoblastic lesions mineralization osteosclerosis

Kitagawa et al., 2005, Cancer Res.
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VEGF autocrine signaling a survival factor

• Expression of autocrine motility factor
  correlates with the angiogenic phenotype of and
  poor prognosis for human gastric cancer. Clin
  Cancer Res. 2005, 115778.
• A hypoxia-driven vascular endothelial growth
  factor/Flt1 autocrine loop interacts with
  hypoxia-inducible factor-1alpha through
  mitogen-activated protein kinase/extracellular
  signal-regulated kinase 1/2 pathway in
  neuroblastoma. Cancer Res. 2005, 657267.
• Overexpression of neuropilin-1 promotes
  constitutive MAPK signalling and chemoresistance
  in pancreatic cancer cells.Br J Cancer. 2005,
  93233.
• Semaphorin 3B induces apoptosis in lung and
  breast cancer, whereas VEGF165 antagonizes this
  effect.Proc Natl Acad Sci U S A. 2004,
  10111432.
• Internal and external autocrine VEGF/KDR loops
  regulate survival of subsets of acute leukemia
  through distinct signaling pathways. Blood. 2004,
  1033883.
• Targeting autocrine and paracrine VEGF receptor
  pathways inhibits human lymphoma xenografts in
  vivo.Blood. 2004, 1042893.
• Competing autocrine pathways involving
  alternative neuropilin-1 ligands regulate
  chemotaxis of carcinoma cells.Cancer Res. 2003,
  635230.
• Overexpression of vascular endothelial growth
  factor by MCF-7 breast cancer cells promotes
  estrogen-independent tumor growth in vivo. Cancer
  Res. 2003, 634684.
• Vascular endothelial growth factor promotes
  breast carcinoma invasion in an autocrine manner
  by regulating the chemokine receptor CXCR4.
  Cancer Res. 2002, 627203.

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Plasma VEGF levels VEGF are associated with lymph
node and skeletal metastases of prostate cancer

• Plasma levels of VEGF are increased in patients
  with metastatic prostate cancer. Urology, 1999,
  54, 523.
• Prognostic significance of plasma vascular
  endothelial growth factor levels in patients with
  hormone-refractory prostate cancer treated on
  Cancer and Leukemia Group B 9480. Clin Cancer
  Res., 2001, 7, 1932.
• Metastatic properties of prostate cancer cells
  are controlled by VEGF. Cell Commun Adhes., 2004,
  11, 1.
• Association of preoperative plasma levels of
  vascular endothelial growth factor and soluble
  vascular cell adhesion molecule-1 with lymph node
  status and biochemical progression after radical
  prostatectomy. J Clin Oncol., 2004, 22, 1655.

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Serum VEGF levels are associated with clinical
bone metastasis
mean values group 1 309.2 pg/ml group 2 156.9
pg/ml (p0.0017)
Biostatistical analysis of the distribution of
VEGF in PCa patients with (group 1 n24) and
without (group 2 n34) bone metastasis.
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VEGF regulation

• Hypoxia stress HIF-1a.
• Growth factors EGF, TGF-a, TGF-ß, M-CSF, IGF-1,
  FGF, PDGF.
• Cytokines IL-1, IL-6, IL-8.
• Hormones GHRH, TSH, ACTH, estradiol, androgen.
• Oncogenic signals neu, k-ras, PI3-kinase/Akt,
  Wnt, c-Src, PKC, STAT3, c-myc, COX, cyclin A1,
  Bcl-2, Id-1.
• Tumor supressor p53, PTEN.
• GPCRs the Kaposi's sarcoma-associated herpes
  virus GPCR, CXCR4.
• Tumor microenvironment SDF-1, SPARC

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ARCaP a prostate cancer model for EMT and bone
metastasis
A
B
ARCaPE
ARCaPM

• H/E histopathology
• gene expression profiling
• migration and invasion assays
• validation in clinical specimens

ARCaP
C
D
E M
Western blotting
IHC
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EMT is implicated in tumor metastasis

