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MP99-05 ELL2 REGULATES DNA DOUBLE-STRAND BREAK REPAIR IN PROSTATE CANCER CELLS
THE JOURNAL OF UROLOGYâ
Vol. 197, No. 4S, Supplement, Tuesday, May 16, 2017
exploited this link to identify actionable targets by performing a shRNA genomic screen in obese and lean mice targeting the entire kinome. Our functional screen identified multiple kinases, which appear to be essential for obesity-driven PC growth including kinases previously implicated in PC and others not previously studied such as Right Open Reading Frame Kinase 2 (RIOK2). METHODS: LAPC-4 cells were inoculated with an shRNA library of ~5000 lentivirus targeting 513 kinases. 5x106 cells (~1,000 cells per shRNA) were grafted to chronically obese mice. Tumors were established to ~200 mm3 and a portion collected for reference. Remaining mice were randomized to continue on ad lib WD or 25% CR diet. Genome-integrated shRNA inserts were amplified using nested barcoded primers and sequenced using Illumina Hi-Seq 2000 and quantified. A virtual screen based on a RIOK2 homology model generated using MODELLER based on two RIOK2 crystal structures. Global gene expression analysis of RNA from scramble control and RIOK2 knockdown with two shRNAs in 22RV1 cells was conducted with Affymetrix U133A Plus Array. RESULTS: RIOK2 expression correlates with Gleason grade in radical prostatectomy tissue and RIOK2 kinase activity is elevated in metastatic vs localized PCs. ENCODE ChIP-seq data shows Androgen Receptor and Myc bind to the RIOK2 promoter and regulate expression. Targeting RIOK2, via newly identified small molecule inhibitors reduces cell viability and soft agar colony growth. Gene set enrichment analysis of RIOK2 depleted PC cells showed reduction of cell cycle, adipogenesis, EMT and cancer stem cell genes. RIOK2 also regulates Neuropeptide Y2 Receptor (NYP2R), which is part of the NPY obesogenic signaling axis that correlates with obesity and worse PC outcomes. CONCLUSIONS: Our in vivo screen highlighted RIOK2 as an actionable PC target in obese hosts. Targeting RIOK2, pharmacologically with our lead compounds or genetically, drastically reduces PC cell viability. RIOK2 may regulate NPY2R expression, which when coupled with elevated NPY in both obese hosts and PCs can amplify NPY protumorigenic signaling. Source of Funding: The Stewart Rahr e Prostate Cancer Foundation Young Investigator Award and NIH Research Supplement to Promote Diversity in Health-Related Research from the NCI 3R01-CA125618-08S1 (EM) as well as NCI awards CA131235 and 5K24CA160653-03 (SF)
MP99-04 TRUNCATED-CRMP4 BY CALPAIN-2 SUPPRESSES CRMP4 TO PROMOTE METASTASIS OF PROSTATE CANCER VIA PROMOTER METHYLATION THROUGH E2F1/ NF-?B/DNMT1 SIGNALING Xin Gao*, Yunhua Mao, Zheng Chen, Jun Pang, Ke Li, Guangzhou, China, People’s Republic of INTRODUCTION AND OBJECTIVES: Metastasis is the primary cause of cancer-specific death in patients with prostate cancer (PCa). Previous studies identified promoter methylation is responsible for the repression of the tumor metastasis-suppressor gene collapsing response mediator protein-4 (CRMP4) in metastatic PCa. However, the underlying mechanisms for promoter methylation remain unknown. METHODS: In this study, calpain-2 expression in prostatic benign and cancer tissues was determined using immunohistochemistry (IHC). The effects of truncated-CRMP4 by calpain-2 in migration and invasion of PCa cells and the underlying mechanisms were explored. The role of nuclear factor-kappaB (NF-kB) in CRMP4 promoter methylation was evaluated. In addition, the downstream signaling regulated by CRMP4 were determined. RESULTS: Calpain-2 was differentially upregulated in metastatic PCa Calpain-2 was differentially upregulated in metastatic PCa
e1321
tissues. N-terminally truncated-CRMP4 by calpain-2 enhanced the ability of migration and invasion via nuclear translocation and subsequently activation of E2F1-mediated DNA methyltransferase 1 (DNMT1) expression. In addition, NF-kB RelA/p65 recruited DNMT1 to directly bind to and methylate the CRMP4 promoter in which Serine276 phosphorylation of p65 was essential. Furthermore, CRMP4 CRMP4 exhibited anti-metastatic function by inhibiting the expression of vascular endothelial growth factor C (VEGFC) via Sema3B- Neuropilin2 signaling. CONCLUSIONS: Calpain-mediated cleavage of CRMP4 promotes PCa metastasis by suppression of CRMP4 transcription via nuclear translocation of N-terminally truncated fragment and subsequent activation of E2F1/NF-kB/DNMT1 signaling which in turn enhanced CRMP4 promoter hypermethylation. Targeting re-expression of CRMP4 may be of great significance in the treatment of patients with metastatic PCa.
