MP31-07 ESTROGEN RECEPTOR α REGULATES PROLIFERATION IN PTEN NULL PROSTATE CANCER
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transcription factor FOXA1, and HOX cofactors, PBX2 and PREP1 as novel binding partners of HOXB13, at least in androgen responsive prostate cancer cell lines. CONCLUSIONS: Differential expression of genes involved in steroid and polyamine metabolizing pathways might explain the ability of HOXB13 G84E to promote prostate carcinogenesis. Co-IP experi- ments demonstrate novel complex formation between HOXB13 and AR, FOXA1, PBX1, and PREP1. Further work is necessary to deter- mine if alterations in this complex formation as a result of HOXB13 mutations are responsible for the altered gene expression pat- terns observed. Source of Funding: Patrick C Walsh Prostate Cancer Research Fund MP31-04 ACSL4 IN PROSTATE CANCER GROWTH, INVASION AND HORMONAL RESISTANCE Xinyu Wu*, Yirong Li, Xinxin Du, Qinghu Ren, Max X. Kong, Jinhua wang, LingHang Wang, Yang Yang, Valerio Zhang, David Zhang, Fei Ye, Garrett Daniels, Fangming Deng, New york, NY; Jianjun Wei, Chicago, IL; Jonathan Melamed, Marie E. Monaco, Peng Lee, New york, NY INTRODUCTION AND OBJECTIVES: Previous studies have shown that the fatty acid biosynthetic enzymes are important in a variety of cancers including prostate cancer (PCa). However, the precise role played by dysregulated expression of lipid metabolic enzymes and altered lipid homeostasis in carcinogenesis remains to be establish- ed.The present study is to examine the role of the understudied lipid metabolic enzyme, long-chain fatty acyl-CoA synthetase 4 (ACSL4), in PCa. METHODS: LNCaP, LNCaP-AI, PC3 and C4-2B cells were utilized to assess the consequences of either increased or decreased levels of ACSL4 expression in cell proliferation, migration, invasion and apoptosis. Signal network proteins targeted by ACSL4 were analyzed by proteomic pathway array analysis. The effect of ACSL4 on tumor growth was examined in nude mice tumor xeno- graft models. RESULTS: Forced expression of ACSL4 in LNCaP cells pro- motes cell proliferation and invasion, while ACSL4 knockdown by siRNA in PCa cells including LNCaP-AI, C4-2B and PC3, reduces cell proliferation. These effects were observed in both the absence and presence of androgens and/or estrogens, hormone-free media, although to varying degrees. The growth enhancing effect of ACSL4 is confirmed in vivo using nude mice xenografts to compare the growth of LNCaP vector control with LNCaP-ACSL4 cells. Consistently, ACSL4 expression is increased in human PCa compared to benign epithelial cells. Meanwhile, there is a reciprocal inverse relationship between ACSL4 and the androgen receptor (AR), with decreased AR expression in LNCaP-ACSL4 compared to LNCaP cells and increased AR levels in LNCaP-AI cells with ACSL4 knockdown using siRNA. Conversely, ACSL4 levels are decreased in PC3 cells with AR overexpression. Interestingly, there is an un-coupling of the inverse relationship between AR and ACSL4 in LNCaP-AI cells. Consistently, ACSL4 is also increased in human castrate resistant prostate cancer. Importantly, ACSL4 expression induces resistance to Casodex as evidenced by flow and apoptosis assays. The resistance to Casodex is mediated by BAX and Caspase 8. CONCLUSIONS: Our study indicates that ACSL4 expression may serve as a biomarker with potential utility in the diagnosis and treatment of PCa. Source of Funding: NIH 1U01CA149556-01, DOD PCRP (PC080010 and PC11624) MP31-05 WITHDRAWN MUTUAL REGULATION BETWEEN RAF/MEK/ERK SIGNALING AND Y-BOX-BINDING PROTEIN-1 PROMOTES PROSTATE CANCER PROGRESSION Kenjiro Imada*, Masaki Shiota, Kenichi Kohashi, Fukuoka, Japan; Kentaro Kuroiwa, Miyazaki, Japan; YooHyun Song, Masaaki Sugimote, Yoshinao Oda, Seiji Naito, Fukuoka, Japan MP31-06 DIET INDUCED ALTERATION OF FATTY ACID SYNTHASE IS INVOLVED IN PROSTATE CANCER PROGRESSION Mingguo Huang*, Shintaro Narita, Norihiko Tsuchiya, Takamitsu Inoue, Shigeru Satoh, Hiroshi Nanjo, Takehiko Sasaki, Tomonori Habuchi, Akita, Japan INTRODUCTION AND OBJECTIVES: High-fat diet (HFD) or obesity is strongly suggested to influence prostate cancer (PCa) pro- gression, but the underlying mechanism is not well defined. Fatty acid synthase (FASN) is a cytosolic metabolic enzyme catalyzing de novo