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1315 EVEN-SKIPPED HOMEOBOX 1 (EVX1) IS FREQUENTLY HYPERMETHYLATED IN PROSTATE CANCER AND PREDICTS PSA RECURRENCE
Vol. 187, No. 4S, Supplement, Monday, May 21, 2012
tome RNA-seq, and Sanger sequencing from multiple independent cohorts of prostate cancer comprising over 400 patients. ERG rearrangement and PTEN deletion status were determined by FISH, somatic copy number aberrations were analyzed by Affymetrix 6.0 SNP arrays, and the association of SPOP mutation with these molecular characteristics was determined. RESULTS: SPOP was the most frequently mutated gene in whole exome sequencing of 111 primary prostate cancers, with missense mutations in 13% of cases. Recurrent missense mutations were found in SPOP in 6-15% of human prostate cancers in multiple independent cohorts. Mutations were found exclusively in the structurallydefined substrate-binding cleft of SPOP; structural analysis suggests that these mutations will affect SPOP function by disrupting SPOPsubstrate interaction. Importantly, all SPOP mutations occurred in primary tumors that lacked ERG rearrangement and inactivation of the p53 and PTEN tumor suppressors. Furthermore, these SPOP mutant cancers displayed characteristic somatic copy number aberrations, including deletions at 5q21 and in the 6q14-22 region. CONCLUSIONS: Recurrent missense mutations occur in substrate-binding residues of SPOP in 6-15% of human prostate cancer. SPOP mutant tumors lack ERG rearrangement, and lesions in the TP53 and PTEN genes, but harbor characteristic somatic copy number aberrations. These data suggest that mutation of SPOP is a driver lesion that may underlie a specific molecular class of prostate cancer. Source of Funding: Department of Defense Synergy Award PC101020 (FD, LG, MAR) and New Investigator Award PC094516 (FD), the National Cancer Institute, Early Detection Research Network U01CA111275, NCI EDRN (FD, AMC, MAR), and National Cancer Institute R01 CA125612 (FD, MAR). This work was supported by the Prostate Cancer Foundation.
1315 EVEN-SKIPPED HOMEOBOX 1 (EVX1) IS FREQUENTLY HYPERMETHYLATED IN PROSTATE CANCER AND PREDICTS PSA RECURRENCE Matthew Truong*, Bing Yang, Madison, WI; Yuya Kobayashi, James Brooks, Stanford, CA; David Jarrard, Madison, WI INTRODUCTION AND OBJECTIVES: DNA methylation is an important epigenetic mechanism of gene regulation that plays a role in prostate cancer (PCa) progression. Even-skipped homeobox 1 (Evx1), located at 7p15, contains several CpG islands and functions as a transcriptional repressor during embryogenesis. Given its role in the regulation of multiple genes, we postulated that methylation patterns might be altered during PCa progression. METHODS: DNA methylation mapping across 83 CpGs within the Evx1 promoter and exon 1 was performed using bisulfite sequencing on human prostate epithelial cells (HPEC) and PCa cell lines. A MethyLight probe was designed near the transcription start site (TSS) and its performance analyzed. It was then used for quantitation of methylation in 15 intermediate and 8 high-grade tumors, non-tumor associated prostate tissue, and normal spleen, liver, lung, and kidney. PC3, Du145, and PPC-1 PCa cell lines were treated with various doses of the demethylating agent 5-azacytidine (5-aza) and/or the histone deacetylase inhibitor trichostatin A (TSA) and the expression of several mRNA transcripts and variants was analyzed using quantitative realtime PCR (qPCR). In a separate validation cohort of 56 patients with long term follow-up, DNA methylation levels were assessed by microarray and methylation statistically compared to clinicopathologic features using a Kaplan Meier analysis. RESULTS: Evx1 contains a 700 bp CpG island spanning the TSS. Increased methylation of the Evx1 promoter was found in PCa cell lines (83-100%) compared to HPECs (0-47%) using bisulfite sequencing. Expression of Evx1 mRNA in PCa cell lines was induced after exposure to 5-aza and/or TSA. Quantitative analysis using MethyLight confirmed the hypermethylation of PCa cell lines, and was linear across
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a wide range of methylation levels. Evx1 was found to be significantly hypermethylated in high-grade tumor specimens compared to normal prostate (p⬍0.05). Using our validation set, increased methylation was demonstrated in 40 (71%) of tumor samples. Tumor hypermethylation was seen in 93% of patients with stage 3 disease compared to 64% of patients with stage 2 (p⬍0.05). Patients with hypermethylated tumors had a significantly worse recurrence-free survival compared to patients without hypermethylation (51% vs 90%; p⬍0.05). CONCLUSIONS: This is the first study to examine Evx1 methylation in any cancer and we demonstrate frequent hypermethylation in PCa. Evx1 promoter hypermethylation predicts biochemical recurrence following prostatectomy suggesting an important role in PCa progression. Source of Funding: 1) University of Wisconsin School of Medicine and Public Health’s Institute for Clinical and Translational Research-Shapiro Medical Student Research Award 2) RO1: 5RO1CA 097131
