Generation of Function Generation of Function Based Biomarkers in Based Biomarkers in

Prostate CancerProstate CancerK.C. Balaji, MDK.C. Balaji, MD

Wake Forest University School of Medicine,Wake Forest University School of Medicine,Winston Salem, NCWinston Salem, NC

Substance used as an indicator of a Substance used as an indicator of a biological statebiological state

Can be used to monitor a normal physiologic Can be used to monitor a normal physiologic state, a pathologic process, a pharmacologic state, a pathologic process, a pharmacologic response to therapeutic intervention, or a response to therapeutic intervention, or a toxic exposuretoxic exposure

Examples: antibodies, radioactive isotopes, Examples: antibodies, radioactive isotopes, specific DNA sequences, specific DNA sequences, PSAPSA

Phases of Biomarker DevelopmentPhases of Biomarker Development

Fig. 1 PSA clinical course and biomarker uses.

Prensner J R et al. Sci Transl Med 2012;4:127rv3-127rv3Published by AAAS

Prostate Specific Antigen (PSA) as Prostate Specific Antigen (PSA) as a Biomarker in Prostate Cancera Biomarker in Prostate Cancer

United States Cancer statistics, 2012United States Cancer statistics, 2012

CA: A Cancer Journal for Clinicians

Volume 62, Issue 1, pages 10-29, 4 JAN 2012 DOI: 10.3322/caac.20138 http://onlinelibrary.wiley.com/doi/10.3322/caac.20138/full#fig1

Cumulative Hazard of Death from Prostate Cancer among Men 55 to 69 Years of Age.

Schröder

FH et al. N Engl

J Med 2012;366:981-990.

Number of Diagnoses of All Prostate Cancers (Panel A) and Number

of Prostate-Cancer Deaths (Panel B).

Andriole

GL et al. N Engl

J Med 2009;360:1310-

1319.

Why is PSA Screening for Why is PSA Screening for Prostate Cancer Prostate Cancer Controversial?Controversial?

For startersFor starters………….PSA is NOT a .PSA is NOT a rationale based biomarkerrationale based biomarker

Prostate Specific Antigen (hK3)Prostate Specific Antigen (hK3)PSA made by prostate tissue PSA made by prostate tissue –– benign and benign and malignant tissuemalignant tissueSink effect Sink effect –– performance to monitor malignant performance to monitor malignant progression improves when prostate tissue is progression improves when prostate tissue is minimalminimalSerine protease of the human Serine protease of the human kallikreinkallikrein familyfamilyLiquefies the coagulum by acting on Liquefies the coagulum by acting on semenogellinsemenogellin I & II and FibronectinI & II and FibronectinProduced as a precursor moleculeProduced as a precursor molecule-- the prothe pro--PSAPSA

HYPOTHESISHYPOTHESIS

Function based biomarkers Function based biomarkers may demonstrate improved may demonstrate improved clinical performance clinical performance characteristicscharacteristics

Cancer PhenotypeCancer Phenotype

Benign Cells

Cancer Cells

Proliferation

Migration

Invasion

Cancer Biology Cancer Biology --

Hallmarks of CancerHallmarks of Cancer

Hanahan

D, Weinberg RA. 2011

Future challenges and unmet needs in prostate cancer biomarker research.

Prensner J R et al. Sci Transl Med 2012;4:127rv3-127rv3

Published by AAAS

OPPORTUNITIES IN PROSTATE OPPORTUNITIES IN PROSTATE CANCERCANCER

Metastatic CascadeMetastatic Cascade

Castration Resistant Prostate Castration Resistant Prostate Cancer (CRPC)Cancer (CRPC)

Schulman et al., European Urology, Vol 58, 1, pages e1 – e18, July 2010

Differential gene expressionDifferential gene expression The modelThe model

Derived from human Derived from human prostate cancer prostate cancer lymph nodal lymph nodal metastasismetastasisAndrogen Androgen dependentdependentProduce PSAProduce PSA

Derived from LNCaP Derived from LNCaP cells grown in presence cells grown in presence of bone stromal cellsof bone stromal cellsLow steady state of Low steady state of androgen receptorandrogen receptorAndrogen independentAndrogen independentHighly metastatic and Highly metastatic and produces osteoblastic produces osteoblastic lesions / produce PSAlesions / produce PSA

LNCaP cells C4-2 cells

Wu et al., Int J Ca, 57, 406-12, 1994

Differential gene expressionDifferential gene expression Experimental designExperimental design

LNCaP tRNA C4-2 tRNA

mRNA mRNA

MICROARRAY

1: RPA Data

2: Western Blotting

3: Kinase activity

4: PKD1 -

down regulated in advanced human prostate cancer

LNCaP C4-2

Protein Kinase D1 (PKD1) is Down Regulated in Protein Kinase D1 (PKD1) is Down Regulated in Advanced Prostate CancerAdvanced Prostate Cancer

PKD1 is downregulated in breast cancer Eiseler et al. Breast Cancer Res, 2009 and in gastric cancer Kim et al. Carcinogenesis. 2008

Varambally et al. Cancer Cell 2005

Phosphatidylserine, Ca2+, Diacylglycerol/phorbol ester,

DAG + PKC

ACTIVATORS:- Regulatory peptides (neurotensin, bombesin)- Lysophosphatidic acid, - Thrombin that act through Gq, G12, Gi, and Rho, - Growth factors, such as PDGF and IGF- B/T cell antigen engagement,- Oxidative stress

Tissues and Cells: - Fibroblasts - Intestinal and kidney epithelial cells - Smooth muscle cells- Cardiac myocytes- Neuronal cells - Osteoblasts- B and T lymphocytes,- Mast cells and platelets, - Several human cancers

Protein Kinase D1 (PKD1)Protein Kinase D1 (PKD1)

LNCaP cells were stained with antibody specific for PKD1 (A) is DIC image (B) is showing immunolocalization of PKD1. (C) is merge of image (A) and (B) showing perinuclear and junctional staining ( Jaggi et al., 2003).