biosynthesis of fatty acid. METHODS: In the present study, we investigated the role of FASN on PCa progression in the LNCaP xenograft mouse fed HFD or low-fat diet (LFD). RESULTS: The tumor growth was increased in the HFD group than LFD group (p ¼ 0.025). The mRNA expression of FASN and SREBP-1, which encodes Sterol regulatory element-binding protein-1 (SREBP-1) were increased in the xenograft tumor by 1.8- and 2.1-fold, respectively (p < 0.05). The serum level of FASN was significantly lower in the HFD group than in the LFD group (p ¼ 0.026) and correlated inversely with the tumor volumes (total, r ¼ 0.642, p ¼ 0.022; HFD group, r ¼0.618, p ¼ 0.024; LFD group, r ¼0.439, p ¼ 0.154; respectively). Upregulation of P-AKT and P-ERK, and downregulation of P-AMPK were observed in xenograft tumors of the HFD group than LFD group. The extracellular release of FASN protein in vitro and AMPK activation was induced in the PCa cells by inhibition of the PI3K and MAPK signaling. In addition, pharmacological acti- vation of AMPK stimulated the extracellular FASN release along with the downregulation of FASN and SREBP-1 in the PCa cells in dose- dependent manner. Cell proliferation was significantly decreased in the PCa cells by inhibition of FASN, and the decreased effect was rescued by addition of palmitic acid. Clinically, imuunohistological evaluation of surgical specimens showed that the expression of FASN was markedly decreased in the PCa response to hormone and chemotherapy. CONCLUSIONS: In conclusion, HFD increased the FASN expression and inhibited the extracellular release of FASN, which may be one of important mechanism in HFD or obesity associated PCa progression. Source of Funding: none MP31-07 ESTROGEN RECEPTOR a REGULATES PROLIFERATION IN PTEN NULL PROSTATE CANCER Itsuhiro Takizawa*, Niigata, Japan; Mitchell Lawrence, Clayton, Australia; Helen Pearson, John Pedersen, Normand Pouliot, Australian Prostate, Cancer BioResource, Patrick Humbert, Melbourne, Australia; Luc Furic, Gail Risbridger, Clayton, Australia INTRODUCTION AND OBJECTIVES: High doses of estrogen, in combination with androgens, can initiate prostate cancer. This effect is attributed to the activity of the estrogen receptor a (ERa). Although it is well established that ERa stimulates proliferation via genomic and non-genomic activities in breast and ovarian cancer, relatively little is known about the functions of ERa in prostate cancer cells. We observed that ERa expression is up-regulated in high Gleason Score specimens of human prostate cancer. We also noted that the incidence e324 THE JOURNAL OF UROLOGY â Vol. 191, No. 4S, Supplement, Sunday, May 18, 2014
Transcript of MP31-07 ESTROGEN RECEPTOR α REGULATES PROLIFERATION IN PTEN NULL PROSTATE CANCER
e324 THE JOURNAL OF UROLOGY� Vol. 191, No. 4S, Supplement, Sunday, May 18, 2014
transcription factor FOXA1, and HOX cofactors, PBX2 and PREP1 asnovel binding partners of HOXB13, at least in androgen responsiveprostate cancer cell lines.
CONCLUSIONS: Differential expression of genes involved insteroid and polyamine metabolizing pathways might explain the abilityof HOXB13 G84E to promote prostate carcinogenesis. Co-IP experi-ments demonstrate novel complex formation between HOXB13 andAR, FOXA1, PBX1, and PREP1. Further work is necessary to deter-mine if alterations in this complex formation as a result of HOXB13mutations are responsible for the altered gene expression pat-terns observed.
Source of Funding:Patrick CWalsh Prostate Cancer ResearchFund
MP31-04ACSL4 IN PROSTATE CANCER GROWTH, INVASION ANDHORMONAL RESISTANCE
Xinyu Wu*, Yirong Li, Xinxin Du, Qinghu Ren, Max X. Kong,Jinhua wang, LingHang Wang, Yang Yang, Valerio Zhang,David Zhang, Fei Ye, Garrett Daniels, Fangming Deng, New york, NY;Jianjun Wei, Chicago, IL; Jonathan Melamed, Marie E. Monaco,Peng Lee, New york, NY
INTRODUCTION AND OBJECTIVES: Previous studies haveshown that the fatty acid biosynthetic enzymes are important in a varietyof cancers including prostate cancer (PCa). However, the precise roleplayed by dysregulated expression of lipid metabolic enzymes andaltered lipid homeostasis in carcinogenesis remains to be establish-ed.The present study is to examine the role of the understudied lipidmetabolic enzyme, long-chain fatty acyl-CoA synthetase 4 (ACSL4),in PCa.
