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EXPRESSION OF THE EXTRACELLULAR MATRIX ASSOCIATED PROTEIN CYR61 IS ASSOCIATED WITH THE DEVELOPMENT OF PROSTATE CANCER
Vol. 179, No. 4, Supplement, Tuesday, May 20, 2008
THE JOURNAL OF UROLOGY®
459
CONCLUSIONS: Changes in Cyr61 expression have been found to contribute to the development and progression of various types of cancer. Cyr61 is clearly associated with the development of prostate cancer. As a protein involved in interactions with the extracellular matrix it is situated to be an important microenviromental regulator of prostate cancer cells. These studies suggest the potential for a novel therapeutic approach for prostate cancer focused on Cyr61. Source of Funding: Patana Fund of the Brady Urological Institute.
1340
Source of Funding: VA Merit Review Career Development Grant.
1339 EXPRESSION OF THE EXTRACELLULAR MATRIX ASSOCIATED PROTEIN CYR61 IS ASSOCIATED WITH THE DEVELOPMENT OF PROSTATE CANCER Katherine D’Antonio*, Antoun Toubaji, Roula Albadine, George J Netto, Robert H Getzenberg. Baltimore, MD. INTRODUCTION AND OBJECTIVE: Prostate cancer is a disease that comprises more than the cancer cells themselves. The microenvironment in which the cancer cells exist is central to the disease process. Genomic studies of cancer have revealed that the genes encoding the CCN family of extracellular matrix associated proteins consistently exhibit altered expression. One of the changes most frequently associated with cancer development is an integrin binding extracellular matrix protein, cystein rich angiogenic inducer 61 (Cyr61), also known as CCN1. The objective of these studies is to examine the expression of Cyr61 in prostate cancer, normal prostate, and the associated tissues as well as to study its role in prostate cancer development. METHODS: Cyr61 gene expression in prostate cancer and normal tissues was evaluated by microarray analysis. Immunoblot analysis was used to assess endogenous Cyr61 protein expression in prostate cell lines and further substantiated by tissue microarray analysis of prostatectomy samples. 200 radical prostatectomy samples were collected and Cyr61 expression was evaluated by tissue microarray analysis (TMA). In vitro functional studies were also performed on prostate cancer cell lines to gain insight into the role of Cyr61 in prostate cancer, particularly regarding growth and adhesion. RESULTS: Cyr61 expression is up-regulated at both the mRNA DQGSURWHLQOHYHOV&\USURWHLQH[SUHVVLRQLVVLJQL¿FDQWO\XSUHJXODWHG compared to normal epithelium and simple atrophy as determined by TMA. Cyr61 expression progressively increases from normal epithelium to low to high grade PIN with the strongest expression in prostate cancer. Human prostate cancer cells that over-express Cyr61 demonstrate poor adhesion and growth when cultured on several different extracellular matrix substrates and exhibit substantially increased apoptosis.
THE ROLE OF GLUTATHIONE PEROXIDASE 1 CODON 198 (GPX1 Pro198Leu) POLYMORPHISM IN PATIENTS WITH PROSTATE CANCER &DQDQ.XFXNJHUJLQ2QHU6DQOL 7]HYDW7H¿N,VPHW1DQH6XOH Seckin, Elif Ozkok, Makbule Aydin, Murat Gokpinar. Istanbul, Turkey. INTRODUCTION AND OBJECTIVE: Cytosolic glutathione SHUR[LGDVH *3; DVHOHQLXPFRQWDLQLQJDQWLR[LGDQWHQ]\PHKDVEHHQ implicated in the development of cancer. In this study, the association between GPX1 Pro198Leu polymorphism and prostate cancer was investigated. 0(7+2'6 &RQVHFXWLYHSDWLHQWVZLWKKLVWRORJLFDOO\FRQ¿UPHG prostate cancer (n= 90, mean age: 63.5 years) and healthy controls with normal serum total PSA (< 4 ng/ml) and DRE (n= 110, mean age 61.7 years) were prospectively enrolled in this study between 2005 and 2007. All subjects were genotyped for the GPX1 Pro198Leu polymorphism by the PCR followed by agarose gel electrophoresis. The plasma glutathione peroxidase activity (GPX), total plasma thiol (-SH) and plasma malondialdehyde (MDA) levels were used as the markers of oxidative stress. For the statistical analyses chi-square, Kruskal-Wallis and Mann-Whitney U tests were used where appropriate. 