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DEREGULATION OF FOXO3A CONTRIBUTES TO PROSTATE CANCER PROGRESSION
460
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approach was used to study the consequences of talin loss on prostate cancer cell apoptosis, migration, adhesion and invasion. RESULTS: Our results demonstrate that talin overexpression correlated with prostate cancer development and progression to metastasis in the TRAMP mouse model. Stable expression of talin in prostate cancer cells PC-3, enhanced cell adhesion and migration, and promoted invasion prostate cancer cells in vitro. ShRNA-mediated VLOHQFLQJRIWDOLQUHVXOWHGLQDVLJQL¿FDQWVXSSUHVVLRQRIWKHLQYDVLRQ potential of prostate cancer cells. Mechanistic dissection revealed that talin regulates cell survival signals through activation/phosphorylation of focal adhesion kinase (FAK) and AKT. &21&/86,216 7KHVH¿QGLQJVLGHQWL¿HGWDOLQDVDSRWHQW mediator of prostate tumor cell migration, invasion and intracellular survival signaling. Thus talin emerges as a potential therapeutic target for advanced prostate cancer, as well as a tumor marker that might predict aggressive prostate tumors during progression to metastasis. Source of Funding: NIH/NCI CA 107757-01 Grant.
1342 ABERRANT ERG EXPRESSION COOPERATES WITH PTEN LOSS TO PROMOTE PROSTATE TUMORIGENESIS Brett S Carver*, Jennifer Tran, Anuhadra Gopalan, Peter T Scardino, :LOOLDP*HUDOG3LHU33DQGRO¿1HZ
1343 DIFFERENTIAL GENE EXPRESSION IN NORMAL PROSTATE EPITHELIUM OF MEN WITH AND WITHOUT PROSTATE CANCER: EVIDENCE FOR A PROSTATE CANCER FIELD EFFECT Michael C Risk*, Ilsa Coleman, Ruthy Dumpit, Robert C Gentleman, Alan R Kristal, Beatrice S Knudsen, Peter S Nelson, Daniel W Lin. Seattle, WA. INTRODUCTION AND OBJECTIVE: Several solid tumors are associated with molecular changes in adjacent, histologically normal FHOOVWKHVRFDOOHG³¿HOGHIIHFW´7KLV¿HOGHIIHFWPD\SUHGLVSRVHWKH
Vol. 179, No. 4, Supplement, Tuesday, May 20, 2008
normal cells to the development of frank malignancy. We sought to determine if normal epithelium from prostates harboring prostatic adenocarcinoma harbor changes in gene expression suggestive of SUHPDOLJQDQF\RU¿HOGHIIHFW METHODS: Prostate needle biopsies from 15 men with high grade (Gleason 8-10) prostate cancer and 15 age- and BMI-matched FRQWUROVZHUHLGHQWL¿HGIURPDSRRORIELRSVLHVFROOHFWHGIURP!PHQ since 2003. Normal epithelia from each patient were isolated via laser FDSWXUHPLFURGLVVHFWLRQ /&0 RIIUHVKIUR]HQELRSV\WLVVXH%LRSVLHV were reviewed by a pathologist prior to LCM to ensure only normal HSLWKHOLDZDVFDSWXUHG51$ZDVLVRODWHGIURPHDFKVDPSOHDPSOL¿HG WKHQ XWLOL]HG IRU PLFURDUUD\ DQDO\VLV XVLQJ WKH$JLOHQW +XPDQ [. DUUD\ SODWIRUP7KH GDWD ZHUH DQDO\]HG E\ SDLUHG WZRVDPSOH7WHVW XVLQJ 6LJQL¿FDQFH$QDO\VLV RI 0LFURDUUD\V 6$0 4XDQWLWDWLYH 3&5 (qPCR) was also used to determine the expression of various genes of LQWHUHVWLGHQWL¿HGE\PLFURDUUD\DQDO\VLV RESULTS: One control patient had Gleason 7 prostate cancer on repeat biopsy during the data analysis period and thus was excluded. The remaining 14 control and 15 cancer samples were included in the ¿QDODQDO\VLV0LFURDUUD\DQDO\VLVUHYHDOHGWKDWWKHVHWZRJURXSVKDG very similar patterns of gene expression overall. SAM analysis found VLJQL¿FDQW T YDOXHV T LQ JHQHV SUHYLRXVO\ DVVRFLDWHG ZLWK prostate cancer, namely FOLH1/PSMA and SSTR1. We examined RWKHUSURVWDWHFDQFHUDVVRFLDWHGJHQHVE\T3&5DQGIRXQGVLJQL¿FDQW differential expression changes in ERG, HOXC4, HOXC5 and MME, although other cancer markers (HPN, AMACR, FASN, SPOCK1, MYO6, 73'(=+ ZHUHQRWGLIIHUHQWEHWZHHQWKHWZRJURXSV,QWHUHVWLQJO\ the elevated ERG expression in the normal epithelia adjacent to prostate cancer did not appear to be associated with the TMPRSS2:ERG fusion with the exception of one patient. &21&/86,216 2YHUDOOJHQHH[SUHVVLRQSUR¿OHVEHWZHHQ normal prostatic epithelia of patients with and without prostate cancer are very similar. However, several of the differentially expressed genes found in this study have been implicated in prostate cancer progression and prognosis in prior studies, and thus may represent the earliest events in prostate carcinogenesis. Source of Funding: NIH/NIDDK.