Source of Funding: This work was supported by National Natural Science Foundation of China (81372728, 81572503).
MP99-05 ELL2 REGULATES DNA DOUBLE-STRAND BREAK REPAIR IN PROSTATE CANCER CELLS Yachen zang, Yibin Zhou*, Leizhen Wei, Joel B. Nelson, Lan LI, Pittsburgh, PA; Boxin xue, Yuxi Shan, Suzhou, China, People’s Republic of; Zhou Wang, Pittsburgh, PA INTRODUCTION AND OBJECTIVES: Androgens are known to protect prostate cancer cells from DNA damage. Recent studies showed regulation of DNA repair genes by androgen receptor (AR) signaling in prostate cancers. We recently reported that androgenregulated protein and potential tumor suppressor ELL2-associated factor 2 (EAF2) can enhance DNA repair through Ku70/Ku80 in the prostate. ELL2 (elongation factor, RNA polymerase II, 2), a component of the super elongation complex (SEC), is an important factor for RNA Pol II transiting from promoter-proximal paused state into elongation state. ELL2 is also regulated by androgens and frequently down-regulated in prostate cancer. These observations led to our hypothesis that ELL2 can regulate DNA repair through Ku70/Ku80 in prostate cancer cells. METHODS: Prostate cancer cells, in the presence or absence of siRNA of ELL2, were treated with ?-irradiation or doxorubicin and then collected for detecting the level of DNA damage marker ?H2ax or neutral comet assay. Nonhomologous end-joining (NHEJ) and homologous recombination (HR) assays were used to test the role of ELL2 in these two double-strand break (DSB) repair pathways. Co-immunoprecipitation was used to determine the interaction between ELL2 and NHEJ pathway proteins Ku70 and Ku80. We examined the effect of ELL2 knockdown on Ku70 and Ku80 recruitment in response to laser microirradiation. RESULTS: Knockdown of ELL2 sensitized prostate cancer cells to DNA damage and overexpression of ELL2 protected prostate cancer cells from DNA damage. Knockdown of ELL2 impaired NHEJ repair but not HR. Transfected ELL2 co-immunoprecipitated with both Ku70 and Ku80 proteins. ELL2 could binds to and co-accumulated with Ku70/Ku80 proteins at sites of DNA damage. Knockdown of ELL2 dramatically/significantl inhibited Ku70 and Ku80 accumulation and
THE JOURNAL OF UROLOGYâ
e1322
retention at DSB sites in prostate cancer cells, and also impaired recruitment of Ku70/Ku80 to DSB sites. The impaired recruitment of Ku70 and Ku80 proteins to DNA damage sites upon ELL2 knockdown was rescued by re-expression of an ELL2 transgene insensitive to siELL2. CONCLUSIONS: This study suggests that ELL2 is an important factor mediating androgen protection of DNA damage via Ku70/Ku80 in prostate cancer cells. Source of Funding: 9R01CA186780 and 1P50CA180995
MP99-06 HIGH FAT DIET-INDUCED INFLAMMATION ACCELERATES TUMOR PROGRESSION IN MICE MODEL FOR PROSTATE CANCER Takuji Hayashi*, Kazutoshi Fujita, Yu Ishizuya, Cong Wang, Yoshiyuki Yamamoto, Toshiro Kinouchi, Kyosuke Matsuzaki, Norihiko Kawamura, Atsunari Kawashima, Akira Nagahara, Takeshi Ujike, Motohide Uemura, Satoshi Nojima, Eiichi Morii, Norio Nonomura, Suita, Japan INTRODUCTION AND OBJECTIVES: Inflammation could be associated with progression of prostate cancer. High fat diet (HFD) causes obesity and systemic inflammation, and might be associated with progression of prostate cancer. The aim of this study is to elucidate how HFD affects tumor progression and local immune cells using model mice for prostate cancer. METHODS: HFD or control diet (CD) had been administered to normal mice and model mice until 22 weeks old. Tumor progression was evaluated by prostate weights, H&E staining, and Ki67 staining. The fractions of immune cells in the prostatic tissues were assessed by flow cytometry. We compared these factors of HFD-fed mice to CD-fed mice, and evaluated the changes of tumor progression and immune cells after administration of celecoxib (8mg/kg/day) to HFD-fed and