Skip to main content
NCBI home page
The FASEB Journal logoLink to The FASEB Journal
. 2016 Mar 10;30(6):2360–2369. doi: 10.1096/fj.201500076

12-HETER1/GPR31, a high-affinity 12(S)-hydroxyeicosatetraenoic acid receptor, is significantly up-regulated in prostate cancer and plays a critical role in prostate cancer progression

Kenneth V Honn *,†,‡, Yande Guo *,1, Yinlong Cai *, Menq-Jer Lee *, Gregory Dyson ‡,§, Wenliang Zhang *,2, Stephanie C Tucker *,3
PMCID: PMC4871796  PMID: 26965684

Abstract

Previously we identified and deorphaned G-protein-coupled receptor 31 (GPR31) as the high-affinity 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] receptor (12-HETER1). Here we have determined its distribution in prostate cancer tissue and its role in prostate tumorigenesis using in vitro and in vivo assays. Data-mining studies strongly suggest that 12-HETER1 expression positively correlates with the aggressiveness and progression of prostate tumors. This was corroborated with real-time PCR analysis of human prostate tumor tissue arrays that revealed the expression of 12-HETER1 positively correlates with the clinical stages of prostate cancers and Gleason scores. Immunohistochemistry analysis also proved that the expression of 12-HETER1 is positively correlated with the grades of prostate cancer. Knockdown of 12-HETER1 in prostate cancer cells markedly reduced colony formation and inhibited tumor growth in animals. To discover the regulatory factors, 5 candidate 12-HETER1 promoter cis elements were assayed as luciferase reporter fusions in Chinese hamster ovary (CHO) cells, where the putative cis element required for gene regulation was mapped 2 kb upstream of the 12-HETER1 transcriptional start site. The data implicate 12-HETER1 in a critical new role in the regulation of prostate cancer progression and offer a novel alternative target for therapeutic intervention.—Honn, K. V., Guo, Y., Cai, Y., Lee, M.-J., Dyson, G., Zhang, W., Tucker, S. C. 12-HETER1/GPR31, a high-affinity 12(S)-hydroxyeicosatetraenoic acid receptor, is significantly up-regulated in prostate cancer and plays a critical role in prostate cancer progression.

Keywords: 12(S)-HETE, arachidonate metabolism in malignancy, inflammatory pathway

12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] is an eicosanoid product of 12-lipoxygenase (12-LOX) metabolism of arachidonate, which was first demonstrated by Hamberg and Samuelsson (1). 12(S)-HETE is involved in many physiologic and pathologic processes, such as cell growth, adhesion, differentiation, angiogenesis, inflammation, atherosclerosis, and cancer promotion (2–6). Previously our laboratory demonstrated that 12(S)-HETE enhances metastatic capacity during tumor progression by evoking a wide variety of cellular responses (7–11). It stimulates tumor invasion and motility by inducing alterations in the cancer cell cytoskeleton (12, 13), thereby enhancing tumor cell motility (5). Exogenous addition of 12(S)-HETE induces cancer cells to overexpress proteinases (14–16), vascular endothelial growth factor (17), integrins (12, 18), and fibronectin (19), which prolongs cell survival (10, 20). In endothelial cells, 12(S)-HETE induces the nondestructive retraction of monolayers (21) and promotes tumor cell adhesion (22). The motility of isolated endothelial cells and tube formation is also enhanced by 12(S)-HETE (23). The diverse biologic effects of this important proinflammatory metabolite suggested that 12(S)-HETE likely elicited its function through a cognate receptor, which we identified as G-protein-coupled receptor 31 (GPR31) (24). In this study, we present evidence for the high-affinity 12(S)-HETE receptor (12-HETER1) in prostate cancer progression and examine its gene regulation, distribution in cancer tissues, and its function in tumorigenesis in vitro and in vivo.

MATERIALS AND METHODS

Cell lines

Chinese hamster ovary (CHO), human embryonic kidney 293 (HEK293), RWPE1, human prostate carcinoma PC3 (a human prostate cancer cell line derived from patient bone metastasis), and DU145 (a human prostate cancer cell line derived from patient brain metastasis) cells were obtained from the American Type Culture Collection (Manassas, VA, USA), and PC3M cells were obtained from Dr. Isaiah J. Fidler and Dr. Katherine Stemke-Hale (M. D. Anderson Cancer, Houston, TX, USA) via Material Transfer Agreement). These were routinely cultured in RPMI 1640 supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY, USA), 2 mM l-glutamine, and 100 μg/ml penicillin–streptomycin, and maintained in a humidified atmosphere of 5% CO2 at 37°C.

Reagents

Reagents included BMD122 [biomide compound 122, N-benzyl-N-hydroxy-5-phenylpentamide (BHPP); Biomide, Grosse Pointe Farms, MI, USA]; 12(S)-HETE (Cayman Chemicals, Ann Arbor, MI, USA).

Quantitative real-time PCR

Prostate tumor tissue arrays were purchased from OriGene Technologies (Rockville, MD, USA; HPRT101 and HPRT102). 12-HETER1 mRNA levels were determined by quantitative real-time PCR (qPCR) on an ABI PRISM 7000 system (Applied Biosystems, Foster City, CA, USA) using the Taqman Gene expression assay for 12-HETER1 (Hs00271094; Applied Biosystems).