1316 FUNCTIONAL ANALYSIS OF THE MIRNA FAMILY LET-7 IN PROSTATE CANCER CELL LINES Maria Schubert*, Martin Spahn, Hubertus Riedmiller, Burkhard Kneitz, Wuerzburg, Germany INTRODUCTION AND OBJECTIVES: The microRNA (miRNA) family let-7 encodes for 9 different miRNAs and plays a pivotal role in the pathogenesis of some solid tumours, e.g. lung cancer. We previously found 2 members of the let-7 family, let-7b/c, to be downregulated in a large study cohort of high risk prostate cancer (PCa) patients. Let-7b down-regulation was associated with clinical failure (CF) and cancer related death. Moreover, let-7b expression was an independent predictor for CF. Aim of this study was to analyze the molecular mechanisms behind these findings. METHODS: We analyzed expression status of members of the let-7 family in PCa cell lines by qRT-PCR. We then transiently transfected androgen-dependent LNCaP cells with siRNA for androgen receptor (AR). After AR suppression expression of let-7 was compared to that in cells stimulated with DHT (AR up-regulation). We further transfected LNCaP and androgen-independent PC-3 cells with precursor and antigo let-7a/b/c and performed proliferation assays. Microarray experiments on mRNA isolated from LNCaP transfected with pre- and anti-let-7b were carried out, followed by association studies (qRT-PCR, West. Blot, luciferase assay). RESULTS: Let-7 expression was highest in androgen-dependent PCa cell lines. Furthermore, when AR-expression was suppressed by siRNA, let-7b/c expression was diminished. Contrary, upregulation of let-7b was seen in cells after stimulation with DHT (p⫽0.01). Let-7 over-expression was linked to lowest proliferation rate in LNCaP and PC-3 cells. Microarray experiment revealed members of the MAP-kinase pathway to be regulated after let-7b suppression. HMGA1, a non-histone protein was also significantly up-regulated in the array. Association studies suggest an inverse correlation of let-7b and HMGA1. CONCLUSIONS: Our results show that let-7 family members play an important role in cancer cell proliferation and that re-expression in PCa cells lead to anti-proliferative effects. Since we saw let-7b expression levels to be highest in androgen-dependent PCa cells and an up-regulation of let-7 after AR-stimulation, a certain androgendependency can be claimed. Let-7b plays an important role in the MAP-kinase pathway, whose increased activation is linked to cancer progression. HMGA-1, a nuclear protein with oncogenic properties, was shown to be contrarily expressed to let-7b, though active complementary sites still have to be found.
tome RNA-seq, and Sanger sequencing from multiple independent cohorts of prostate cancer comprising over 400 patients. ERG rearrangement and PTEN deletion status were determined by FISH, somatic copy number aberrations were analyzed by Affymetrix 6.0 SNP arrays, and the association of SPOP mutation with these molecular characteristics was determined. RESULTS: SPOP was the most frequently mutated gene in whole exome sequencing of 111 primary prostate cancers, with missense mutations in 13% of cases. Recurrent missense mutations were found in SPOP in 6-15% of human prostate cancers in multiple independent cohorts. Mutations were found exclusively in the structurallydefined substrate-binding cleft of SPOP; structural analysis suggests that these mutations will affect SPOP function by disrupting SPOPsubstrate interaction. Importantly, all SPOP mutations occurred in primary tumors that lacked ERG rearrangement and inactivation of the p53 and PTEN tumor suppressors. Furthermore, these SPOP mutant cancers displayed characteristic somatic copy number aberrations, including deletions at 5q21 and in the 6q14-22 region. CONCLUSIONS: Recurrent missense mutations occur in substrate-binding residues of SPOP in 6-15% of human prostate cancer. SPOP mutant tumors lack ERG rearrangement, and lesions in the TP53 and PTEN genes, but harbor characteristic somatic copy number aberrations. These data suggest that mutation of SPOP is a driver lesion that may underlie a specific molecular class of prostate cancer. Source of Funding: Department of Defense Synergy Award PC101020 (FD, LG, MAR) and New Investigator Award PC094516 (FD), the National Cancer Institute, Early Detection Research Network U01CA111275, NCI EDRN (FD, AMC, MAR), and National Cancer Institute R01 CA125612 (FD, MAR). This work was supported by the Prostate Cancer Foundation.