• EMT is required for invasive phenotypes in cell
  culture models and transgenic mouse models of
  epithelial tumors
• EMT-associated signals has been implicated in the
  distinct steps of tumor metastasis invasive
  growth, intravasation, extravasation and
  colonization at distant sites
• Interaction between tumors and their
  microenvironment is also involved in the
  modulation of EMT
• The EMT concept provides a novel means of
  identifying signal transduction pathways that are
  important for cancer progression, and presents a
  great opportunity to prevent metastasis

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EMT is associated with VEGF overexpression
RT-PCR
ELISA
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Activation of CREB signaling is associated with
EMT
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CREB regulates VEGF expression in ARCaP cells
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A model for CREB regulation of VEGF transcription
in normoxia
CREB
nucleus
VEGF mRNA
1
-2kb
-5kb
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CREB regulation of VEGF expression in
normoxiaHIF1a-dependent?
Relative luciferase activity (X 10-4)
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CREB regulation of VEGF expression in normoxia
HIF1a-dependent?
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CREB regulation of VEGF expression in
normoxiaHIF1a-dependent?
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CREB phosphorylation is associated with clinical
prostate cancer bone metastasis
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Summary 1 CREB regulates VEGF expression in
normoxia

• Activation of VEGF and CREB signaling is
  associated with clinical PCa bone metastasis, and
  with EMT in the ARCaP cells
• CREB induces VEGF transcription in normoxia
• CREB-mediated recruitment and formation of
  transcriptional complex regulate VEGF expression,
  presumably through a HIF-1-dependent mechanism

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Autocrine VEGF signaling in prostate cancer
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Expression of VEGF signaling components in ARCaP
cells
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VEGF signaling switch model in EMT
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Neuropilin-1 (NRP1) in cancer

• NRP1 was originally identified in neuronal cells
  as a receptor for the class 3 semaphorin (sema3)
  subfamily mediating neuronal guidance and axonal
  growth. It is also an isoform-specific receptor
  for VEGF165 but not VEGF121 on endothelial cells
  and tumor cells.
• NRP1 is a transmembrane protein with a large
  extracellular domain and a short intracytoplasmic
  tail.
• The VEGF/NRP1 uses VEGFRs to signal, whereas the
  sema3/NRP1 uses plexin-As.
• Aberrant up-regulation of NRP1 has been observed
  in high-grade metastatic tumors in a variety of
  cancers, including breast cancer, lung cancer,
  gastrointestinal adenocarcinoma, colon cancer,
  pancreatic cancer, neuroblastoma, glioma and
  prostate cancer.
• Prostate cancer cells do not express Flt-1 or
  Flk-1. VEGF165 binding is mediated solely by NRP1.

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NRP1 Expression is associated with bone
metastasis
Human PCa
ARCaPM model
1
3
5
2
4
6
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NRP1/plexinA1 signaling is a survival pathwayin
bone-metastatic cancer cells
Control siRNA
NRP1 siRNA
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NRP1/plexinA1 signaling is a survival pathwayin
bone-metastatic cancer cells
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Proposed NRP1 signaling in prostate cancer cells
VEGF
PlexinA1
NRP1
Fyn
Src
JAK
Cdk5
P
Ser727
nucleus
P
Mcl-1 survivin
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Summary 2 NRP1 signaling in prostate cancer

• VEGF is a survival factor for prostate cancer
  cells
• A VEGF signaling switch, from Flt-1-dependent
  to NRP1-dependent may be implicated in tumor
  progression and EMT
• NRP1 is predominantly expressed by prostate
  cancer cell lines and its overexpression is
  associated with EMT and bone metastasis status in
  ARCaP model and in human tumor tissues
• NRP1 may use plexinA1 to transduce survival
  signals of VEGF
• The anti-apoptotic factors, Mcl-1 and survivin,
  are novel downstream targets of NRP1 signaling
• Blockade of NRP1/plexinA1 signaling may induce
  apoptosis

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Acknowledgements

• Emory Urology
• Dr. Leland Chung
• Dr. Haiyen Zhau
• Dr. Jianchun Xu
• Dr. Zhihui Xie
• Dr. Valerie Odero-Marah
• Dr. Wen-Chin Huang
• Dr. Shian-Ying Sung
• Dr. Fray F. Marshall
• Western General Hospital, Edinburgh, UK
• Dr. Fouad Habib
• Louisiana State University School of Medicine
• Dr. Oliver Sartor