Cloning and functional assay of 12-HETER1 promoter cis elements in pMetLuc2 reporter vector

Specific oligonucleotide primers were designed based on GenBank accession number NT_025741.15. Primers for P1Luc, P2Luc, P3Luc, P4Luc, and P5Luc were as follows: P1Luc+: 5′-AAT TCT GAC CAG TGT GTA AAA TCA AGC CCC ATG CAG AAG GAT CCC AGC AAG CGT CG-3′; P1Luc−: 5′-GAT CCG ACG CTT GCT GGG ATC CTT CTG CAT GGG GCT TGA TTT TAC ACA CTG GTC AG-3′; P2Luc+: 5′-AAT TCC AAA GTA CTT TAA AAA GGG GCA GAA GTT TTT AAA AAG GAA AAT ACG TAA TTA TTA G-3′; P2Luc−: 5′-GAT CCT AAT AAT TAC GTA TTT TCC TTT TTA AAA ACT TCT GCC CCT TTT TAA AGT ACT TTG G-3′; P3Luc+: 5′-AAT TCG AAA TAA TCA TAA AAA TAG CCA ACT AGC AGG CCT TGG GCC TGC TCT GCC TG-3′; P3Luc−: 5′-GAT CCA GGC AGA GCA GGC CCA AGG CCT GCT AGT TGG CTA TTT TTA TGA TTA TTT CG-3′; P4Luc+: 5′-AAT TCC CCA TAT GGT CTA AAA AGG GGA GGA ACC CTC AGT TTC AGG AAT TAT CCA CG-3′; P4Luc−: 5′-GAT CCG TGG ATA ATT CCT GAA ACT GAG GGT TCC TCC CCT TTT TAG ACC ATA TGG GG-3′; P5Luc+: 5′-AAT TCC ACT GTG AGT TTA CAA ATG CCA TGG CAA CAT CAG GAA GTT ACC CCA TAT GG-3′; P5Luc−: 5′-GAT CCC ATA TGG GGT AAC TTC CTG ATG TTG CCA TGG CAT TTG TAA ACT CAC AGT GG-3′. The PCR reactions with these primers were performed on genomic DNA isolated from human PC3 cells. Amplification was carried out for 30 cycles (30 s at 94°C, 30 s at 53°C, and 2 min at 72°C), followed by an additional 7 min at 72°C. The resulting DNA fragments were cloned directionally via EcoRI-BamHI sites (underlined) into the pMetLuc2-Reporter vector (631729; Clontech Laboratories, Mountain View, CA, USA) and confirmed by sequencing. The constructs were then transfected into CHO or prostate cells. After 24 h, the luciferase activities were measured using the Ready-To-Glow Secreted Luciferase Reporter System (631728; Clontech Laboratories).

Transfection

Cells were seeded in 6-well plates (2 × 105 cells/well) and cultured in RPMI 1640 plus 10% fetal bovine serum at 37°C for 20 h in a humidified atmosphere of 5% CO2. The cells were transfected with pcDNA3.1-GPR31 or other constructs with GenePorter reagent (Genlantis, San Diego, CA, USA) or TransIT-Prostate Transfection kit (2130; Mirus Bio, Madison, WI, USA) according to the manufacturers’ protocols.

Immunocytochemistry

The cells grown on glass coverslips were fixed for 10 min in 3.7% paraformaldehyde solution, washed, and blocked with 3% bovine serum albumin (BSA) in PBS for 30 min at room temperature. After washing in PBS–1% BSA 3 times, the cells were incubated with anti-12-HETER1 antibodies (Sigma-Aldrich, St. Louis, MO, USA) for 40 min at room temperature. Then the cells were stained using the ABC kit (PK6200; Vector Laboratories, Burlingame, CA, USA) and mounted in Permount (15-500; Fisher Scientific, Waltham, MA, USA).

Immunohistochemistry

For tissue immunostaining for 12-HETER1 expression, tissue array ( prostate cancer tissue stage II, III A222III section 105, barcode 122120707312; Isu Abxis, Seoul, Korea;) or paraffin-embedded tissue sections of tumor specimens from patients who presented with clinically localized prostate cancer were deparaffinized, rehydrated, and permeabilized at 95°C for 20 min in 1 mM EDTA (tissues) or 1% H2O2 for 15 min (array) followed by staining with 12-HETER1 antibody (B2849 bleed 2 rabbit anti-GPR31 against second extracellular loop; or SAB4501269; Sigma-Aldrich) using the ABC kit (PK-6200; Vector Laboratories) according to the manufacturer’s instruction.

Cell colony formation

Based on the method of Liu (25), 400 transfected cells were plated into each well of a 6-well dish or into 60 mm tissue culture dishes (3 dishes for each treatment) overnight; media were replaced the next day with 12(S)-HETE or BMD122 as indicated. Cells were grown for 12 d with medium changes every 3 d. Subsequently, cells were fixed with 4% paraformaldehyde and stained with 0.05% crystal violet. Colonies in excess of 50 cells were scored.

Tumor growth in athymic mice

All animal procedures were in compliance with the U.S. National Institutes of Health and with the institutional guidelines established for the Department of Laboratory Animal Research facility at Wayne State University. Male athymic NCr nude mice were purchased from Taconic Biosciences (Hudson, NY, USA). PC3M cells stably transfected with different 12-HETER1 shRNA constructs were grown for 4 d, washed, suspended in HBSS, and injected subcutaneously in the right flank of 5- to 6–wk-old NCr nude mice at a density of 2 × 106 viable cells per 100 µl (26). Six mice were used in each group. Tumor growth was measured using a vernier caliper 3 times a week for 4 wk (30 d), and tumor volume was calculated using the following formula: Tumor volume (mm3) = 0.5 × a × b2, where a is the longest diameter and b is the shortest diameter (27). Mice were humanely killed 4 wk after injection. Tumors were removed, weighed, and photographed.