1344 DEREGULATION OF FOXO3A CONTRIBUTES TO PROSTATE CANCER PROGRESSION Sanjeev Shukla*, Gregory T MacLennan, Sanjay Gupta. Cleveland, OH. INTRODUCTION AND OBJECTIVE: Forkhead box (Fox) proteins are a super-family of evolutionary conserved transcriptional regulators, which control a wide spectrum of biological processes. The IDPLO\LVFKDUDFWHUL]HGE\DFRQVHUYHG'1$ELQGLQJGRPDLQFRPSULVLQJ PRUHWKDQPHPEHUVLQKXPDQVFODVVL¿HGIURP)2;$WR)2;5 on the basis of sequence similarity. In particular, FOX subfamily O family has been implicated in the regulation of cell cycle and apoptosis and are the major substrates for the protein kinase B/Akt. Earlier we demonstrated the involvement of Akt/PKB during prostate cancer SURJUHVVLRQ KRZHYHUWKHUROHRI)2;2IDPLO\LQSURVWDWHWXPRULJHQHVLV has not been elucidated. 0(7+2'6 7KHH[SUHVVLRQRIPHPEHUVRI)2;2IDPLO\YL] FOXO1A, FOXO3A, FOXO4 were examined in human prostate cancer specimens of various Gleason grades, various human prostate cancer cells and transgenic adenocarcinoma of the mouse prostate (TRAMP) model representing various stages of disease progression along with non-tumorigenic counterparts. The interactions between FOXO, Akt/ PKB and 14-3-3 chaperone and associated signaling were determined by immunoprecipitation, immunoblotting and immunohistochemistry. RESULTS: Basal protein levels of FOXO1A was higher in androgen-refractory human prostate cancer cells, DU145 and PC-3 compared to androgen-responsive LNCaP and 22Rv1 cells, demonstrating its possible involvement in disease transition. No changes were observed in the protein levels of FOXO4 in all these cell lines. Interestingly, FOXO3A levels were downregulated in all human prostate cancer cell lines and redistributed in the cellular compartments. Compared WREHQLJQWLVVXHV)2;2$DFFXPXODWLRQZDVVLJQL¿FDQWO\KLJKHULQWKH cytosolic fraction than in the nuclear fraction and this difference was
Vol. 179, No. 4, Supplement, Tuesday, May 20, 2008
highest in high-grade tumor specimens. Similar observations were noted in TRAMP mice prostates where increased cytoplasmic FOXO3A levels correlated with phosphorylation at Ser253. The interactions between )2;2$ DQG$NW ZDV FRQ¿UPHG DIWHU LPPXQRSUHFLSLWDWLRQ RI HLWKHU proteins and increased binding with 14-3-3 and its accumulation in the cytoplasm of TRAMP mice prostates, compared to non-transgenic littermates at 20-28 weeks of age. Furthermore, decreased FOXO3A levels correlated with downregulation of the basal levels of p21/WAF1, 0Q62'DQG&X=Q62'LQWKHSURVWDWHVRI75$03PLFH &21&/86,216 7KHVH ¿QGLQJV GHPRQVWUDWH WKDW WXPRU associated reductions and redistribution of FOXO3A and FOXO1A proteins are frequent events in the etiology of prostate cancer. Source of Funding: USPHS grants NIH RO1CA10851, and The James & Eilleen Dicke Research Endowment funds.