5(68/76 3ODVPDWRWDO6+OHYHOVZHUHVLJQL¿FDQWO\ORZHULQ the patients with prostate cancer while MDA level and GPX activity were VLJQL¿FDQWO\KLJKHUFRPSDUHGZLWKWKHKHDOWK\FRQWUROV7KHGLVWULEXWLRQ of GPX1 genotypes was different comparing the prostate cancer with FRQWUROV,QGLYLGXDOVZLWKWKHKRPR]\JRXVYDULDQWJHQRW\SH /HX/HX ZHUH DW JUHDWHU ULVN IRU SURVWDWH FDQFHU FRPSDUHG ZLWK KRPR]\JRXV ZLOG W\SH 3UR3UR DQG KHWHUR]\JRXV JHQRW\SH &7 %\ DQDO\]LQJ the frequency of a polymorphism within the GPX1 gene resulting in a leucin (T) or prolin(C) at codon 198, it was determined that the leucincontaining allele was more frequently associated with prostate cancer than the proline- containing allele. *3; DFWLYLW\ ZDV QRW VLJQL¿FDQWO\ different in Pro/Pro (CC) genotype whereas Pro/Leu (CT) and Leu/Leu 77 JHQRW\SHVZHUHVLJQL¿FDQWO\KLJKHULQSURVWDWHFDQFHUSDWLHQWVDV compared with healthy controls. &21&/86,216 $FFRUGLQJWRRXU¿QGLQJV*3;/HX/HX77 genotype is associated with an increase in prostate cancer. In addition, GPX1 polymorphism is correlated with higher GPX activity and this may be a risk factor for prostate cancer in Turkish men. Source of Funding: None
1341 TALIN AS A NOVEL MEDIATOR OF PROSTATE CANCER CELL MIGRATION, INVASION AND METASTASIS Shinichi Sakamoto*, Richard McCann, Natasha Kyprianou. Lexington, KY. INTRODUCTION AND OBJECTIVE: Talin is an integrin binding protein that regulates integrin activation towards mediating cell signaling interactions with the extracellular matrix (ECM). The role of talin in human cancer metastasis has not been yet explored. This VWXG\LQYHVWLJDWHGWKHIXQFWLRQDOVLJQL¿FDQFHRIWKLVNH\IRFDODGKHVLRQ component in the regulation of prostate cancer cell invasion in the context of the tumor microenvironment and metastatic behavior in vitro and in vivo. METHODS: In vivo expression of talin was determined by immunohistochemical analysis using talin polyclonal antibody in prostate tissue obtained from transgenic adenocarcinoma prostate (TRAMP) mouse model of increasing ages (6-28 weeks). Cell viability was assessed by the MTT assay and TUNEL and Annexin-V analysis were used to determine the incidence of apoptosis. ShRNA silencing
THE JOURNAL OF UROLOGY®
459
CONCLUSIONS: Changes in Cyr61 expression have been found to contribute to the development and progression of various types of cancer. Cyr61 is clearly associated with the development of prostate cancer. As a protein involved in interactions with the extracellular matrix it is situated to be an important microenviromental regulator of prostate cancer cells. These studies suggest the potential for a novel therapeutic approach for prostate cancer focused on Cyr61. Source of Funding: Patana Fund of the Brady Urological Institute.
1340
Source of Funding: VA Merit Review Career Development Grant.
1339 EXPRESSION OF THE EXTRACELLULAR MATRIX ASSOCIATED PROTEIN CYR61 IS ASSOCIATED WITH THE DEVELOPMENT OF PROSTATE CANCER Katherine D’Antonio*, Antoun Toubaji, Roula Albadine, George J Netto, Robert H Getzenberg. Baltimore, MD. INTRODUCTION AND OBJECTIVE: Prostate cancer is a disease that comprises more than the cancer cells themselves. The microenvironment in which the cancer cells exist is central to the disease process. Genomic studies of cancer have revealed that the genes encoding the CCN family of extracellular matrix associated proteins consistently exhibit altered expression. One of the changes most frequently associated with cancer development is an integrin binding extracellular matrix protein, cystein rich angiogenic inducer 61 (Cyr61), also known as CCN1. The objective of these studies is to examine the expression of Cyr61 in prostate cancer, normal prostate, and the associated tissues as well as to study its role in prostate cancer development. METHODS: Cyr61 gene expression in prostate cancer and normal tissues was evaluated by microarray analysis. Immunoblot analysis was used to assess endogenous Cyr61 protein expression in prostate cell lines and further substantiated by tissue microarray analysis of prostatectomy samples. 200 radical prostatectomy samples were collected and Cyr61 expression was evaluated by tissue microarray analysis (TMA). In vitro functional studies were also performed on prostate cancer cell lines to gain insight into the role of Cyr61 in prostate cancer, particularly regarding growth and adhesion. RESULTS: Cyr61 expression is up-regulated at both the mRNA DQGSURWHLQOHYHOV&\USURWHLQH[SUHVVLRQLVVLJQL¿FDQWO\XSUHJXODWHG compared to normal epithelium and simple atrophy as determined by TMA. Cyr61 expression progressively increases from normal epithelium to low to high grade PIN with the strongest expression in prostate cancer. Human prostate cancer cells that over-express Cyr61 demonstrate poor adhesion and growth when cultured on several different extracellular matrix substrates and exhibit substantially increased apoptosis.