Computational mining of gene expression profiling data

Meta-analysis of relative 12-HETER1 gene expression was performed on multiple normal tissue and cancer types by queries against normalized, microarray profiling data sets of clinical samples, stored in the Oncomine 4.3 database (Compendia Bioscience, Ann Arbor, MI, USA) (28), the Memorial Sloan Kettering Cancer Center’s cBioPortal Database (29, 30), or an Omnibus data set (31) from the U.S. National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) repository. Box plot analysis was used to display the distributions of normalized gene expression intensities, either for clinically annotated descriptor classes or for normal tissue/cancer types. Two-class differential gene expression analyses of paired conditions were performed within the Oncomine 4.3 platform by Student’s t test on Z score–normalized intensity values.

RESULTS

12-HETER1 is up-regulated in prostate cancer and plays a critical role in prostate cancer progression

It has been shown that the expression of 12-LOX, a rate-limiting enzyme for 12(S)-HETE biosynthesis, is significantly up-regulated in prostate cancer tissues and correlates positively with the aggressiveness of prostate cancers (32, 33). It is also well established that 12(S)-HETE plays a critical role in tumor invasion and metastasis (5, 7, 12, 13, 15, 16, 20–22, 34). The critical functions executed by this metabolite were thought to occur through a cognate receptor that has been sought for many years. GPR31 was identified as the high-affinity 12(S)-HETE receptor, which we named 12-HETER1 (24). In order to establish the relationship between GPR31/12-HETER1 and cancer progression, we conducted extensive data-mining analyses for 12-HETER1 expression in global cancer–type surveys and in 6 separate microarray profiling data sets representing various pathologic stages of prostate cancer (28, 35–38). We observed that 12-HETER1 is markedly elevated in prostate cancer (Fig. 1A) and that the trend toward higher levels of 12-HETER1 in metastatic prostate cancer is significant. In one study, 12-HETER1 was elevated (fold change = 2.056, P = 0.052) in those patients with metastatic disease (n = 6) compared to patients with clinically localized disease (n = 7) (Fig. 1B). In another study of primary prostate carcinoma, 12-HETER1 was elevated (fold change = 2.618, P = 0.003) in patients experiencing biochemical recurrence (n = 5) in 1 yr compared to those who had no recurrence (n = 17) (Fig. 1C). 12-HETER1 is elevated in higher-grade, more aggressive prostate cancer. In 4 independent analyses, 12-HETER1 was found to be associated with a higher Gleason score (GS) or stage. For example, 12-HETER1 was elevated (fold change = 1.202, P = 0.076) in GS-7 (n = 48) compared to GS-6 (n = 17) prostate adenocarcinoma (Fig. 1D). Finally, we assessed data in the NCBI GEO data sets (39, 40) by generating box plot analyses for 12-HETER1 stratified by tumor type—normal (n = 29), primary (n = 131), and metastasis (n = 19) (Fig. 1E)—and stratified by GS (including 9 metastatic and 130 primary cancers): GS-6 (n = 41), GS-7 (3 + 4) (n = 53), and GS-7 (4 + 3) (n = 25) (Fig. 1F) [(primary tumor vs. normal: fold change = 1.239, P < 0.001); (metastasis vs. normal: fold change = 1.280, P = 0.001)]. These results are in agreement with our own analyses of protein and mRNA/qPCR. However, it should be noted that many of the publicly available data sets have surprisingly small n values, particularly for high-grade samples and metastases, compared to normal tissue. We took advantage of the largest public expression data set available, which is that of Taylor et al. (31) with approximately 200 samples. A greater level of statistical power will be possible when expression data sets approach the size of the available survival data sets such as in the Surveillance, Epidemiology, and End Results database, which has data from more than 4000 cases (41).

Figure 1.

Figure 1.

Open in a new tab

12-HETER1 expression correlates with aggressiveness of human prostate tumors. A) Analysis of 12-HETER1expression in the Memorial Sloan Kettering Cancer Center cBioPortal Cancer Genomics database. Receptor is markedly up-regulated in 27.2% of human prostate tumors. B–D) Oncomine 4.3 database data correlate expression level of 12-HETER1 with aggressiveness of human prostate tumors. E, F) NCBI GEO data (GSE21032) show 12-HETER1 becomes elevated in high-grade tumors.

Next we used qPCR to determine 12-HETER1 expression in 2 commercially available human prostate tissue microarrays from OriGene Technologies, HPRT101 and HPRT102 (Fig. 2). The results indicated that the expression of 12-HETER1 is positively correlated with the aggressiveness (GS; Fig. 2A) and clinical stages (Fig. 2B) of human prostate tumors, which is statistically significant.

Figure 2.

Figure 2.

Open in a new tab

qPCR quantitation of 12-HETER1 in commercially available prostate tumor tissue arrays (HPRT101 and HPRT102, OriGene Technologies). 12-HETER1 expression positively correlates with GS (A) and cliniical stage (B) of prostate tumors. m, mean value.