METHODS: LNCaP, LNCaP-AI, PC3 and C4-2B cells wereutilized to assess the consequences of either increased ordecreased levels of ACSL4 expression in cell proliferation, migration,invasion and apoptosis. Signal network proteins targeted by ACSL4were analyzed by proteomic pathway array analysis. The effect ofACSL4 on tumor growth was examined in nude mice tumor xeno-graft models.
RESULTS: Forced expression of ACSL4 in LNCaP cells pro-motes cell proliferation and invasion, while ACSL4 knockdown bysiRNA in PCa cells including LNCaP-AI, C4-2B and PC3, reduces cellproliferation. These effects were observed in both the absence andpresence of androgens and/or estrogens, hormone-free media,although to varying degrees. The growth enhancing effect of ACSL4 isconfirmed in vivo using nude mice xenografts to compare the growth ofLNCaP vector control with LNCaP-ACSL4 cells. Consistently, ACSL4expression is increased in human PCa compared to benign epithelialcells. Meanwhile, there is a reciprocal inverse relationship betweenACSL4 and the androgen receptor (AR), with decreased AR expressionin LNCaP-ACSL4 compared to LNCaP cells and increased AR levels inLNCaP-AI cells with ACSL4 knockdown using siRNA. Conversely,ACSL4 levels are decreased in PC3 cells with AR overexpression.Interestingly, there is an un-coupling of the inverse relationship betweenAR and ACSL4 in LNCaP-AI cells. Consistently, ACSL4 is alsoincreased in human castrate resistant prostate cancer. Importantly,ACSL4 expression induces resistance to Casodex as evidenced by flowand apoptosis assays. The resistance to Casodex is mediated by BAXand Caspase 8.
CONCLUSIONS: Our study indicates that ACSL4 expressionmay serve as a biomarker with potential utility in the diagnosis andtreatment of PCa.
Source of Funding: NIH 1U01CA149556-01, DOD PCRP(PC080010 and PC11624)
MP31-05WITHDRAWNMUTUAL REGULATION BETWEEN RAF/MEK/ERK SIGNALINGAND Y-BOX-BINDING PROTEIN-1 PROMOTES PROSTATECANCER PROGRESSION
Kenjiro Imada*, Masaki Shiota, Kenichi Kohashi, Fukuoka, Japan;Kentaro Kuroiwa, Miyazaki, Japan; YooHyun Song, Masaaki Sugimote,Yoshinao Oda, Seiji Naito, Fukuoka, Japan
MP31-06DIET INDUCED ALTERATION OF FATTY ACID SYNTHASE ISINVOLVED IN PROSTATE CANCER PROGRESSION
Mingguo Huang*, Shintaro Narita, Norihiko Tsuchiya, Takamitsu Inoue,Shigeru Satoh, Hiroshi Nanjo, Takehiko Sasaki, Tomonori Habuchi,Akita, Japan
INTRODUCTION AND OBJECTIVES: High-fat diet (HFD) orobesity is strongly suggested to influence prostate cancer (PCa) pro-gression, but the underlying mechanism is not well defined. Fatty acidsynthase (FASN) is a cytosolic metabolic enzyme catalyzing de novobiosynthesis of fatty acid.
METHODS: In the present study, we investigated the role ofFASN on PCa progression in the LNCaP xenograft mouse fed HFD orlow-fat diet (LFD).
RESULTS: The tumor growth was increased in the HFD groupthan LFD group (p ¼ 0.025). The mRNA expression of FASN andSREBP-1, which encodes Sterol regulatory element-binding protein-1(SREBP-1) were increased in the xenograft tumor by 1.8- and 2.1-fold,respectively (p < 0.05). The serum level of FASN was significantlylower in the HFD group than in the LFD group (p ¼ 0.026) andcorrelated inversely with the tumor volumes (total, r ¼ 0.642,p ¼ 0.022; HFD group, r ¼ �0.618, p ¼ 0.024; LFD group, r ¼ �0.439,p ¼ 0.154; respectively). Upregulation of P-AKT and P-ERK, anddownregulation of P-AMPK were observed in xenograft tumors of theHFD group than LFD group. The extracellular release of FASN proteinin vitro and AMPK activation was induced in the PCa cells by inhibitionof the PI3K and MAPK signaling. In addition, pharmacological acti-vation of AMPK stimulated the extracellular FASN release along withthe downregulation of FASN and SREBP-1 in the PCa cells in dose-dependent manner. Cell proliferation was significantly decreased inthe PCa cells by inhibition of FASN, and the decreased effect wasrescued by addition of palmitic acid. Clinically, imuunohistologicalevaluation of surgical specimens showed that the expression of FASNwas markedly decreased in the PCa response to hormone andchemotherapy.