1345 RELATIVE FREQUENCIES OF ETV1 AND ERG OVEREXPRESSION IN PROSTATE CANCER Ying Hu*, Lakshmi Ravindranath, Bungo Furusato, Chen Sun, Albert Dobi, Isabell A Sesterhenn, David G McLeod, Gyorgy Petrovics, Shiv Srivastava. Rockville, MD, and Washington, DC. INTRODUCTION AND OBJECTIVE: Recent studies have established that high proportion (>70%) of prostate cancers (CaP) harbor overexpression of the ETS related genes (ERG, ETV1 or ETV4) due to fusion of an androgen receptor regulated promoter element of the TMPRSS2 gene to the protein coding sequence of the ETS related genes. While TMPRSS2-ERG fusions are the most common in CaP, a recent report showed the fusion of distinct regulatory sequences to ETV1 in some prostate tumors overexpressing ETV1. The frequency of ETV1 alterations is not well understood in CaP. Here we have performed comparative evaluations of the ERG and ETV1 in CaP. 0(7+2'6 :HKDYHDQDO\]HGWKHTXDQWLWDWLYHH[SUHVVLRQ of ETV1 in benign and tumor cells laser capture microdissected from SULPDU\ SURVWDWH WXPRUV RI SDWLHQWV WKDW ZHUH DOUHDG\ DQDO\]HG for the quantitative expression of ERG, as well as TMPRSS2-ERG fusion. 5(68/76 )RUW\ ¿YH RI SDWLHQWV ZLWK QR GHWHFWDEOH TMPRSS2-ERGIXVLRQLQWKHLUWXPRUVZHUHDQDO\]HGIRUTXDQWLWDWLYH expression of ETV1 in paired tumor and benign cells. Four of 45 patients UHYHDOHGVLJQL¿FDQWRYHUH[SUHVVLRQRI ETV1 in tumor cells in comparison to matched benign cells. None of the recently reported ETV1 fusions were detected suggesting for the presence of new fusions or other mechanism of ETV1 overexpression in these tumors. CONCLUSIONS: In comparison to the ERG overxpression, ETV1 overxpression is detectable in a very small subset of prostate tumors. In order to more accurately assess the overall frequency of ETS related gene alterations in CaP, careful evaluations of ERG, ETV1 and other ETS factors in the same cohort of patients is warranted. Source of Funding: CPDR, NIH.
1346 METHYLATION OF GSTP1, RARB AND RASSF1A IN FREE CIRCULATING DNA AND BUFFY COAT DNA IN PROSTATE CANCER PATIENTS AND CONTROLS Rakesh Singal*, Kavitha Ramachandran, Isildinha Reis, Merce Jorda, Murugesan Manoharan. Miami, FL. INTRODUCTION AND OBJECTIVE: Prostate cancer is one of the most frequently diagnosed neoplasm and the second leading cause of cancer related mortality in men in the United States. The FXUUHQWO\XVHGGHWHFWLRQPHWKRGV SURVWDWHVSHFL¿FDQWLJHQDQGGLJLWDO UHFWDO H[DPLQDWLRQ KDYH OLPLWHG VSHFL¿FLW\ DQG VHQVLWLYLW\$OWKRXJK not presently used in routine clinical settings, DNA based molecular WXPRU PDUNHUV KDYH LQFUHDVHG VHQVLWLYLW\ DQG VSHFL¿FLW\ RI WXPRU detection. Previous studies have used DNA from plasma, serum and buffycoat to detect tumor-derived methylation changes as potential molecular markers for cancer diagnosis. In this study, we compared gene methylation patterns in serum and buffycoat of prostate cancer patients and patients with benign prostatic conditions. METHODS: DNA extracted from serum and buffycoat of prostate cancer patients and patients with benign prostatic conditions
THE JOURNAL OF UROLOGY®
461
