Peng Lee, M.D., Ph.D.

Department of Pathology and UrologyNew York University School of MedicineNew York Harbor Healthcare System

A pathway based signature

for

prostate cancer racial disparity

• Risk factors: Aging, race, genetics, heredity, milk

• Most common cancer and second leading cause of cancer death in men in the US

Prostate Cancer Racial Disparity

• Decreasing gap (between 1990 and 2000)

• Higher incidence

• Higher mortality

Berger et al. Urology. 2006 67(1):120-4

• Younger age

Biology in Prostate Cancer Racial Disparity

Hatcher et al. Am J Transl Res. 2009, 1(3):235-48

Powell IJ. J Rrol 2007, 177:444-9

• Androgen

• EGFR

• BCL-2

• EphB2

• Androgen metabolism

• Androgen receptor

Aim 1: To identify signal transduction proteins that are associated with racialdifferences in prostate cancer between AA and CA men using a novel ProteomicPathway Array System:

A) We will screen a total of 40 pairs of AA and 40 pairs of CA prostate samples(cancer and matched benign tissues) with 320 antibodies in order to identifydifferentially-expressed proteins and phospho-proteins between the twogroups. The AA and CA cancer samples will be matched by clinicopathologicalparameters including age, PSA, tumor grade, and stage.

B) We will integrate our previously obtained genomic information (gene expression)on PCa of AA vs CA with PPAA data using bioinformatics tools to uncover additionalsignal transduction proteins involved in racial disparity. We expect 30-50differentially expressed proteins with statistical significance (p<0.05) using SAMand t-test analysis.

Aim 1: Marker Discovery Phase

Pathway array based approaches for biomarker discovery

DiscoveryPathway Array analysis (~200 abs): Proteomics (~3,000 proteins)

Validation

Function

Therapy

Differentially expressedproteins/phosphorylation

Source:Cell Lines and Tissue

Clinically significantproteins/phosphorylation

Potential therapeutic targets

Biologicallyimportant regulators

Diagnostic/Prognostic Assay

Pathway Specific Drug

Zhang et al. Cell Division 2009, 4:20

Pathway Array for Protein analysis

Protein extraction

Gel electrophoresis

Nylon membrane/plate

Immunoblot

Image analysis

Data acquisition

Signaling network

Beads

Binding of antibodies

Samples (cells, tissues, FFPE)

Genomic array information

Pathway Array

Nature 1 January 2009 Volume 457 Number 7225 pp7 - 122

• Broad coverage of cellular pathways: – Angiogenesis– Apoptosis– Inflammatory-response– cytokines– Hormone receptor signaling– Cell proliferation pathways– Cancer drug resistance – Chemokines and receptors– Cell damage and repair– Drug metabolism– Extracellular matrix and adhesion– Hypoxia signaling pathway– Stem cells– Epithelial-mesenchymal transition – Stress response– Tumor metastasis

Ratio of differentially expressed proteins and phosphoproteinsbetween aggressive (AA) and (less aggressive) CA PCa

Stem cell markers: Notch 4CD44

Tumor suppressors: Nkx3.1NEP

BCL2Apoptosis:

Invasion: L-selectin

Cell cycle genes: CDK6FoxM1

Ratio of differentially expressed proteins and phosphoproteinsbetween aggressive (AA) and (less aggressive) CA PCa

Notch4 is overexpressed in PCa vs normal tissue.

Notch4 overexpression in PCa is greater in AA than CA: Potential basis for racial disparity?

Notch4 is a receptor for membrane bound ligand (delta). Upon ligand binding Notch is cleaved and goes to the nucleus to affect transcription.

Aim 2: Marker Validation Phase

Aim 2. To determine the association of signal transduction proteins with theclinical outcome of more aggressive prostate cancer in AA using highthroughput TMAs:

A)We will perform IHC on our racial disparity Tissue Microarrays (rTMA1) (n=150AA and n=150 CA cases) to confirm that the proteins identified by PPAA in Aim 1are indeed associated with prostate cancer racial disparity between AA and CA.The patterns of expression will be correlated with clinical and pathologicalparameters, including grade, stage, and PSA levels, etc.

B)We will then evaluate these proteins on the second prognostic TMA (rTMA2)(n=300) to determine their association with aggressive PCa in AA. The levels ofexpression of the selected markers will be correlated with clinical outcomesincluding recurrence, metastasis, and survival. Nested case-control design withconditional logistic regression analysis will be used for recurrence and metastasis.Cox proportional hazard models will be used for survival. We expect to narrow thelist down to approximately 25 proteins.

0.000.501.001.502.002.503.003.504.004.505.00

1 2

Cytoplasmic Notch 4 is increased in PCa among AA

P =0.0011

1=CA, n=70

2=AA, n=19

Combi

ned

inte

nsity

and

perc

enta

ge

Allred score (0-8)=intensity (0-3) + percentage (1-5)

Aim 3: Functional Relevance: Pathway Based Target

Aim 3. To determine whether the altered expression of signal transduction proteins between AA and CA is responsible for the more aggressive behavior of AA prostate cancer using in vitro (cell proliferation, migration, and invasion) and in vivo (nude mice tumor xenograft) experiments:

A)We will investigate the functions (cell proliferation, migration, and invasion) of the differentially expressed proteins identified in Aims 1 and 2 with xCELLigence high throughput instrument using benign (hTert immortalized) and malignant (primary) cell lines from different races.

B)The proteins important in controlling cell behaviors determined by in vitro studies will be further confirmed in orthotopic intra-prostate nude mice xenograft models. We expect to identify 5-10 functionally important proteins

ctrlsiRNA Notch4siRNA

Function of Notch 4 in prostate cancer cells

Cell

prol

ifer

atio

n in

dex

Notch4siRNA

ctrlsiRNA

Notch4siRNA - +

Notch4β-actin

Num

ber

of in

vasi

ve c

ells

Notch4

β-actin

LNCa

P

LNCa

P-AI

PC3

PC3

LNCaP-AI

0

20

40

60

80

100

Conclusions:Depletion of Notch4 decreases cell proliferation & invasion of LNCaP-AI cells.Thus, high levels of Notch4, as observed in AI cells, promote proliferation and invasion.

LNCaP: androgen-dependent PCa, positive for AR expressionLNCaP-AI: androgen-independent PCa, higher levels of AR expressionPC3: androgen-independent PCa, negative for AR

ACKNOWLEDGEMENT

Jonathan Melamed

Yirong LiGarrett Daniels

Lee Lab Collaborators

Iman Osman

Jinhua Wang

Liantian Tian

NYUSOM Urologic Diseases COE Seed FundNYUCTSI Pilot Fund

DOD PCRP

Xinyu Wu

NIH CRCHD (1U01CA149556-01)

Harry Ostrer

David Zhang (MSSM)

Thanks

for

Coming !