CONCLUSIONS: In conclusion, HFD increased the FASNexpression and inhibited the extracellular release of FASN, which maybe one of important mechanism in HFD or obesity associated PCaprogression.
Source of Funding: none
MP31-07ESTROGEN RECEPTOR a REGULATES PROLIFERATION IN PTENNULL PROSTATE CANCER
Itsuhiro Takizawa*, Niigata, Japan; Mitchell Lawrence, Clayton,Australia; Helen Pearson, John Pedersen, Normand Pouliot,Australian Prostate, Cancer BioResource, Patrick Humbert, Melbourne,Australia; Luc Furic, Gail Risbridger, Clayton, Australia
INTRODUCTION AND OBJECTIVES: High doses of estrogen,in combination with androgens, can initiate prostate cancer. This effectis attributed to the activity of the estrogen receptor a (ERa). Although itis well established that ERa stimulates proliferation via genomic andnon-genomic activities in breast and ovarian cancer, relatively little isknown about the functions of ERa in prostate cancer cells. Weobserved that ERa expression is up-regulated in high Gleason Scorespecimens of human prostate cancer. We also noted that the incidence
Vol. 191, No. 4S, Supplement, Sunday, May 18, 2014 THE JOURNAL OF UROLOGY� e325
and severity of tumours in PTEN conditional knockout mice withprostate-specific deletion of PTEN, correlates with the estrogensensitivity of each lobe of the prostate. Therefore, we hypothesized thatthis model could be used to study the role of ERa in prostate cancerprogression.
METHODS: Immunohistochemistry and stereology were usedto quantify ERa and Ki67 expression in PTEN null mice. To assessthe functional role of ERa, a cell line derived from a PTEN nulltumour was treated with shRNA or TPSF, a non-competitive ERaantagonist. Rescue experiments with expression constructs for eitherfull length ERa, capable of genomic and non-genomic actions, ormembrane-only ERa, only able to trigger rapid non-genomic signal-ling, were used to determine the mechanism underlying ERa-regu-lated proliferation.
RESULTS: There was a dramatic increase in ERa expressionin prostate tumours of PTEN null mice compared with normal prostatesof control animals. Within the PTEN null prostate, there was a consis-tent pattern of ERa expression: low in benign glands, moderate in tu-mours within the dorsal, lateral and ventral lobes, and high in tumourswithin the anterior prostate. This pattern significantly correlated with thelevels of the proliferative marker Ki67. There was also a significantcorrelation between ERa and Ki67 within individual malignant glands inthe anterior prostate. In vitro knockdown of ERa attenuated the prolif-eration of PTEN null cells as did treatment with TPSF. Loss of ERareduced the activity of both the PI3K and MAPK pathways anddecreased MYC levels. This effect was reversed by re-expressing full-length or membrane-only ERa.
CONCLUSIONS: Collectively, these results demonstrate thatERa drives the proliferation of prostate cancer cells through classicalgenomic and rapid non-genomic signalling.
Source of Funding: ML and LF are Movember YoungInvestigators funded by the Prostate Cancer Foundation ofAustralia’s Research Program.
MP31-08SEMENOGELIN I PROMOTES PROSTATE CANCER CELLGROWTH VIA FUNCTIONING AS AN ANDROGEN RECEPTORCOACTIVATOR AND PROTECTING AGAINST ZINCCYTOTOXICITY
Hitoshi Ishiguro*, Baltimore, MD; Koji Izumi, Yi Li, Rochester, NY;Yichun Zheng, Eiji Kashiwagi, Takashi Kawahara, Hiroshi Miyamoto,Baltimore, MD
INTRODUCTION AND OBJECTIVES: A seminal plasma pro-tein, semenogelin I (SgI), contributes to semen clotting, upon binding toZn2+, and can be proteolyzed by prostate-specific antigen (PSA) torelease the encased spermatozoa after ejaculation. In contrast to thewell-recognized physiological actions of semenogelins, their role inhuman malignancies remains poorly understood. We have demon-strated that SgI is overexpressed in prostate cancer tissues and itsexpression is enhanced by zinc treatment in LNCaP cells. In the currentstudy, using cell lines stably expressing SgI, we investigated itsbiological functions in prostate cancer.