ZHUH ELVXOILWH WUHDWHG DQG DQDO\]HG XVLQJ PHWK\ODWLRQVSHFLILF polymerase chain reaction (MSPCR). Age-adjusted odds ratios (OR) and corresponding p-values are reported. RESULTS: serum, higher risk of prostate cancer was associated with methylation of the following genes GSTP1 (OR=3.77, p=0.080), RARB (OR=6.59, p=0.094), RASSF1A (OR= 13.10, p=0.020). When methylation of the same genes in DNA from buffycoat was DQDO\]HGZLWKH[FHSWLRQRI5$5%RGGVUDWLRHVWLPDWHVEHFDPHVPDOOHU though the direction of the association did not change: GSTP1 (OR=1.49, p=0.506), RARB (OR=6.45, p=0.010), RASSF1A (OR= 5.84, p=0.014). We calculated the methylation index ( MI) based on the GSTP1, RARB and RASSF1A using serum only, buffycoat only and both (serum and/ or buffycoat). Age-adjusted OR for MI1+ vs. MI=0 was 4.417 for serum only (p=0.017), 2.252 for buffycoat only (p=0.146) and 3.279 for serum and/or buffycoat (p=0.03). CONCLUSIONS: Methylation changes derived from serum are more discriminatory between prostate cancer patients and controls as compared to methylation changes derived from buffycoat.Moreover, evaluation of methylation of set of genes may be a better predictor of tumor than analysis of a single gene. Source of Funding: None
1347 ECTOPIC EXPRESSION OF MKK4/JNKK1 CONTROLS PROSTATE CANCER METASTATIC COLONIZATION BY CONTROLLING GROWTH OF DISSEMINATED CANCER CELLS AT SECONDARY SITES Russell Z Szmulewitz*, Tamara Lotan, Jennifer Taylor, Shaheena Khan, Nkechiyere Nwani, Carrie Rinker-Schaeffer. Chicago, IL, and Baltimore, MD. INTRODUCTION AND OBJECTIVE: The process by which disseminated prostate cancer cells persist as dormant lesions and then initiate growth is largely unknown. Metastasis suppressor proteins are helping to dissect the molecular mechanisms regulating this clinically important event. MKK4/JNKK1 is a stress-activated signaling protein which is a metastasis suppressor in experimental models of prostate and ovarian cancers. Ectopic expression of the MKK4/JNKK1 protein in AT6.1 rat prostate cancer cells reduced the number of spontaneous metastases in the lungs by 90% and increased survival by 63%. %LRFKHPLFDOVWXGLHVVKRZHGWKDWWKH0..-1..NLQDVHLVVSHFL¿FDOO\ activated in disseminated cells, supporting a role for context-dependent activation of the protein within the microenviroment of the metastatic site. We are now examining the cellular mechanism of MKK4/JNKK1PHGLDWHGVXSSUHVVLRQRIPHWDVWDWLFFRORQL]DWLRQ METHODS: Spontaneous and experimental (using tail vein LQMHFWLRQVRIFDQFHUFHOOV PHWDVWDVLVDVVD\VLQLPPXQRGL¿FLHQWPLFH using the AT6.1 prostate cancer model were employed. The numbers RI FHOOV SUHVHQW LQ WKH OXQJ DW VSHFL¿F WLPH SRLQWV GXULQJ PHWDVWDWLF FRORQL]DWLRQZDVGHWHUPLQHGXVLQJTXDQWLWDWLYHUHDOWLPH3&5 T573&5 ZLWKPRXVHDQGUDWVSHFL¿FSULPHUV7KHSHUFHQWDJHRIGLVVHPLQDWHG cells undergoing apoptosis was determined by TUNEL, cleaved caspase 3 and morphologic changes. Proliferation was assessed using phosphohistone-H3 and BrdU incorporation. RESULTS: qRT-PCR showed similar numbers of AT6.1-control and AT6.1-MKK4/JNKK1 lodge within the lung during spontaneous metastasis. Immunohistochemical studies found no increase in the percentage of cells undergoing apoptosis. Experimental metastasis assays are now being employed to further dissect the proliferative events, and preliminary studies show that MKK4/ JNKK1 does indeed suppress experimental metastasis. CONCLUSIONS: MKK4/JNKK1 suppresses metastasis E\ LQKLELWLQJ PHWDVWDWLF FRORQL]DWLRQ 2XU SUHOLPLQDU\ VWXGLHV LQGLFDWH that this is due to decreased cell proliferation and not decreased number of cells lodging at secondary sites or increased numbers of cell undergoing apoptosis. Determining how MKK4/JNKK1 activation regulates proliferation could provide new clues for controlling the growth of disseminated cancer cells at metastatic sites, and present new therapeutic considerations for men with metastatic prostate cancer. Source of Funding: NIH RO1 CA 89569, W81XWH-06-1-0041, Arthur Foundation, University of Chicago RESCUE Fund.