CD-fed model mice. RESULTS: Prostate weights were significantly increased in HFD-fed model mice compared to CD-fed model mice (0.72 0.57g vs 0.30 0.14g, p ¼0.029), while there were no significant changes in between HFD-fed and CD-fed normal mice. Prostate cancer was pathologically more invasive, and the ratio of Ki67-positive cells to tumor cells of these areas was significantly increased in HFD-fed model mice compared to CD-fed model mice (30.0 13.8% vs 7.8 2.9%, p ¼0.002). The fraction of Myeloid-Derived Suppressor Cells (MDSCs) was significantly increased in HFD-fed model mice compared to CD-fed model mice (p ¼0.044). The M2/M1 macrophage ratio was significantly increased in HFD-fed compared to CD-fed model mice (p ¼0.011), while the ratio was significantly decreased in HFD-fed compared to CD-fed normal mice (p ¼0.037). Administration of celecoxib to HFD-fed model mice significantly decreased the prostate weights (0.28 0.10g, p ¼0.040) and the ratio of Ki67-positive cells (8.3 5.6%, p ¼0.005), and also decreased the fraction of MDSCs significantly (p ¼0.035) and the M2/M1 ratio (p ¼0.114). Administration of celecoxib to CD-fed model mice did not decrease the prostate weights. CONCLUSIONS: HFD-induced pro-tumor changes of immune cells could accelerate tumor progression of prostate cancer, which was suppressed by celecoxib. Inflammation could be one of the key regulators for progression of prostate cancer. Source of Funding: none
Vol. 197, No. 4S, Supplement, Tuesday, May 16, 2017
MP99-07 AP4 PROMOTES PROLIFERATION AND METASTASIS OF CASTRATION-RESISTANT PROSTATE CANCER THROUGH BINDING AND UP-REGULATION OF L-PLASTIN Changhao Chen*, TIanxin Lin, Guangzhou, China, People’s Republic of; Thomas B Lam, Aberdeen, United Kingdom; Yue Zhao, Jian Huang, Guangzhou, China, People’s Republic of INTRODUCTION AND OBJECTIVES: The transition from androgen-dependent to castration-resistant prostate cancer (CRPC) is a lethal event of uncertain molecular etiology. The aim of this study was to identify the mechanism that AP4 promotes the proliferation and metastasis of CRPC by upreguating of L-plastin. METHODS: A total of 136 paired PCa and adjacent normal tissues were collected from patients who underwent prostatectomy between 2005 and 2015. The univariate and multivariate Cox regression analyses showed that AP4 expression was independent prognostic factor in patients with prostate cancer (PCa). The EMSA, supershift assays and CHIP-qPCR experiments noted that AP4 directly binds to L-plastin promoter. The in vitro and in vivo experiments were performed. RESULTS: Previously, we reported that L-plastin is involved in the metastasis of PCa and up-regulated by androgen. Recently, we found that L-plastin is activated even after androgen deprivation, suggesting that androgen-independent factors might regulate its expression. Here, we noted that an androgen-independent factor, which locates in the area close to the transcription initiation site (-216 to +118) of L-plastin promoter, might facilitate the up-regulation of L-plastin. AP4 was then identified as a key transcription activator, which directly binds to L-plastin promoter. The microarray analysis noted that L-plastin is the differentially expressed downstream target gene of AP4. Furthermore, we demonstrated that AP4 upregulated L-plastin expression, which promotes the proliferation and metastatic of CRPC in vitro and in vivo. Importantly, AP4 level was increased in CRPC tissues compared with ASPC. Overexpression of AP4 was significantly correlated with poor survival, Gleason score over 7 and lymph node metastasis in a large cohort of PCa tissues (n¼136). CONCLUSIONS: Our study characterizes a new mechanism in CRPC, in which AP4 increases the progression of CRPC through binding L-plastin promoter. Strategies designed to target AP4 may provide novel therapeutic agents for the management of CRPC.