THE ROLE OF GLUTATHIONE PEROXIDASE 1 CODON 198 (GPX1 Pro198Leu) POLYMORPHISM IN PATIENTS WITH PROSTATE CANCER &DQDQ.XFXNJHUJLQ2QHU6DQOL 7]HYDW7H¿N,VPHW1DQH6XOH Seckin, Elif Ozkok, Makbule Aydin, Murat Gokpinar. Istanbul, Turkey. INTRODUCTION AND OBJECTIVE: Cytosolic glutathione SHUR[LGDVH *3; DVHOHQLXPFRQWDLQLQJDQWLR[LGDQWHQ]\PHKDVEHHQ implicated in the development of cancer. In this study, the association between GPX1 Pro198Leu polymorphism and prostate cancer was investigated. 0(7+2'6 &RQVHFXWLYHSDWLHQWVZLWKKLVWRORJLFDOO\FRQ¿UPHG prostate cancer (n= 90, mean age: 63.5 years) and healthy controls with normal serum total PSA (< 4 ng/ml) and DRE (n= 110, mean age 61.7 years) were prospectively enrolled in this study between 2005 and 2007. All subjects were genotyped for the GPX1 Pro198Leu polymorphism by the PCR followed by agarose gel electrophoresis. The plasma glutathione peroxidase activity (GPX), total plasma thiol (-SH) and plasma malondialdehyde (MDA) levels were used as the markers of oxidative stress. For the statistical analyses chi-square, Kruskal-Wallis and Mann-Whitney U tests were used where appropriate. 5(68/76 3ODVPDWRWDO6+OHYHOVZHUHVLJQL¿FDQWO\ORZHULQ the patients with prostate cancer while MDA level and GPX activity were VLJQL¿FDQWO\KLJKHUFRPSDUHGZLWKWKHKHDOWK\FRQWUROV7KHGLVWULEXWLRQ of GPX1 genotypes was different comparing the prostate cancer with FRQWUROV,QGLYLGXDOVZLWKWKHKRPR]\JRXVYDULDQWJHQRW\SH /HX/HX ZHUH DW JUHDWHU ULVN IRU SURVWDWH FDQFHU FRPSDUHG ZLWK KRPR]\JRXV ZLOG W\SH 3UR3UR DQG KHWHUR]\JRXV JHQRW\SH &7 %\ DQDO\]LQJ the frequency of a polymorphism within the GPX1 gene resulting in a leucin (T) or prolin(C) at codon 198, it was determined that the leucincontaining allele was more frequently associated with prostate cancer than the proline- containing allele. *3; DFWLYLW\ ZDV QRW VLJQL¿FDQWO\ different in Pro/Pro (CC) genotype whereas Pro/Leu (CT) and Leu/Leu 77 JHQRW\SHVZHUHVLJQL¿FDQWO\KLJKHULQSURVWDWHFDQFHUSDWLHQWVDV compared with healthy controls. &21&/86,216 $FFRUGLQJWRRXU¿QGLQJV*3;/HX/HX77 genotype is associated with an increase in prostate cancer. In addition, GPX1 polymorphism is correlated with higher GPX activity and this may be a risk factor for prostate cancer in Turkish men. Source of Funding: None
1341 TALIN AS A NOVEL MEDIATOR OF PROSTATE CANCER CELL MIGRATION, INVASION AND METASTASIS Shinichi Sakamoto*, Richard McCann, Natasha Kyprianou. Lexington, KY. INTRODUCTION AND OBJECTIVE: Talin is an integrin binding protein that regulates integrin activation towards mediating cell signaling interactions with the extracellular matrix (ECM). The role of talin in human cancer metastasis has not been yet explored. This VWXG\LQYHVWLJDWHGWKHIXQFWLRQDOVLJQL¿FDQFHRIWKLVNH\IRFDODGKHVLRQ component in the regulation of prostate cancer cell invasion in the context of the tumor microenvironment and metastatic behavior in vitro and in vivo. METHODS: In vivo expression of talin was determined by immunohistochemical analysis using talin polyclonal antibody in prostate tissue obtained from transgenic adenocarcinoma prostate (TRAMP) mouse model of increasing ages (6-28 weeks). Cell viability was assessed by the MTT assay and TUNEL and Annexin-V analysis were used to determine the incidence of apoptosis. ShRNA silencing