THE JOURNAL OF UROLOGY®
approach was used to study the consequences of talin loss on prostate cancer cell apoptosis, migration, adhesion and invasion. RESULTS: Our results demonstrate that talin overexpression correlated with prostate cancer development and progression to metastasis in the TRAMP mouse model. Stable expression of talin in prostate cancer cells PC-3, enhanced cell adhesion and migration, and promoted invasion prostate cancer cells in vitro. ShRNA-mediated VLOHQFLQJRIWDOLQUHVXOWHGLQDVLJQL¿FDQWVXSSUHVVLRQRIWKHLQYDVLRQ potential of prostate cancer cells. Mechanistic dissection revealed that talin regulates cell survival signals through activation/phosphorylation of focal adhesion kinase (FAK) and AKT. &21&/86,216 7KHVH¿QGLQJVLGHQWL¿HGWDOLQDVDSRWHQW mediator of prostate tumor cell migration, invasion and intracellular survival signaling. Thus talin emerges as a potential therapeutic target for advanced prostate cancer, as well as a tumor marker that might predict aggressive prostate tumors during progression to metastasis. Source of Funding: NIH/NCI CA 107757-01 Grant.
1342 ABERRANT ERG EXPRESSION COOPERATES WITH PTEN LOSS TO PROMOTE PROSTATE TUMORIGENESIS Brett S Carver*, Jennifer Tran, Anuhadra Gopalan, Peter T Scardino, :LOOLDP*HUDOG3LHU33DQGRO¿1HZ
1343 DIFFERENTIAL GENE EXPRESSION IN NORMAL PROSTATE EPITHELIUM OF MEN WITH AND WITHOUT PROSTATE CANCER: EVIDENCE FOR A PROSTATE CANCER FIELD EFFECT Michael C Risk*, Ilsa Coleman, Ruthy Dumpit, Robert C Gentleman, Alan R Kristal, Beatrice S Knudsen, Peter S Nelson, Daniel W Lin. Seattle, WA. INTRODUCTION AND OBJECTIVE: Several solid tumors are associated with molecular changes in adjacent, histologically normal FHOOVWKHVRFDOOHG³¿HOGHIIHFW´7KLV¿HOGHIIHFWPD\SUHGLVSRVHWKH
Vol. 179, No. 4, Supplement, Tuesday, May 20, 2008
normal cells to the development of frank malignancy. We sought to determine if normal epithelium from prostates harboring prostatic adenocarcinoma harbor changes in gene expression suggestive of SUHPDOLJQDQF\RU¿HOGHIIHFW METHODS: Prostate needle biopsies from 15 men with high grade (Gleason 8-10) prostate cancer and 15 age- and BMI-matched FRQWUROVZHUHLGHQWL¿HGIURPDSRRORIELRSVLHVFROOHFWHGIURP!PHQ since 2003. Normal epithelia from each patient were isolated via laser FDSWXUHPLFURGLVVHFWLRQ /&0 RIIUHVKIUR]HQELRSV\WLVVXH%LRSVLHV were reviewed by a pathologist prior to LCM to ensure only normal HSLWKHOLDZDVFDSWXUHG51$ZDVLVRODWHGIURPHDFKVDPSOHDPSOL¿HG WKHQ XWLOL]HG IRU PLFURDUUD\ DQDO\VLV XVLQJ WKH$JLOHQW +XPDQ [. DUUD\ SODWIRUP7KH GDWD ZHUH DQDO\]HG E\ SDLUHG WZRVDPSOH7WHVW XVLQJ 6LJQL¿FDQFH$QDO\VLV RI 0LFURDUUD\V 6$0 4XDQWLWDWLYH 3&5 (qPCR) was also used to determine the expression of various genes of LQWHUHVWLGHQWL¿HGE\PLFURDUUD\DQDO\VLV RESULTS: One control patient had Gleason 7 prostate cancer on repeat biopsy during the data analysis period and thus was excluded. The remaining 14 control and 15 cancer samples were included in the ¿QDODQDO\VLV0LFURDUUD\DQDO\VLVUHYHDOHGWKDWWKHVHWZRJURXSVKDG very similar patterns of gene expression overall. SAM analysis found VLJQL¿FDQW T YDOXHV T LQ JHQHV SUHYLRXVO\ DVVRFLDWHG ZLWK prostate cancer, namely FOLH1/PSMA and SSTR1. We examined RWKHUSURVWDWHFDQFHUDVVRFLDWHGJHQHVE\T3&5DQGIRXQGVLJQL¿FDQW differential expression changes in ERG, HOXC4, HOXC5 and MME, although other cancer markers (HPN, AMACR, FASN, SPOCK1, MYO6, 73'(=+ ZHUHQRWGLIIHUHQWEHWZHHQWKHWZRJURXSV,QWHUHVWLQJO\ the elevated ERG expression in the normal epithelia adjacent to prostate cancer did not appear to be associated with the TMPRSS2:ERG fusion with the exception of one patient. &21&/86,216 2YHUDOOJHQHH[SUHVVLRQSUR¿OHVEHWZHHQ normal prostatic epithelia of patients with and without prostate cancer are very similar. However, several of the differentially expressed genes found in this study have been implicated in prostate cancer progression and prognosis in prior studies, and thus may represent the earliest events in prostate carcinogenesis. Source of Funding: NIH/NIDDK.