Vol. 197, No. 4S, Supplement, Tuesday, May 16, 2017
exploited this link to identify actionable targets by performing a shRNA genomic screen in obese and lean mice targeting the entire kinome. Our functional screen identified multiple kinases, which appear to be essential for obesity-driven PC growth including kinases previously implicated in PC and others not previously studied such as Right Open Reading Frame Kinase 2 (RIOK2). METHODS: LAPC-4 cells were inoculated with an shRNA library of ~5000 lentivirus targeting 513 kinases. 5x106 cells (~1,000 cells per shRNA) were grafted to chronically obese mice. Tumors were established to ~200 mm3 and a portion collected for reference. Remaining mice were randomized to continue on ad lib WD or 25% CR diet. Genome-integrated shRNA inserts were amplified using nested barcoded primers and sequenced using Illumina Hi-Seq 2000 and quantified. A virtual screen based on a RIOK2 homology model generated using MODELLER based on two RIOK2 crystal structures. Global gene expression analysis of RNA from scramble control and RIOK2 knockdown with two shRNAs in 22RV1 cells was conducted with Affymetrix U133A Plus Array. RESULTS: RIOK2 expression correlates with Gleason grade in radical prostatectomy tissue and RIOK2 kinase activity is elevated in metastatic vs localized PCs. ENCODE ChIP-seq data shows Androgen Receptor and Myc bind to the RIOK2 promoter and regulate expression. Targeting RIOK2, via newly identified small molecule inhibitors reduces cell viability and soft agar colony growth. Gene set enrichment analysis of RIOK2 depleted PC cells showed reduction of cell cycle, adipogenesis, EMT and cancer stem cell genes. RIOK2 also regulates Neuropeptide Y2 Receptor (NYP2R), which is part of the NPY obesogenic signaling axis that correlates with obesity and worse PC outcomes. CONCLUSIONS: Our in vivo screen highlighted RIOK2 as an actionable PC target in obese hosts. Targeting RIOK2, pharmacologically with our lead compounds or genetically, drastically reduces PC cell viability. RIOK2 may regulate NPY2R expression, which when coupled with elevated NPY in both obese hosts and PCs can amplify NPY protumorigenic signaling. Source of Funding: The Stewart Rahr e Prostate Cancer Foundation Young Investigator Award and NIH Research Supplement to Promote Diversity in Health-Related Research from the NCI 3R01-CA125618-08S1 (EM) as well as NCI awards CA131235 and 5K24CA160653-03 (SF)
MP99-04 TRUNCATED-CRMP4 BY CALPAIN-2 SUPPRESSES CRMP4 TO PROMOTE METASTASIS OF PROSTATE CANCER VIA PROMOTER METHYLATION THROUGH E2F1/ NF-?B/DNMT1 SIGNALING Xin Gao*, Yunhua Mao, Zheng Chen, Jun Pang, Ke Li, Guangzhou, China, People’s Republic of INTRODUCTION AND OBJECTIVES: Metastasis is the primary cause of cancer-specific death in patients with prostate cancer (PCa). Previous studies identified promoter methylation is responsible for the repression of the tumor metastasis-suppressor gene collapsing response mediator protein-4 (CRMP4) in metastatic PCa. However, the underlying mechanisms for promoter methylation remain unknown. METHODS: In this study, calpain-2 expression in prostatic benign and cancer tissues was determined using immunohistochemistry (IHC). The effects of truncated-CRMP4 by calpain-2 in migration and invasion of PCa cells and the underlying mechanisms were explored. The role of nuclear factor-kappaB (NF-kB) in CRMP4 promoter methylation was evaluated. In addition, the downstream signaling regulated by CRMP4 were determined. RESULTS: Calpain-2 was differentially upregulated in metastatic PCa Calpain-2 was differentially upregulated in metastatic PCa