12-HETER1 is up-regulated in prostate cancer tissues and the expression of 12-HETER1 is positively correlated with tumor grades

We screened 2 commercial 12-HETER1 antibodies, SAB4501269 and HPA014014, from Sigma-Aldrich. We tested SAB4501269 recognition of the receptor and specificity, in the absence of an available competitive peptide from Sigma-Aldrich or their vendor, by testing the immunologic reaction with 12-HETER1 in HEK293 cells transiently transfected with 12-HETER1 cDNA (Fig. 3Aa). In contrast, no specific immunoreactivity was detected in HEK293 cells transfected with pcDNA control vector (Fig. 3Ab). Additionally we performed a competitive inhibition of the antibody by incubation with membranes from cell preparations from cells either overexpressing 12-HETER1 (GPR31) or not (Supplemental Fig. 1) We then examined the expression of 12-HETER1 in human prostate tumor specimens provide by the Wayne State University Pathology Research Service. Representative images from 7 human prostate tumor specimens are shown in Fig. 3Ac, d. For example, immunoreactivity was rare in normal glands (Fig. 3Ac, arrow). However, in the neoplastic glands (GS-3/4), intense brown staining was observed (Fig. 3Ac, arrowhead). Furthermore, strong staining was observed in a GS-3/4 tumor area (Fig. 3Ad, arrow) and weak staining in an area with a GS-3 tumor area (Fig. 3Ad, arrowhead).

Figure 3.

Figure 3.

Open in a new tab

12-HETER1 expression positively correlates with aggressiveness and progression of prostate tumors. A) Expression of 12-HETER1 in prostate carcinoma cells. Immunocytochemistry on HEK293 cells expressing 12-HETER1. a, b) Staining with 12-HETER1 antibody was optimized on HEK293 cells transfected with 12-HETER1 (a) or pcDNA (b) vector. Strong brown staining was detected in HEK293/12-HETER1 cells (a) compared to negative control (b). c, d) Immunohistochemistry to detect 12-HETER1 in 2 cases of human prostate cancer tissue. c) Benign gland (N, left corner, arrow) with rare cells showing positive staining, and neoplastic glands (T, GS-3/4, arrowhead) showing intense brown staining (arrowhead); original magnification, ×10. d) Strong staining in area (upper left, arrow) with mixed GS-3/4 tumor area, weak staining in area (lower right, arrowhead) with GS-3 tumor; original magnification, ×10. Images are representatives from 7 samples. B) Array analysis of 12-HETER1 expression. a, b) Immunohistochemistry to assay 12-HETER1 expression on 2 human prostate tissue microarrays. Expression of 12-HETER1 is significantly up-regulated in disease stage II/III human prostate tumor specimens (n = 8, P = 0.007, Student’s t test. c, d) Representative images of normal (c) and prostate tumor (d) specimens.

Next, we determined 12-HETER1 expression in 2 commercially available human prostate tissue microarrays. The results indicated that the expression of 12-HETER1 is significantly up-regulated in disease stage II/III human prostate tumor specimens (n = 8; P = 0.007, Student’s t test) (Fig. 3Ba, b). Representative images of normal and prostate tumor specimen are shown in Fig. 3Bc, d. These protein data are in agreement with our data-mining analysis and qPCR quantification, and they suggest that 12-HETER1 is up-regulated in prostate cancer tissues where the expression of 12-HETER1 positively correlated with tumor grades.

Expression of 12-HETER1 is controlled by special cis element

Upstream of the 12-HETER1 transcriptional initiation site within P1 (−8AGGATCCCAGCA+9), analysis using CpG Island Searcher software (http://cpgislands.usc.edu) returned no potential CpG islands for DNA methylation within 5 kb of the 5′ end. However, the Cis-element Cluster Finder software (http://zlab.bu.edu/~mfrith/cister.shtml) identified several possible cis elements, including TBP (TATA binding protein), Sp1 (transcription factor, Specificity protein 1), B1/B2 (transcription factor, mitochondrial), Ets (transcription factor, E26 transformation specific), and ERE (estrogen response element). Thus cis element–driven transcriptional activation may be the mechanism for the enhanced expression of 12-HETER1 in prostate tumors. Using a neural network promoter prediction software developed by the Berkeley Drosophila Genome Project, and successfully applied by others to identify oncogene promoters (42, 43) (http://www.fruitfly.org/seq_tools/promoter.html), we found 5 candidates spanning from −41 to +9, 2050 to −2000, −3517 to −3467, −3612 to −3570, and −3662 to −3620 nucleotides (Fig. 4A). As a first approximation, and before pursuing an extended nested deletion strategy to determine the core regulatory sequence, these gross segments were cloned into the pMetLuc2-Reporter vector and named P1, P2, P3, P4, and P5, respectively. These constructs were transfected into CHO cells and luciferase activities measured. The results indicated that P2 has highest luciferase activity compared to the other constructs. However, P3, P4, and P5 also have partial promoter functions (Fig. 4B). Next, we transfected the P2, P3, and P4 constructs into DU145, PC3, and RWPE (normal human prostate epithelial cells immortalized with HPV18) cells, and the luciferase activities were measured 2 d later. The luciferase activity was normalized to transfection efficiency in a parallel experiment. The results indicated that P2 in the PC3 background had the strongest effect (Fig. 4C). The relative expression of P2-driven luciferase correlated well with the expression of 12-HETER1 in PC3, DU145, and RWPE cells (Fig. 4D).

Figure 4.

Figure 4.

Open in a new tab

Novel cis element in 12-HETER1 promoter. A) Candidate promoter regions, P1-P5, of 12-HETER1. B) P1-P5 fragments were subcloned into pMET-LUC2 vector. Luciferase activity was measured in transfected CHO cells, with P2 fragment exhibiting highest luciferase activity. C) PC3, DU145, and RWPE cells were transfected with P2-, P3-, P4-Luciferase vectors. Luciferase activity was normalized to transfection efficiency in parallel experiment. D) 12-HETER1 expression in PC3, DU145, and RWPE cells. Levels of P2-driven luciferase expression (C) correlates well with corresponding expression levels of 12-HETER1 in PC3, DU145, and RWPE cells.