1344 DEREGULATION OF FOXO3A CONTRIBUTES TO PROSTATE CANCER PROGRESSION Sanjeev Shukla*, Gregory T MacLennan, Sanjay Gupta. Cleveland, OH. INTRODUCTION AND OBJECTIVE: Forkhead box (Fox) proteins are a super-family of evolutionary conserved transcriptional regulators, which control a wide spectrum of biological processes. The IDPLO\LVFKDUDFWHUL]HGE\DFRQVHUYHG'1$ELQGLQJGRPDLQFRPSULVLQJ PRUHWKDQPHPEHUVLQKXPDQVFODVVL¿HGIURP)2;$WR)2;5 on the basis of sequence similarity. In particular, FOX subfamily O family has been implicated in the regulation of cell cycle and apoptosis and are the major substrates for the protein kinase B/Akt. Earlier we demonstrated the involvement of Akt/PKB during prostate cancer SURJUHVVLRQ KRZHYHUWKHUROHRI)2;2IDPLO\LQSURVWDWHWXPRULJHQHVLV has not been elucidated. 0(7+2'6 7KHH[SUHVVLRQRIPHPEHUVRI)2;2IDPLO\YL] FOXO1A, FOXO3A, FOXO4 were examined in human prostate cancer specimens of various Gleason grades, various human prostate cancer cells and transgenic adenocarcinoma of the mouse prostate (TRAMP) model representing various stages of disease progression along with non-tumorigenic counterparts. The interactions between FOXO, Akt/ PKB and 14-3-3 chaperone and associated signaling were determined by immunoprecipitation, immunoblotting and immunohistochemistry. RESULTS: Basal protein levels of FOXO1A was higher in androgen-refractory human prostate cancer cells, DU145 and PC-3 compared to androgen-responsive LNCaP and 22Rv1 cells, demonstrating its possible involvement in disease transition. No changes were observed in the protein levels of FOXO4 in all these cell lines. Interestingly, FOXO3A levels were downregulated in all human prostate cancer cell lines and redistributed in the cellular compartments. Compared WREHQLJQWLVVXHV)2;2$DFFXPXODWLRQZDVVLJQL¿FDQWO\KLJKHULQWKH cytosolic fraction than in the nuclear fraction and this difference was
Vol. 179, No. 4, Supplement, Tuesday, May 20, 2008
highest in high-grade tumor specimens. Similar observations were noted in TRAMP mice prostates where increased cytoplasmic FOXO3A levels correlated with phosphorylation at Ser253. The interactions between )2;2$ DQG$NW ZDV FRQ¿UPHG DIWHU LPPXQRSUHFLSLWDWLRQ RI HLWKHU proteins and increased binding with 14-3-3 and its accumulation in the cytoplasm of TRAMP mice prostates, compared to non-transgenic littermates at 20-28 weeks of age. Furthermore, decreased FOXO3A levels correlated with downregulation of the basal levels of p21/WAF1, 0Q62'DQG&X=Q62'LQWKHSURVWDWHVRI75$03PLFH &21&/86,216 7KHVH ¿QGLQJV GHPRQVWUDWH WKDW WXPRU associated reductions and redistribution of FOXO3A and FOXO1A proteins are frequent events in the etiology of prostate cancer. Source of Funding: USPHS grants NIH RO1CA10851, and The James & Eilleen Dicke Research Endowment funds.
