Generation of Function Based Biomarkers in Prostate Cancer. K.C. Balaji, MD Wake Forest University School of Medicine, Winston Salem, NC

Transcription

1 Generation of Function Based Biomarkers in Prostate Cancer K.C. Balaji, MD Wake Forest University School of Medicine, Winston Salem, NC

2 Substance used as an indicator of a biological state Can be used to monitor a normal physiologic state, a pathologic process, a pharmacologic response to therapeutic intervention, or a toxic exposure Examples: antibodies, radioactive isotopes, specific DNA sequences, PSA

3 Phases of Biomarker Development

4 Prostate Specific Antigen (PSA) as Prostate Fig. Specific 1 PSA clinical course Antigen and biomarker uses. (PSA) as a Biomarker in Prostate Cancer Published by AAAS Prensner J R et al. Sci Transl Med 2012;4:127rv3-127rv3

5 CA: A Cancer Journal for Clinicians Volume 62, Issue 1, pages 10-29, 4 JAN 2012 DOI: /caac United States Cancer statistics, 2012

6 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:

7 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:

8 Why is PSA Screening for Prostate Cancer Controversial? For starters.psa is NOT a rationale based biomarker

9 Prostate Specific Antigen (hk3) PSA made by prostate tissue benign and malignant tissue Sink effect performance to monitor malignant progression improves when prostate tissue is minimal Serine protease of the human kallikrein family Liquefies the coagulum by acting on semenogellin I & II and Fibronectin Produced as a precursor molecule- the pro-psa

10 HYPOTHESIS Function based biomarkers may demonstrate improved clinical performance characteristics

11 Cancer Phenotype Benign Cells Migration Invasion Proliferation Cancer Cells

12 Cancer Biology - Hallmarks of Cancer Hanahan D, Weinberg RA. 2011

13 OPPORTUNITIES IN PROSTATE OPPORTUNITIES Future challenges and unmet needs in prostate IN cancer PROSTATE biomarker research. CANCER Prensner J R et al. Sci Transl Med 2012;4:127rv3-127rv3 Published by AAAS

14 Metastatic Cascade

15 Castration Resistant Prostate Cancer (CRPC) Schulman et al., European Urology, Vol 58, 1, pages e1 e18, July 2010

16 Differential gene expression The model LNCaP cells Derived from human prostate cancer lymph nodal metastasis Androgen dependent Produce PSA C4-2 cells Derived from LNCaP cells grown in presence of bone stromal cells Low steady state of androgen receptor Androgen independent Highly metastatic and produces osteoblastic lesions / produce PSA Wu et al., Int J Ca, 57, , 1994

17 Differential gene expression Experimental design LNCaP trna C4-2 trna mrna mrna MICROARRAY

18 Protein Kinase D1 (PKD1) is Down Regulated in Advanced Prostate Cancer 1: RPA Data LNCaP C4-2 4: PKD1 - down regulated in advanced human prostate cancer 2: Western Blotting 3: Kinase activity Varambally et al. Cancer Cell 2005 PKD1 is downregulated in breast cancer Eiseler et al. Breast Cancer Res, 2009 and in gastric cancer Kim et al. Carcinogenesis. 2008

19 Protein Kinase D1 (PKD1) Phosphatidylserine, Ca 2+, Diacylglycerol/phorbol ester, 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 DAG + PKC 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

20 Localization of PKD1 in LNCaP cells 7 DIC PKD1 A MERGE B C 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).

21 Epithelial cells microvilli tight junction adherens junction band of actin filaments desmosome keratin filaments gap junction hemi-desmosome basal lamina Molecular Biology of the Cell 3rd Edition, Figure 19-18

22 Cadherin Catenin Protein Complex p120 ctn Cadherin ZO-1 β-catenin/ plakoglobin α-catenin vinculin α-actinin Actin filaments Wheelock et al. Current Topics in Membranes (1996)

23 LNCaP PKD1 interacts and phosphorylates E-cadherin

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

25 PKD-1 and E-Cadherin regulate PC cells proliferation and soft agar colony formation Corroboration with molecular markers

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

27 Cadherin Catenin Protein Complex p120 ctn Cadherin ZO-1 β-catenin/ plakoglobin α-catenin vinculin α-actinin Actin filaments Wheelock et al. Current Topics in Membranes (1996)

28 PKD1 promotes β-catenin membrane trafficking

29 PKD1 is associated with membrane trafficking of β-catenin

30 C4-2 C4-2/PKD1 C4-2 C4-2/PKD1 PKD1 phosphorylates β-catenin at Thr120 active PKD dead PKD autoradio graph Staining Raamfp etldegmqip stqfdaahpt nvqr C4-2/PKD1 3T3 - + Bryostatin WT pt120 β-catenin total β-catenin ps916 PKD1 total PKD1 C4-2/PKD1 T102I /T112R /T120I T120I β-catenin α-catenin pt120 H102 pt pt normal peptide phosphopeptide Total β catenine (α HA tag) β-cat H102 Ab p230 β-cat pt120 Ab p230