12-HETER1 expression affects tumorigenesis of prostate cancer cells in vitro and in vivo

To address the role of 12-HETER1 in prostate cancer progression, PC3M (human prostate cancer cell line derived from mouse liver metastasis of PC3 cells grown in NCr nude mouse spleen) cells expressing various levels of receptor were used (24). Specifically, 12-HETER1 was efficiently knocked down in sh-GPR31#4 cells compared to sh-GPR31#2 and sh-GPR31#5 (scrambled negative control) cells (Fig. 5A). However, sh-GPR31#2 cells formed strikingly more colonies (3-fold) than sh-GPR31#4 cells in a soft agar colony formation assay (62 ± 25 vs. 21 ± 3; P < 0.01, Student’s t test) (Fig. 5B) and also bound more 12(S)-HETE (Fig 5A, inset). 12-HETER1 knockdown cells (sh-GPR31#4) formed fewer colonies in response to exogenous 12-HETER1 ligand, namely 12(S)-HETE, and fewer still with the inhibition of the 12LOX enzyme that produces the ligand naturally (Fig. 5C). However, exogenous addition of the mono HETE did not yield greater colony formation in the in vitro assay.

Figure 5.

Figure 5.

Open in a new tab

12-HETER1 expression affects colony formation in vitro. A) qPCR confirmation of 12-HETER1 knockdown (inset) and 12(S)-HETE binding assay as per Guo et al. (24) on sh-GPR31#2, -#4, and -#5 (negative control, scrambled). B) Clonogenic assay on PC3M cells stably transfected with sh-GPR31#2 formed 3-fold more colonies than those with sh-GPR31#4 constructs. Numbers in parentheses are means ± sd of triplicate determinations. **P < 0.01 (Student’s t test). C) Clonogenic assay on PC3M cells with either sh-GPR31#4 (knockdown) or sh-GPR31#5 (scrambled) either untreated, treated with receptor agonist 12(S)-HETE and 12-LOX inhibitor BMD122, or treated with receptor agonist 12(S)-HETE alone. P values for Student’s t test are indicated.

The role of 12-HETER1 in tumor growth in animals was examined using a subcutaneous tumor implant model in athymic mice. As shown in Fig. 6, knockdown of 12-HETER1 markedly inhibited tumor growth in animals. All animals injected with PC3M carrying the sh-GPR31#5 scrambled construct developed visible, large tumors. Five of 6 mice implanted with PC3M cells carrying sh-GPR31#2 constructs developed large tumors (Fig. 6, #2), and 1 mouse developed a noticeable but smaller tumor. In contrast, the tumors were completely resolved in 3 of 6 mice injected with the PC3M cells carrying the sh-GPR31#4 construct. The remaining 3 of 6 mice from the latter set developed very minor tumors. The tumor masses in these 3 mice were ∼4 times smaller than those from mice injected with PC3M carrying #2 or #5 constructs.

Figure 6.

Figure 6.

Open in a new tab

12-HETER1 knockdown inhibits prostate tumor progression in vivo. A) Mice were inoculated with prostate tumor cells transfected with either sh-scrambled (#5) or sh-12-HETER1 (#2, #4). B) Tumor growth of mice inoculated with sh-GPR31#2, -#4, and -#5 cells. Knockdown of 12-HETER1 (sh-GPR31#4) markedly inhibits tumor growth in animals. Tumors excised from mice implanted with PC3M cells carrying sh-GPR31#4 constructs are ∼4 times smaller than those from mice implanted with PC3M cells carrying sh-GPR31#2 and -#5 constructs. **P < 0.01; *P < 0.05, Student’s t test. Tumors and minute tumor nodules are circled. Inset: weights of excised tumors are shown. Data are presented as means ± sd.

DISCUSSION

12(S)-HETE is a fundamentally important proinflammatory signaling molecule that acts before cytokines come online and is a contributing factor in cancer pathology, where it has been shown to increase the invasiveness and metastatic potential of prostate tumors, among others (20, 21, 23, 44). It is also involved in carcinogenesis and is accompanied by an elevation in the 12-LOX enzyme (20, 23, 32, 33, 45). Numerous studies by us and others have placed emphasis on disabling the enzyme responsible for its synthesis, which has met with success (46, 47). However, evidence indicated that a membrane G-protein-coupled receptor for 12(S)-HETE plays an important role in signal initiation in diverse settings from prostate cancer to psoriasis, where in the latter condition the epidermal isoform of 12-LOX leads to 12(S)-HETE synthesis (11, 48–50). This presented a potentially novel therapeutic target in addition to the enzyme. Previously, we deorphaned GPR31, located on chromosome 6, as a high-affinity receptor of 12(S)-HETE and renamed it 12-HETER1 (24, 51). In the present study, we conducted extensive data-mining analyses and found that 12-HETER1 expression may positively correlate with the aggressiveness, progression, and recurrence of prostate tumors. Indeed, we demonstrated that 12-HETER1 is markedly up-regulated in the neoplastic prostate glands, whereas 12-HETER1 is rarely detected in normal prostate gland. Moreover, the expression of 12-HETER1 receptor positively correlates with the grades of prostate cancer tissues.

This is particularly relevant as the receptor is encoded at 6q27 on chromosome 6, an area that has been determined to be susceptible to alterations in prostate cancer and that constitutes a novel susceptibility locus (52, 53).