1345 RELATIVE FREQUENCIES OF ETV1 AND ERG OVEREXPRESSION IN PROSTATE CANCER Ying Hu*, Lakshmi Ravindranath, Bungo Furusato, Chen Sun, Albert Dobi, Isabell A Sesterhenn, David G McLeod, Gyorgy Petrovics, Shiv Srivastava. Rockville, MD, and Washington, DC. INTRODUCTION AND OBJECTIVE: Recent studies have established that high proportion (>70%) of prostate cancers (CaP) harbor overexpression of the ETS related genes (ERG, ETV1 or ETV4) due to fusion of an androgen receptor regulated promoter element of the TMPRSS2 gene to the protein coding sequence of the ETS related genes. While TMPRSS2-ERG fusions are the most common in CaP, a recent report showed the fusion of distinct regulatory sequences to ETV1 in some prostate tumors overexpressing ETV1. The frequency of ETV1 alterations is not well understood in CaP. Here we have performed comparative evaluations of the ERG and ETV1 in CaP. 0(7+2'6 :HKDYHDQDO\]HGWKHTXDQWLWDWLYHH[SUHVVLRQ of ETV1 in benign and tumor cells laser capture microdissected from SULPDU\ SURVWDWH WXPRUV RI SDWLHQWV WKDW ZHUH DOUHDG\ DQDO\]HG for the quantitative expression of ERG, as well as TMPRSS2-ERG fusion. 5(68/76 )RUW\ ¿YH RI SDWLHQWV ZLWK QR GHWHFWDEOH TMPRSS2-ERGIXVLRQLQWKHLUWXPRUVZHUHDQDO\]HGIRUTXDQWLWDWLYH expression of ETV1 in paired tumor and benign cells. Four of 45 patients UHYHDOHGVLJQL¿FDQWRYHUH[SUHVVLRQRI ETV1 in tumor cells in comparison to matched benign cells. None of the recently reported ETV1 fusions were detected suggesting for the presence of new fusions or other mechanism of ETV1 overexpression in these tumors. CONCLUSIONS: In comparison to the ERG overxpression, ETV1 overxpression is detectable in a very small subset of prostate tumors. In order to more accurately assess the overall frequency of ETS related gene alterations in CaP, careful evaluations of ERG, ETV1 and other ETS factors in the same cohort of patients is warranted. Source of Funding: CPDR, NIH.
1346 METHYLATION OF GSTP1, RARB AND RASSF1A IN FREE CIRCULATING DNA AND BUFFY COAT DNA IN PROSTATE CANCER PATIENTS AND CONTROLS Rakesh Singal*, Kavitha Ramachandran, Isildinha Reis, Merce Jorda, Murugesan Manoharan. Miami, FL. INTRODUCTION AND OBJECTIVE: Prostate cancer is one of the most frequently diagnosed neoplasm and the second leading cause of cancer related mortality in men in the United States. The FXUUHQWO\XVHGGHWHFWLRQPHWKRGV SURVWDWHVSHFL¿FDQWLJHQDQGGLJLWDO UHFWDO H[DPLQDWLRQ KDYH OLPLWHG VSHFL¿FLW\ DQG VHQVLWLYLW\$OWKRXJK not presently used in routine clinical settings, DNA based molecular WXPRU PDUNHUV KDYH LQFUHDVHG VHQVLWLYLW\ DQG VSHFL¿FLW\ RI WXPRU detection. Previous studies have used DNA from plasma, serum and buffycoat to detect tumor-derived methylation changes as potential molecular markers for cancer diagnosis. In this study, we compared gene methylation patterns in serum and buffycoat of prostate cancer patients and patients with benign prostatic conditions. METHODS: DNA extracted from serum and buffycoat of prostate cancer patients and patients with benign prostatic conditions
THE JOURNAL OF UROLOGY®
461