Localization of PKD1 in LNCaP cells7

A B

C

DIC PKD1

MERGE

Molecular Biology of the Cell 3rd Edition, Figure 19-18

tight junction

adherens

junction

desmosome

gap junction

hemi-desmosome

microvilli

keratin filaments

basal lamina

band of actin filaments

Epithelial cellsEpithelial cells

Cadherin

β-catenin/plakoglobin

α-catenin

Actin filaments

ZO-1

p120ctn

vinculinα-actinin

Cadherin–Catenin Protein Complex

Wheelock et al. Current Topics in Membranes (1996)

PKD1 interacts and phosphorylates E-cadherinLNCaP

PKD-1 and E-Cadherin regulate PC cells proliferation and soft agar colony formation

PKD-1 and E-Cadherin regulate PC cells proliferation and soft agar colony formation –

Corroboration with molecular markers

β-Catenin mediates the PKD1 and E-cadherin functions in PC cells

Cadherin

β-catenin/plakoglobin

α-catenin

Actin filaments

ZO-1

p120ctn

vinculinα-actinin

Cadherin–Catenin Protein Complex

Wheelock et al. Current Topics in Membranes (1996)

PKD1 promotes β-catenin membrane trafficking

PKD1 is associated with membrane trafficking of β-catenin

Totalβ−catenine(α HA tag)

pT120

3T3

WT

T102

I/T

112R

/T12

0I

T120

I

pS916 PKD1

pT120 β-catenin

total β-catenin

total PKD1

- + Bryostatin

C4-2/PKD1

C4-

2C

4-2/

PKD

1C

4-2

C4-

2/PK

D1

+ + + pT120-

-

+ normal peptide-

+ -

phosphopeptide

C4-2/PKD1

H102 pT120

autoradiographStaining

active PKD1 - + -dead PKD1 - - +

Raamfp etldegmqipsTqfdaahpT nvqR

PKD1 phosphorylates β-catenin at Thr120

β-cat H102 Ab

p230

β-cat pT120 Ab

p230

β-catenin

α-catenin

Active BetaActive Beta--catenin catenin (unphosphorylated S37/T41) in the Nucleus(unphosphorylated S37/T41) in the Nucleus

Maher et al., PLoS One. 2010; 5(4): e10184

PKD1 represses generation of ABCPKD1 represses generation of ABC

Table 1. Analysis of pT120 antibody staining in prostate cancer

TransGolgi

network staining

Two-sample t test (P value)

n positive (%) negative (%)

Normal vs. tumor Gleason 3-6 vs. 7-10

Normal

22 16 (72.7%)

6 (27.3%)

Total tumor 200 15 (7.5%) 185 (92.5%)

P<0.01

Gleason3-6

27 4 (14.8%) 23 (85.2%)7-10

173 11 (6.3%) 162 (93.7%)

P<0.05

Table 2. Comparison of H102 and pT120 staining patterns

Total

H102 staining

pT120 staining

Normal tissue

membranous β-catenin 9

8 (88.9%)TGN β-catenin

9

8 (88.9%)

Tumor tissue

increased

expression

56

18 (32.1%)

9 (16.1%)decreased expression 56

5 (8.9%)

5 (8.9%)membranous

54

42 (77.8%)

8 (14.3%)cytoplasma/nuclear

54

12 (22.2%)

6 (10.7%)

EE--cadherin and Betacadherin and Beta--catenin Expression are catenin Expression are Down Regulated in High Risk Prostate Cancer Down Regulated in High Risk Prostate Cancer

(6F9 monoclonal anti(6F9 monoclonal anti--beta catenin COOH terminal antibody)beta catenin COOH terminal antibody)

E-cadh Beta-cat

PIN

Nuc-Beta Cat

TGN staining of pT120 BetaTGN staining of pT120 Beta-- catenin antibodycatenin antibody

OPPORTUNITIESOPPORTUNITIESPhosphoPhospho--specific Betaspecific Beta--catenin characterize catenin characterize functional changes in cellsfunctional changes in cellsUnderstanding postUnderstanding post--translational modifications translational modifications and implications on biological functions can and implications on biological functions can facilitate development of rationale based facilitate development of rationale based biomarkersbiomarkersHYPOTHESISHYPOTHESIS: Generation of function based : Generation of function based biomarkers will improve performance biomarkers will improve performance characteristics of biomarkers in clinical settingcharacteristics of biomarkers in clinical setting

AcknowledgementsAcknowledgements Mentors, Staff and ColleaguesMentors, Staff and Colleagues

University of Nebraska Medical Center, University of Nebraska Medical Center, Omaha, NEOmaha, NEUniversity of Massachusetts Medical University of Massachusetts Medical School, Worcester, MASchool, Worcester, MAWake Forest University School of Wake Forest University School of Medicine, Winston Salem, NCMedicine, Winston Salem, NC

Funding: DoD, VA, Prostate Cancer Foundation –

Univ

of Neb Med Ctr, UMass Med Sch, Wake Forest Univ

Sch

of Med Cancer Center, WFU Institute of Regen

Med

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