1315 EVEN-SKIPPED HOMEOBOX 1 (EVX1) IS FREQUENTLY HYPERMETHYLATED IN PROSTATE CANCER AND PREDICTS PSA RECURRENCE Matthew Truong*, Bing Yang, Madison, WI; Yuya Kobayashi, James Brooks, Stanford, CA; David Jarrard, Madison, WI INTRODUCTION AND OBJECTIVES: DNA methylation is an important epigenetic mechanism of gene regulation that plays a role in prostate cancer (PCa) progression. Even-skipped homeobox 1 (Evx1), located at 7p15, contains several CpG islands and functions as a transcriptional repressor during embryogenesis. Given its role in the regulation of multiple genes, we postulated that methylation patterns might be altered during PCa progression. METHODS: DNA methylation mapping across 83 CpGs within the Evx1 promoter and exon 1 was performed using bisulfite sequencing on human prostate epithelial cells (HPEC) and PCa cell lines. A MethyLight probe was designed near the transcription start site (TSS) and its performance analyzed. It was then used for quantitation of methylation in 15 intermediate and 8 high-grade tumors, non-tumor associated prostate tissue, and normal spleen, liver, lung, and kidney. PC3, Du145, and PPC-1 PCa cell lines were treated with various doses of the demethylating agent 5-azacytidine (5-aza) and/or the histone deacetylase inhibitor trichostatin A (TSA) and the expression of several mRNA transcripts and variants was analyzed using quantitative realtime PCR (qPCR). In a separate validation cohort of 56 patients with long term follow-up, DNA methylation levels were assessed by microarray and methylation statistically compared to clinicopathologic features using a Kaplan Meier analysis. RESULTS: Evx1 contains a 700 bp CpG island spanning the TSS. Increased methylation of the Evx1 promoter was found in PCa cell lines (83-100%) compared to HPECs (0-47%) using bisulfite sequencing. Expression of Evx1 mRNA in PCa cell lines was induced after exposure to 5-aza and/or TSA. Quantitative analysis using MethyLight confirmed the hypermethylation of PCa cell lines, and was linear across
THE JOURNAL OF UROLOGY姞
e533
a wide range of methylation levels. Evx1 was found to be significantly hypermethylated in high-grade tumor specimens compared to normal prostate (p⬍0.05). Using our validation set, increased methylation was demonstrated in 40 (71%) of tumor samples. Tumor hypermethylation was seen in 93% of patients with stage 3 disease compared to 64% of patients with stage 2 (p⬍0.05). Patients with hypermethylated tumors had a significantly worse recurrence-free survival compared to patients without hypermethylation (51% vs 90%; p⬍0.05). CONCLUSIONS: This is the first study to examine Evx1 methylation in any cancer and we demonstrate frequent hypermethylation in PCa. Evx1 promoter hypermethylation predicts biochemical recurrence following prostatectomy suggesting an important role in PCa progression. Source of Funding: 1) University of Wisconsin School of Medicine and Public Health’s Institute for Clinical and Translational Research-Shapiro Medical Student Research Award 2) RO1: 5RO1CA 097131
1316 FUNCTIONAL ANALYSIS OF THE MIRNA FAMILY LET-7 IN PROSTATE CANCER CELL LINES Maria Schubert*, Martin Spahn, Hubertus Riedmiller, Burkhard Kneitz, Wuerzburg, Germany INTRODUCTION AND OBJECTIVES: The microRNA (miRNA) family let-7 encodes for 9 different miRNAs and plays a pivotal role in the pathogenesis of some solid tumours, e.g. lung cancer. We previously found 2 members of the let-7 family, let-7b/c, to be downregulated in a large study cohort of high risk prostate cancer (PCa) patients. Let-7b down-regulation was associated with clinical failure (CF) and cancer related death. Moreover, let-7b expression was an independent predictor for CF. Aim of this study was to analyze the molecular mechanisms behind these findings. METHODS: We analyzed expression status of members of the let-7 family in PCa cell lines by qRT-PCR. We then transiently transfected androgen-dependent LNCaP cells with siRNA for androgen receptor (AR). After AR suppression expression of let-7 was compared to that in cells stimulated with DHT (AR up-regulation). We further transfected LNCaP and androgen-independent PC-3 cells with precursor and antigo let-7a/b/c and performed proliferation assays. Microarray experiments on mRNA isolated from LNCaP transfected with pre- and anti-let-7b were carried out, followed by association studies (qRT-PCR, West. Blot, luciferase assay). RESULTS: Let-7 expression was highest in androgen-dependent PCa cell lines. Furthermore, when AR-expression was suppressed by siRNA, let-7b/c expression was diminished. Contrary, upregulation of let-7b was seen in cells after stimulation with DHT (p⫽0.01). Let-7 over-expression was linked to lowest proliferation rate in LNCaP and PC-3 cells. Microarray experiment revealed members of the MAP-kinase pathway to be regulated after let-7b suppression. HMGA1, a non-histone protein was also significantly up-regulated in the array. Association studies suggest an inverse correlation of let-7b and HMGA1. CONCLUSIONS: Our results show that let-7 family members play an important role in cancer cell proliferation and that re-expression in PCa cells lead to anti-proliferative effects. Since we saw let-7b expression levels to be highest in androgen-dependent PCa cells and an up-regulation of let-7 after AR-stimulation, a certain androgendependency can be claimed. Let-7b plays an important role in the MAP-kinase pathway, whose increased activation is linked to cancer progression. HMGA-1, a nuclear protein with oncogenic properties, was shown to be contrarily expressed to let-7b, though active complementary sites still have to be found.