ZHUH ELVXOILWH WUHDWHG DQG DQDO\]HG XVLQJ PHWK\ODWLRQVSHFLILF polymerase chain reaction (MSPCR). Age-adjusted odds ratios (OR) and corresponding p-values are reported. RESULTS: serum, higher risk of prostate cancer was associated with methylation of the following genes GSTP1 (OR=3.77, p=0.080), RARB (OR=6.59, p=0.094), RASSF1A (OR= 13.10, p=0.020). When methylation of the same genes in DNA from buffycoat was DQDO\]HGZLWKH[FHSWLRQRI5$5%RGGVUDWLRHVWLPDWHVEHFDPHVPDOOHU though the direction of the association did not change: GSTP1 (OR=1.49, p=0.506), RARB (OR=6.45, p=0.010), RASSF1A (OR= 5.84, p=0.014). We calculated the methylation index ( MI) based on the GSTP1, RARB and RASSF1A using serum only, buffycoat only and both (serum and/ or buffycoat). Age-adjusted OR for MI1+ vs. MI=0 was 4.417 for serum only (p=0.017), 2.252 for buffycoat only (p=0.146) and 3.279 for serum and/or buffycoat (p=0.03). CONCLUSIONS: Methylation changes derived from serum are more discriminatory between prostate cancer patients and controls as compared to methylation changes derived from buffycoat.Moreover, evaluation of methylation of set of genes may be a better predictor of tumor than analysis of a single gene. Source of Funding: None
1347 ECTOPIC EXPRESSION OF MKK4/JNKK1 CONTROLS PROSTATE CANCER METASTATIC COLONIZATION BY CONTROLLING GROWTH OF DISSEMINATED CANCER CELLS AT SECONDARY SITES Russell Z Szmulewitz*, Tamara Lotan, Jennifer Taylor, Shaheena Khan, Nkechiyere Nwani, Carrie Rinker-Schaeffer. Chicago, IL, and Baltimore, MD. INTRODUCTION AND OBJECTIVE: The process by which disseminated prostate cancer cells persist as dormant lesions and then initiate growth is largely unknown. Metastasis suppressor proteins are helping to dissect the molecular mechanisms regulating this clinically important event. MKK4/JNKK1 is a stress-activated signaling protein which is a metastasis suppressor in experimental models of prostate and ovarian cancers. Ectopic expression of the MKK4/JNKK1 protein in AT6.1 rat prostate cancer cells reduced the number of spontaneous metastases in the lungs by 90% and increased survival by 63%. %LRFKHPLFDOVWXGLHVVKRZHGWKDWWKH0..-1..NLQDVHLVVSHFL¿FDOO\ activated in disseminated cells, supporting a role for context-dependent activation of the protein within the microenviroment of the metastatic site. We are now examining the cellular mechanism of MKK4/JNKK1PHGLDWHGVXSSUHVVLRQRIPHWDVWDWLFFRORQL]DWLRQ METHODS: Spontaneous and experimental (using tail vein LQMHFWLRQVRIFDQFHUFHOOV PHWDVWDVLVDVVD\VLQLPPXQRGL¿FLHQWPLFH using the AT6.1 prostate cancer model were employed. The numbers RI FHOOV SUHVHQW LQ WKH OXQJ DW VSHFL¿F WLPH SRLQWV GXULQJ PHWDVWDWLF FRORQL]DWLRQZDVGHWHUPLQHGXVLQJTXDQWLWDWLYHUHDOWLPH3&5 T573&5 ZLWKPRXVHDQGUDWVSHFL¿FSULPHUV7KHSHUFHQWDJHRIGLVVHPLQDWHG cells undergoing apoptosis was determined by TUNEL, cleaved caspase 3 and morphologic changes. Proliferation was assessed using phosphohistone-H3 and BrdU incorporation. RESULTS: qRT-PCR showed similar numbers of AT6.1-control and AT6.1-MKK4/JNKK1 lodge within the lung during spontaneous metastasis. Immunohistochemical studies found no increase in the percentage of cells undergoing apoptosis. Experimental metastasis assays are now being employed to further dissect the proliferative events, and preliminary studies show that MKK4/ JNKK1 does indeed suppress experimental metastasis. CONCLUSIONS: MKK4/JNKK1 suppresses metastasis E\ LQKLELWLQJ PHWDVWDWLF FRORQL]DWLRQ 2XU SUHOLPLQDU\ VWXGLHV LQGLFDWH that this is due to decreased cell proliferation and not decreased number of cells lodging at secondary sites or increased numbers of cell undergoing apoptosis. Determining how MKK4/JNKK1 activation regulates proliferation could provide new clues for controlling the growth of disseminated cancer cells at metastatic sites, and present new therapeutic considerations for men with metastatic prostate cancer. Source of Funding: NIH RO1 CA 89569, W81XWH-06-1-0041, Arthur Foundation, University of Chicago RESCUE Fund.