Much has come to light in recent years regarding regulation of transcription in prostate cancer and the importance of gene fusions in such factors as Ets, for example (54–61). Therefore, because 12-HETER1 is differentially expressed between normal and cancer cells, we strove to begin to characterize the cis-active elements located upstream of its coding sequence by examining individual gross fragments. We thus analyzed the 5 kb DNA sequence upstream of the transcriptional start site and identified a potential regulatory segment, P2, located at −2050 to −2000 bp that increased expression of a luciferase reporter. We demonstrated that 12-HETER1 was expressed in cancer cells (PC3 and DU145) and was not expressed in normal cells (RWPE), which correlated well with P2-driven luciferase activities in the parallel experiments. Therefore, work is underway to characterize those core transcription binding sites in proximity to this element and in the context of the full upstream region using nested deletions. However, determining the true role of these elements in proper context will require future animal knockdown studies, as has been suggested elsewhere (62, 63).

Since the early studies and as interest in the area of eicosanoids in cancer progresses, the contribution of 12(S)-HETE to cancer, colony formation, and spread continues to be validated (64–67). However, the receptor was always inferred. We determined that in the absence of 12-HETER1, PC3M cells were curtailed from forming colonies in soft agar or growing into tumors in athymic mice. Colonies were attenuated even more efficiently when the lipoxygenase inhibitor BMD122 was added in addition to knocking down the receptor. The addition of exogenous 12(S)-HETE did not appear to increase the colonies formed in vitro by parental strains, as we might have expected on the basis of previous in vivo studies where we and others have found increased colony formation and migration in response to the mono HETE (24, 65, 66). Future in vivo studies with the knockdown strains will allow us to examine the capacity for distal colony formation in the absence of potentially confounding in vitro, issues such as the need for serum that may still contain fatty acids that affect the assay despite charcoal stripping. These findings close a major signaling loop that includes the lipoxygenase enzyme, its downstream signaling metabolite, and now the 12-HETER1 high-affinity receptor that transduces the signals necessary to advance the disease. Collectively, these data strongly suggest that 12-HETER1 is a critical component in the tumorigenesis of prostate tumors, tested here, and is likely relevant in numerous other cancers. There is also a general sense that this receptor is likely to play an important role in other pathologies, as 12(S)-HETE is a master regulator of inflammatory processes that affect diverse disease states such as diabetes and neurologic disorders (68–72).

Supplementary Material

Supplemental Data
supp_30_6_2360__index.html (941B, html)

Acknowledgments

This work was supported in part by U.S. National Institutes of Health (NIH) National Cancer Institute Grants CA029997, Department of Defense Grants W81XWH-06-1-0226 and W81XWH-11-1-0519 (to K.V.H.), and NIH National Heart, Lung, and Blood Institute Grant HL071071 (to M.-J.L.). Results are in part based on data generated by The Cancer Genome Atlas Research Network (http://cancergenome.nih.gov/). The authors declare no conflicts of interest.