e1321
tissues. N-terminally truncated-CRMP4 by calpain-2 enhanced the ability of migration and invasion via nuclear translocation and subsequently activation of E2F1-mediated DNA methyltransferase 1 (DNMT1) expression. In addition, NF-kB RelA/p65 recruited DNMT1 to directly bind to and methylate the CRMP4 promoter in which Serine276 phosphorylation of p65 was essential. Furthermore, CRMP4 CRMP4 exhibited anti-metastatic function by inhibiting the expression of vascular endothelial growth factor C (VEGFC) via Sema3B- Neuropilin2 signaling. CONCLUSIONS: Calpain-mediated cleavage of CRMP4 promotes PCa metastasis by suppression of CRMP4 transcription via nuclear translocation of N-terminally truncated fragment and subsequent activation of E2F1/NF-kB/DNMT1 signaling which in turn enhanced CRMP4 promoter hypermethylation. Targeting re-expression of CRMP4 may be of great significance in the treatment of patients with metastatic PCa.
Source of Funding: This work was supported by National Natural Science Foundation of China (81372728, 81572503).
MP99-05 ELL2 REGULATES DNA DOUBLE-STRAND BREAK REPAIR IN PROSTATE CANCER CELLS Yachen zang, Yibin Zhou*, Leizhen Wei, Joel B. Nelson, Lan LI, Pittsburgh, PA; Boxin xue, Yuxi Shan, Suzhou, China, People’s Republic of; Zhou Wang, Pittsburgh, PA INTRODUCTION AND OBJECTIVES: Androgens are known to protect prostate cancer cells from DNA damage. Recent studies showed regulation of DNA repair genes by androgen receptor (AR) signaling in prostate cancers. We recently reported that androgenregulated protein and potential tumor suppressor ELL2-associated factor 2 (EAF2) can enhance DNA repair through Ku70/Ku80 in the prostate. ELL2 (elongation factor, RNA polymerase II, 2), a component of the super elongation complex (SEC), is an important factor for RNA Pol II transiting from promoter-proximal paused state into elongation state. ELL2 is also regulated by androgens and frequently down-regulated in prostate cancer. These observations led to our hypothesis that ELL2 can regulate DNA repair through Ku70/Ku80 in prostate cancer cells. METHODS: Prostate cancer cells, in the presence or absence of siRNA of ELL2, were treated with ?-irradiation or doxorubicin and then collected for detecting the level of DNA damage marker ?H2ax or neutral comet assay. Nonhomologous end-joining (NHEJ) and homologous recombination (HR) assays were used to test the role of ELL2 in these two double-strand break (DSB) repair pathways. Co-immunoprecipitation was used to determine the interaction between ELL2 and NHEJ pathway proteins Ku70 and Ku80. We examined the effect of ELL2 knockdown on Ku70 and Ku80 recruitment in response to laser microirradiation. RESULTS: Knockdown of ELL2 sensitized prostate cancer cells to DNA damage and overexpression of ELL2 protected prostate cancer cells from DNA damage. Knockdown of ELL2 impaired NHEJ repair but not HR. Transfected ELL2 co-immunoprecipitated with both Ku70 and Ku80 proteins. ELL2 could binds to and co-accumulated with Ku70/Ku80 proteins at sites of DNA damage. Knockdown of ELL2 dramatically/significantl inhibited Ku70 and Ku80 accumulation and
THE JOURNAL OF UROLOGYâ
e1322
