Committee Meeting

1
Attenuation of Endoglin in Bone Marrow Stromal
(BMS) Cells Supports the Growth of Prostate
Cancer John C. OConnor
2
How Does Bone Marrow Stroma Preferentially
Support Prostate Cancer Growth??
3
Overall Hypothesis

• Arrival of prostate cancer cells into bone marrow
  causes changes in gene expression in the BMS that
  ultimately facilitates the growth of prostate
  cancer cells within the bone microenvironment.

4
Experimental Approach

• Co-culture experiments with the HS-5/HS-27a BMS
  cells and C4-2B prostate cancer cells
• Cells plated at 200,000 cells/well
• Co-cultures initiated when cells are 80
  confluent
• Total RNA prepared after 6 days in serum free
  DMEM
• TGF Pathway SuperArray
• Experiments repeated with timecourse (1,3,5,6
  days of co-culture) and different concentrations
  of serum (0 and 1 FBS) and with both HS-5 and
  HS-27a cells

5
Endoglin (CD105)

• Decreased 3.6-fold in array
• TGF-b receptor accessory protein
• Transmembrane glycoprotein (homodimer 180 kD)
• Constitutively phosphorylated
• Located on human chromosome 9
• Gene is 40 kb, 14 exons 3.4 kb mRNA
• Two isoforms differing cytoplasmic tail
• Long form (predominant form 633 aa)
• Short form (alternate splicing 600 aa shorter
  cytoplasmic tail)
• Binds TGF-b1 and b3 after associating with
  TGF-bRII
• Subsequently phosphorylates TGF-bRI (ALK5
  ALK1) leading to smad signaling
• Major role in angiogenesis
• High expression in endothelial cells, found in
  other cell types (e.g., stromal cells)

6
Endoglin (CD105)

• Knockout is embryonic lethal in mice (E10.5
  impaired vessel/heart development)
• Similar to knockout of TGF-b1, TGFb-RII, ALK5,
  and ALK1
• Heterozygotes have some vascular defects
  (haploinsufficiency)
• Mutated proteins retained intracellularly
• Observed in human syndrome also
• Human syndrome (Hereditary Hemorrhagic
  Telangiectasia type I) characterized by vascular
  malformations, nose bleeds, etc
• HHT type II due to mutation in ALK1

7
Co-culture Experiments Experimental Approach

• Co-culture experiments with the HS-5 and HS-27a
  BMS and prostate cancer cells
• LNCaP progression model (LNCaP, C4-2, C4-2B)
• PC-3 cells
• Foreskin fibroblast cells (negative control)
• Cells plated at 125,000 cells/well
• Used transwells
• Co-cultures initiated when cells are 80
  confluent
• Total RNA/protein prepared after 1 or 3 days in
  DMEM w/1 FBS
• Westerns for endoglin

8
Endoglin Protein Levels Are Decreased in HS-27a
Cells After Co-Culture with C4-2 Cells
C4-2 Co-culture
Endoglin b-actin
9
Conditioned Media Experiments Experimental
Approach

• Co-culture experiments with the HS-5 and HS-27a
  BMS cells and prostate cancer cell conditioned
  media
• LNCaP progression model (LNCaP, C4-2, C4-2B)
• PC-3 cells
• Foreskin fibroblast cells (negative control)
• Cells plated at 125,000 cells/well in DMEM w/1
  FBS
• Conditioned media exposure initiated when cells
  are 80 confluent
• Total protein prepared after 3 days
• Westerns for endoglin
• Conditioned media saved and evaluated for TGF-b
• Photos of BMS cells on day 3 of exposure
• Apoptosis and cell proliferation assays

10
Endoglin Protein Levels Are Decreased in HS-5 and
HS-27a Cells After Growth in Conditioned Media
From C4-2 and C4-2B Cells
HS-5 Cells
HS-27a Cells
Endoglin b-actin
11
Endoglin Protein Levels Are Decreased in HS-5 and
HS-27a Cells After Growth in Conditioned Media
From DU-145 But Not LNCaP Cells
HS-5 Cells
HS-27a Cells
Endoglin b-actin
12
Endoglin Protein Levels Are Increased in HS-5 and
HS-27a Cells After Growth in Conditioned Media
From PrEC But Not ZR-75-1 Cells
HS-5 Cells
HS-27a Cells
Endoglin b-actin
13
Inside slide with TGF-b/smad signaling pathway
14
Endoglin and Smad Protein Levels Are Not Altered
in Foreskin Fibroblast Cells After Growth in
Conditioned Media From Prostate Cancer Cell Lines
Endoglin
Total Smads
Endoglin tSmad1 tSmad2/3 b-actin
15
Endoglin Protein Levels in HS-5 Cells is
Dependent on the Concentration of Conditioned
Media
Endoglin b-actin
16
Endoglin Protein Levels are Altered in Primary
BMS Cells After Growth in Conditioned Media From
C4-2, C4-2B, and PrEC Cells
Endoglin b-actin
17
Total Smad 1 Protein Expression in HS-5 and
HS-27a Cells is Decreased After Growth in
Conditioned Media From C4-2B Cells
HS-5 Cells
HS-27a Cells
tSmad1
tSmad2/3 b-actin
18
Total Smad Protein Expression in HS-5 and HS-27a
Cells Is Not Altered After Growth in Conditioned
Media From DU-145 or LNCaP Cells
HS-5 Cells
HS-27a Cells
tSmad1
tSmad2/3 b-actin
19
Total Smad 1 Protein Expression in HS-5 and
HS-27a Cells Is Increased After Growth in
Conditioned Media From PrEC Cells
HS-5 Cells
HS-27a Cells
tSmad1
tSmad2/3 b-actin
20
BMP-2 Stimulated pSmad 1 Protein Expression is
Altered in HS -27a Cells After Growth in
Conditioned Media From C4-2B Cells
pSmad1
tSmad1 b-actin
21
Conditioned Media From DU-145, C4-2, C4-2B
Cells Decreases Cell Proliferation in HS-5
HS-27a Cells
HS-5 Cells
HS-27a Cells
 
22
Conditioned Media From PC-3, DU-145, C4-2,
C4-2B Cells Alters Apoptosis in HS-5 HS-27a
Cells
HS-5 Cells
HS-27a Cells
 
23
Endoglin Signaling in Endothelial Cells
Low Endoglin
High Endoglin
ALK5 is usually predominant pathway ALK1 is
highly active in endothelial cells.
24
Path Forward

• Overexpression of endoglin in HS-5 HS-27a BMS
  cells
• How will this affect TGF-b signaling, cell
  proliferation, and apoptosis in BMS cells after
  co-culture with PCa?
• Repeat co-culture (conditioned media)
  experiments
• Transgenic mouse constitutively expressing
  endoglin in bone marrow
• How will overexpression affect growth of PCa?