METHODS: We assessed the effects of SgI, in conjunctionwith zinc and androgen, on cell growth and androgen receptor (AR)in prostate cancer lines, using western blotting, MTT assay, trans-well invasion assay, luciferase assay, and co-immunoprecipitaionassay.
RESULTS: Even though SgI is a secreted protein, immuno-blots detected signals in conditioned medium only after culturing SgI-overexpressing cells, but not control LNCaP with endogenous SgI,suggesting that prostate cancer cells do not generally secrete a largeamount of SgI. Zinc, without SgI, inhibited cell growth of both AR-positive and AR-negative lines. Co-expression of SgI induced dihy-drotestosterone (DHT)-mediated proliferation of AR-positive cellswhen cultured with zinc, whereas SgI and/or DHT showed marginaleffects in AR-negative cells. Similarly, SgI enhanced DHT-induced cell
invasion only in the presence of high-level zinc. Moreover, over-expression of SgI induced DHT-mediated PSA expression in cancercells, whereas SgI showed marginal induction without DHT. In a re-porter gene assay, SgI showed a slight inhibitory effect (15%decrease) at 0 mM zinc, a slight stimulatory effect (31% increase) at 15mM zinc, or a significant stimulatory effect (3.2-fold) at 100 mM zinc onDHT-enhanced AR transactivation. Co-immunoprecipitation thendemonstrated DHT-induced physical interactions between ARand SgI.
CONCLUSIONS: We show molecular evidence indicating thatcellular SgI, as a new AR coactivator, enhances the transcriptionalactivity of the receptor in the presence of high levels of zinc and pro-motes androgen-mediated prostate cancer progression. Our resultsmay also provide an underlying reason why prostate cancer tissuecontains relatively high levels of zinc which by itself shows an inhibitoryeffect on tumor growth.
Source of Funding: Department of Defense
MP31-09IDENTIFICATION OF A RETRO-TRANSPOSON DERIVED GENEASSOCIATED WITH PROGRESSION TO NEUROENDOCRINEPROSTATE CANCER.
Shusuke Akamatsu*, Alexander Wyatt, Dong Lin, Summer Lysakowski,Fan Zhang, Soojin Kim, Ladan Fazli, Vancouver, Canada;Himisha Beltran, Mark Rubin, New York, NY; Amina Zoubeidi,Yuzhuo Wang, Colin Collins, Martin Gleave, Vancouver, Canada
INTRODUCTION AND OBJECTIVES: The treatment ofcastration resistant prostate cancer has dramatically improved with therecent development of potent androgen receptor (AR) pathway in-hibitors. However, stronger AR pathway inhibition appears to bedriving resistance mechanisms that are independent of the AR axis,the most recognized of which is neuroendocrine prostate cancer(NEPC). To date, few genes have been associated with progression toNEPC. We developed a patient-derived xenograft model of NEPCtrans-differentiation: a hormone-naïve adenocarcinoma that upon AR-blockade initially regresses, but rapidly relapses as NEPC. In thisstudy, we carried out longitudinal expression profiling of xenografttumors during the trans-differentiation process to identify genesassociated with tumor cell survival post-castration and the develop-ment NEPC.
METHODS: Gene profiling of xenografts collected at differenttime points during the trans-differentiation were compared to data setsof human NEPC. Immunohistochemistry was performed using clinicalNEPC samples. Loss of function studies were carried out using siRNAand shRNA in cell growth (WST-8), invasion (Boyden chamber) andmigration (scratch) assays.
RESULTS: We identified a retro-transposon derived gene,Paternally Expressed 10 (PEG10), to be highly expressed during theearly trans-differentiation stage and also in clinical NEPC. Weconfirmed at the protein level that PEG10 is up-regulated post-castra-tion and further significantly elevated in terminal NEPC. PEG10 washighly expressed within NEPC foci of clinical samples. Knockdown ofPEG10 in prostate cancer (PC) cells induced apoptosis and G0/G1arrest, and also attenuated invasion and migration. We found PEG10knockdown inhibited invasion and migration induced by TGF-b, andmodulated response of the cells to TGF-b, resulting in decreasedphosphorylation of Smad2 and Smad3, decrease in SBE4 luciferasereporter activity, and inhibition of Snail and Zeb1 induction. Collectively,these data show that PEG10 promotes PC cell growth, and also co-operates with TGF-b to promote invasion and migration of PC cells,conferring aggressive phenotype to these cells.
CONCLUSIONS: PEG10 is a gene associated both with growthand invasion of NEPC, and is a potential novel therapeutic target for thetreatment of NEPC.
Source of Funding: None