Glossary

12(S)-HETE

12(S)-hydroxyeicosatetraenoic acid

12-HETER1

high-affinity 12(S)-HETE receptor

12-LOX

12-lipoxygenase

BMD122

biomide compound 122, N-benzyl-N-hydroxy-5-phenylpentamide

BSA

bovine serum albumin

CHO

Chinese hamster ovary

Ets

transcription factor, E26 transformation specific

GPR31

G-protein-coupled receptor 31

GS

Gleason score

HEK293

human embryonic kidney 293

NCBI GEO

U.S. National Center for Biotechnology Information Gene Expression Omnibus

qPCR

quantitative real-time PCR

Footnotes

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

REFERENCES

  • 1.Hamberg M., Samuelsson B. (1974) Prostaglandin endoperoxides. Novel transformations of arachidonic acid in human platelets. Proc. Natl. Acad. Sci. USA 71, 3400–3404 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Chen Y. Q., Duniec Z. M., Liu B., Hagmann W., Gao X., Shimoji K., Marnett L. J., Johnson C. R., Honn K. V. (1994) Endogenous 12(S)-HETE production by tumor cells and its role in metastasis. Cancer Res. 54, 1574–1579 [PubMed] [Google Scholar]
  • 3.Funk C. D., Chen X.-S., Johnson E. N., Zhao L. (2002) Lipoxygenase genes and their targeted disruption. Prostaglandins Other Lipid Mediat. 68-69, 303–312 [DOI] [PubMed] [Google Scholar]
  • 4.Kamitani H., Ikawa H., Hsi L. C., Watanabe T., DuBois R. N., Eling T. E. (1999) Regulation of 12-lipoxygenase in rat intestinal epithelial cells during differentiation and apoptosis induced by sodium butyrate. Arch. Biochem. Biophys. 368, 45–55 [DOI] [PubMed] [Google Scholar]
  • 5.Timar J., Silletti S., Bazaz R., Raz A., Honn K. V. (1993) Regulation of melanoma-cell motility by the lipoxygenase metabolite 12-(S)-HETE. Int. J. Cancer 55, 1003–1010 [DOI] [PubMed] [Google Scholar]
  • 6.Yoshimoto T., Takahashi Y. (2002) Arachidonate 12-lipoxygenases. Prostaglandins Other Lipid Mediat. 68-69, 245–262 [DOI] [PubMed] [Google Scholar]
  • 7.Honn K. V., Tang D. G., Crissman J. D. (1992) Platelets and cancer metastasis: a causal relationship? Cancer Metastasis Rev. 11, 325–351 [DOI] [PubMed] [Google Scholar]
  • 8.Liu B., Khan W. A., Hannun Y. A., Timar J., Taylor J. D., Lundy S., Butovich I., Honn K. V. (1995) 12(S)-hydroxyeicosatetraenoic acid and 13(S)-hydroxyoctadecadienoic acid regulation of protein kinase C-alpha in melanoma cells: role of receptor-mediated hydrolysis of inositol phospholipids. Proc. Natl. Acad. Sci. USA 92, 9323–9327 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Menter D. G., Tucker S. C., Kopetz S., Sood A. K., Crissman J. D., Honn K. V. (2014) Platelets and cancer: a casual or causal relationship: revisited. Cancer Metastasis Rev. 33, 231–269 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Pidgeon G. P., Lysaght J., Krishnamoorthy S., Reynolds J. V., O’Byrne K., Nie D., Honn K. V. (2007) Lipoxygenase metabolism: roles in tumor progression and survival. Cancer Metastasis Rev. 26, 503–524 [DOI] [PubMed] [Google Scholar]
  • 11.Szekeres C. K., Tang K., Trikha M., Honn K. V. (2000) Eicosanoid activation of extracellular signal-regulated kinase1/2 in human epidermoid carcinoma cells. J. Biol. Chem. 275, 38831–38841 [DOI] [PubMed] [Google Scholar]
  • 12.Chopra H., Timar J., Chen Y. Q., Rong X. H., Grossi I. M., Fitzgerald L. A., Taylor J. D., Honn K. V. (1991) The lipoxygenase metabolite 12(S)-HETE induces a cytoskeleton-dependent increase in surface expression of integrin α IIb β 3 on melanoma cells. Int. J. Cancer 49, 774–786 [DOI] [PubMed] [Google Scholar]
  • 13.Tang, D. G., and Honn, K. V. (1997) Role of protein kinase C and phosphatases in 12(S)-HETE-induced tumor cell cytoskeletal reorganization. In Advances in Experimental Medicine and Biology (Honn, K. V., Nigam, S., and Marnett, L. J., eds.), pp. 349–361, Springer, New York [DOI] [PubMed] [Google Scholar]
  • 14.Dilly A. K., Ekambaram P., Guo Y., Cai Y., Tucker S. C., Fridman R., Kandouz M., Honn K. V. (2013) Platelet-type 12-lipoxygenase induces MMP9 expression and cellular invasion via activation of PI3K/Akt/NF-κB. Int. J. Cancer 133, 1784–1791 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Honn K. V., Timár J., Rozhin J., Bazaz R., Sameni M., Ziegler G., Sloane B. F. (1994) A lipoxygenase metabolite, 12-(S)-HETE, stimulates protein kinase C–mediated release of cathepsin B from malignant cells. Exp. Cell Res. 214, 120–130 [DOI] [PubMed] [Google Scholar]
  • 16.Liu X.-H., Connolly J. M., Rose D. P. (1996) Eicosanoids as mediators of linoleic acid–stimulated invasion and type IV collagenase production by a metastatic human breast cancer cell line. Clin. Exp. Metastasis 14, 145–152 [DOI] [PubMed] [Google Scholar]
  • 17.Natarajan R., Bai W., Lanting L., Gonzales N., Nadler J. (1997) Effects of high glucose on vascular endothelial growth factor expression in vascular smooth muscle cells. Am. J. Physiol. 273, H2224–H2231 [DOI] [PubMed] [Google Scholar]
  • 18.Tang D. G., Diglio C. A., Bazaz R., Honn K. V. (1995) Transcriptional activation of endothelial cell integrin alpha v by protein kinase C activator 12(S)-HETE. J. Cell Sci. 108, 2629–2644 [DOI] [PubMed] [Google Scholar]
  • 19.Natarajan R., Gonzales N., Lanting L., Nadler J. (1994) Role of the lipoxygenase pathway in angiotensin II–induced vascular smooth muscle cell hypertrophy. Hypertension 23(1, Suppl)I142–I147 [DOI] [PubMed] [Google Scholar]
  • 20.Pidgeon G. P., Tang K., Rice R. L., Zacharek A., Li L., Taylor J. D., Honn K. V. (2003) Overexpression of leukocyte-type 12-lipoxygenase promotes W256 tumor cell survival by enhancing alphavbeta5 expression. Int. J. Cancer 105, 459–471 [DOI] [PubMed] [Google Scholar]
  • 21.Honn K. V., Tang D. G., Gao X., Butovich I. A., Liu B., Timar J., Hagmann W. (1994) 12-Lipoxygenases and 12(S)-HETE: role in cancer metastasis. Cancer Metastasis Rev. 13, 365–396 [DOI] [PubMed] [Google Scholar]