retention at DSB sites in prostate cancer cells, and also impaired recruitment of Ku70/Ku80 to DSB sites. The impaired recruitment of Ku70 and Ku80 proteins to DNA damage sites upon ELL2 knockdown was rescued by re-expression of an ELL2 transgene insensitive to siELL2. CONCLUSIONS: This study suggests that ELL2 is an important factor mediating androgen protection of DNA damage via Ku70/Ku80 in prostate cancer cells. Source of Funding: 9R01CA186780 and 1P50CA180995
MP99-06 HIGH FAT DIET-INDUCED INFLAMMATION ACCELERATES TUMOR PROGRESSION IN MICE MODEL FOR PROSTATE CANCER Takuji Hayashi*, Kazutoshi Fujita, Yu Ishizuya, Cong Wang, Yoshiyuki Yamamoto, Toshiro Kinouchi, Kyosuke Matsuzaki, Norihiko Kawamura, Atsunari Kawashima, Akira Nagahara, Takeshi Ujike, Motohide Uemura, Satoshi Nojima, Eiichi Morii, Norio Nonomura, Suita, Japan INTRODUCTION AND OBJECTIVES: Inflammation could be associated with progression of prostate cancer. High fat diet (HFD) causes obesity and systemic inflammation, and might be associated with progression of prostate cancer. The aim of this study is to elucidate how HFD affects tumor progression and local immune cells using model mice for prostate cancer. METHODS: HFD or control diet (CD) had been administered to normal mice and model mice
Vol. 197, No. 4S, Supplement, Tuesday, May 16, 2017
MP99-07 AP4 PROMOTES PROLIFERATION AND METASTASIS OF CASTRATION-RESISTANT PROSTATE CANCER THROUGH BINDING AND UP-REGULATION OF L-PLASTIN Changhao Chen*, TIanxin Lin, Guangzhou, China, People’s Republic of; Thomas B Lam, Aberdeen, United Kingdom; Yue Zhao, Jian Huang, Guangzhou, China, People’s Republic of INTRODUCTION AND OBJECTIVES: The transition from androgen-dependent to castration-resistant prostate cancer (CRPC) is a lethal event of uncertain molecular etiology. The aim of this study was to identify the mechanism that AP4 promotes the proliferation and metastasis of CRPC by upreguating of L-plastin. METHODS: A total of 136 paired PCa and adjacent normal tissues were collected from patients who underwent prostatectomy between 2005 and 2015. The univariate and multivariate Cox regression analyses showed that AP4 expression was independent prognostic factor in patients with prostate cancer (PCa). The EMSA, supershift assays and CHIP-qPCR experiments noted that AP4 directly binds to L-plastin promoter. The in vitro and in vivo experiments were performed. RESULTS: Previously, we reported that L-plastin is involved in the metastasis of PCa and up-regulated by androgen. Recently, we found that L-plastin is activated even after androgen deprivation, suggesting that androgen-independent factors might regulate its expression. Here, we noted that an androgen-independent factor, which locates in the area close to the transcription initiation site (-216 to +118) of L-plastin promoter, might facilitate the up-regulation of L-plastin. AP4 was then identified as a key transcription activator, which directly binds to L-plastin promoter. The microarray analysis noted that L-plastin is the differentially expressed downstream target gene of AP4. Furthermore, we demonstrated that AP4 upregulated L-plastin expression, which promotes the proliferation and metastatic of CRPC in vitro and in vivo. Importantly, AP4 level was increased in CRPC tissues compared with ASPC. Overexpression of AP4 was significantly correlated with poor survival, Gleason score over 7 and lymph node metastasis in a large cohort of PCa tissues (n¼136). CONCLUSIONS: Our study characterizes a new mechanism in CRPC, in which AP4 increases the progression of CRPC through binding L-plastin promoter. Strategies designed to target AP4 may provide novel therapeutic agents for the management of CRPC.