31 Active Beta-catenin (unphosphorylated S37/T41) in the Nucleus Maher et al., PLoS One. 2010; 5(4): e10184

32 PKD1 represses generation of ABC

33 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 Normal (72.7%) 6 (27.3%) Total tumor (7.5%) 185 (92.5%) P<0.01 Gleason (14.8%) 23 (85.2%) (6.3%) 162 (93.7%) P<0.05 Table 2. Comparison of H102 and pt120 staining patterns Normal tissue membranous β-catenin 9 8 (88.9%) Total H102 staining pt120 staining TGN β-catenin 9 8 (88.9%) Tumor tissue increased expression (32.1%) 9 (16.1%) decreased expression 56 5 (8.9%) 5 (8.9%) membranous (77.8%) 8 (14.3%) cytoplasma/nuclear (22.2%) 6 (10.7%)

34 E-cadherin and Beta-catenin Expression are Down Regulated in High Risk Prostate Cancer (6F9 monoclonal anti-beta catenin COOH terminal antibody) E-cadh Beta-cat PIN Nuc-Beta Cat

35 TGN staining of pt120 Beta- catenin antibody

36 OPPORTUNITIES Phospho-specific specific Beta-catenin characterize functional changes in cells Understanding post-translational translational modifications and implications on biological functions can facilitate development of rationale based biomarkers HYPOTHESIS: : Generation of function based biomarkers will improve performance characteristics of biomarkers in clinical setting

37 Acknowledgements Mentors, Staff and Colleagues University of Nebraska Medical Center, Omaha, NE University of Massachusetts Medical School, Worcester, MA Wake Forest University School of Medicine, 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

38 References 1. Cheng Du, Chuanyou Zhang, Zhuo Li, Md. Helal Uddin Biswas and K.C. Balaji: β-catenin Phosphorylated at Threonine 120 Antagonizes Generation of Active β-catenin by Spatial Localization at Trans-Golgi Network. PLoS One. 2012;7(4):e Epub 2012 Apr Cheng Du, Chuanyou Zhang, Sazzad Hassan, Md Helal Uddin Biswas and K.C. Balaji: Protein Kinase D1 Suppresses Epithelial to Mesenchymal Transition through Phosphorylation horylation of Snail. Cancer Research; 2010 Oct 15;70(20): Epub 2010 Oct Md Helal Uddin Biswas,, Cheng Du, Chuanyou Zhang, Juerg Straubhaar,, Lucia R. Languino and K.C. Balaji: Protein Kinase D1 Inhibits Cell Proliferation through Matrix M Metalloproteinases (MMP) -22 and -9 Secretion in Prostate Cancer. Cancer Research, 2010 Mar 1;70(5): Epub 2010 Feb Sazzad Hassan, Md. Helal Uddin Biswas and K.C. Balaji: Heat Shock Protein 27 Mediates Repression of Androgen Receptor Function by Protein Kinase D1 in Prostate Cancer C Cells: Oncogene Dec 10;28(49): PMID: Cheng Du, Meena Jaggi, Chuanyou Zhang and K.C. Balaji; Protein Kinase D1 (PKD1) Mediated Phosphorylation and Subcellular Localization of β-catenin: Cancer Res 2009; 69: (3).February 1, Du, C., et al., Protein kinase D1-mediated phosphorylation and subcellular localization of beta-catenin. Cancer Res, (3): p Jaggi,, M., et al., Bryostatin 1 modulates beta-catenin subcellular localization and transcription activity through protein kinase D1 activation. Mol Cancer Ther,, (9): p Mak,, P., et al., Protein kinase D1 (PKD1) influences androgen receptor (AR) function in prostate cancer cells. Biochem Biophys Res Commun,, (4): p Syed,, V., et al., Beta-catenin mediates alteration in cell proliferation, motility and invasion of prostate cancer cells by differential expression of E-cadherin E and protein kinase D1. J Cell Biochem,, (1): p Jaggi,, M., et al., Protein kinase D1: a protein of emerging translational interest. Front Biosci,, : p Jaggi,, M., et al., E-cadherin phosphorylation by protein kinase D1/protein kinase C{mu} } is associated with altered cellular aggregation and motility in prostate cancer. Cancer Res, (2): p Rao,, P.S., et al., Metallothionein 2A interacts with the kinase domain of PKCmu in prostate cancer. Biochem Biophys Res Commun,, (3): p Jaggi,, M., et al., Protein kinase C mu is down-regulated in androgen-independent prostate cancer. Biochem Biophys Res Commun,, (2): p