  • 22.Honn K. V., Tang D. G., Grossi I., Duniec Z. M., Timar J., Renaud C., Leithauser M., Blair I., Johnson C. R., Diglio C. A. (1994) Tumor cell–derived 12(S)-hydroxyeicosatetraenoic acid induces microvascular endothelial cell retraction. Cancer Res. 54, 565–574 [PubMed] [Google Scholar]
  • 23.Nie D., Hillman G. G., Geddes T., Tang K., Pierson C., Grignon D. J., Honn K. V. (1998) Platelet-type 12-lipoxygenase in a human prostate carcinoma stimulates angiogenesis and tumor growth. Cancer Res. 58, 4047–4051 [PubMed] [Google Scholar]
  • 24.Guo Y., Zhang W., Giroux C., Cai Y., Ekambaram P., Dilly A. K., Hsu A., Zhou S., Maddipati K. R., Liu J., Joshi S., Tucker S. C., Lee M. J., Honn K. V. (2011) Identification of the orphan G protein–coupled receptor GPR31 as a receptor for 12-(S)-hydroxyeicosatetraenoic acid. J. Biol. Chem. 286, 33832–33840 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Liu F. (2012) Soft-agar colony formation assay. Bio Protoc. 2, e220 Available from http://www.bio-protocol.org/e220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Cho Y. S., Kim M. K., Tan L., Srivastava R., Agrawal S., Cho-Chung Y. S. (2002) Protein kinase A RIalpha antisense inhibition of PC3M prostate cancer cell growth: Bcl-2 hyperphosphorylation, Bax up-regulation, and Bad-hypophosphorylation. Clin. Cancer Res. 8, 607–614 [PubMed] [Google Scholar]
  • 27.Yamaoka M., Yamamoto T., Ikeyama S., Sudo K., Fujita T. (1993) Angiogenesis inhibitor TNP-470 (AGM-1470) potently inhibits the tumor growth of hormone-independent human breast and prostate carcinoma cell lines. Cancer Res. 53, 5233–5236 [PubMed] [Google Scholar]
  • 28.Rhodes D. R., Kalyana-Sundaram S., Mahavisno V., Varambally R., Yu J., Briggs B. B., Barrette T. R., Anstet M. J., Kincead-Beal C., Kulkarni P., Varambally S., Ghosh D., Chinnaiyan A. M. (2007) Oncomine 3.0: genes, pathways, and networks in a collection of 18,000 cancer gene expression profiles. Neoplasia 9, 166–180 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Cerami E., Gao J., Dogrusoz U., Gross B. E., Sumer S. O., Aksoy B. A., Jacobsen A., Byrne C. J., Heuer M. L., Larsson E., Antipin Y., Reva B., Goldberg A. P., Sander C., Schultz N. (2012) The cBio Cancer Genomics Portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2, 401–404 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Gao J., Aksoy B. A., Dogrusoz U., Dresdner G., Gross B., Sumer S. O., Sun Y., Jacobsen A., Sinha R., Larsson E., Cerami E., Sander C., Schultz N. (2013) Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci. Signal. 6, pl1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Taylor B. S., Schultz N., Hieronymus H., Gopalan A., Xiao Y., Carver B. S., Arora V. K., Kaushik P., Cerami E., Reva B., Antipin Y., Mitsiades N., Landers T., Dolgalev I., Major J. E., Wilson M., Socci N. D., Lash A. E., Heguy A., Eastham J. A., Scher H. I., Reuter V. E., Scardino P. T., Sander C., Sawyers C. L., Gerald W. L. (2010) Integrative genomic profiling of human prostate cancer. Cancer Cell 18, 11–22 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Gao X., Grignon D. J., Chbihi T., Zacharek A., Chen Y. Q., Sakr W., Porter A. T., Crissman J. D., Pontes J. E., Powell I. J. (1995) Elevated 12-lipoxygenase mRNA expression correlates with advanced stage and poor differentiation of human prostate cancer. Urology 46, 227–237 [DOI] [PubMed] [Google Scholar]
  • 33.Matsuyama M., Yoshimura R., Mitsuhashi M., Hase T., Tsuchida K., Takemoto Y., Kawahito Y., Sano H., Nakatani T. (2004) Expression of lipoxygenase in human prostate cancer and growth reduction by its inhibitors. Int. J. Oncol. 24, 821–827 [PubMed] [Google Scholar]
  • 34.Tang K., Cai Y., Joshi S., Tovar E., Tucker S. C., Maddipati K. R., Crissman J. D., Repaskey W. T., Honn K. V. (2015) Convergence of eicosanoid and integrin biology: 12-lipoxygenase seeks a partner. Mol. Cancer 14, 111 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Kilpinen S., Autio R., Ojala K., Iljin K., Bucher E., Sara H., Pisto T., Saarela M., Skotheim R. I., Björkman M., Mpindi J.-P., Haapa-Paananen S., Vainio P., Edgren H., Wolf M., Astola J., Nees M., Hautaniemi S., Kallioniemi O. (2008) Systematic bioinformatic analysis of expression levels of 17,330 human genes across 9,783 samples from 175 types of healthy and pathological tissues. Genome Biol. 9, R139 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Nanni S., Priolo C., Grasselli A., D’Eletto M., Merola R., Moretti F., Gallucci M., De Carli P., Sentinelli S., Cianciulli A. M., Mottolese M., Carlini P., Arcelli D., Helmer-Citterich M., Gaetano C., Loda M., Pontecorvi A., Bacchetti S., Sacchi A., Farsetti A. (2006) Epithelial-restricted gene profile of primary cultures from human prostate tumors: a molecular approach to predict clinical behavior of prostate cancer. Mol. Cancer Res. 4, 79–92 [DOI] [PubMed] [Google Scholar]
  • 37.Varambally S., Yu J., Laxman B., Rhodes D. R., Mehra R., Tomlins S. A., Shah R. B., Chandran U., Monzon F. A., Becich M. J., Wei J. T., Pienta K. J., Ghosh D., Rubin M. A., Chinnaiyan A. M. (2005) Integrative genomic and proteomic analysis of prostate cancer reveals signatures of metastatic progression. Cancer Cell 8, 393–406 [DOI] [PubMed] [Google Scholar]
  • 38.Wallace T. A., Prueitt R. L., Yi M., Howe T. M., Gillespie J. W., Yfantis H. G., Stephens R. M., Caporaso N. E., Loffredo C. A., Ambs S. (2008) Tumor immunobiological differences in prostate cancer between African-American and European-American men. Cancer Res. 68, 927–936 [DOI] [PubMed] [Google Scholar]
  • 39.Barrett T., Wilhite S. E., Ledoux P., Evangelista C., Kim I. F., Tomashevsky M., Marshall K. A., Phillippy K. H., Sherman P. M., Holko M., Yefanov A., Lee H., Zhang N., Robertson C. L., Serova N., Davis S., Soboleva A. (2013) NCBI GEO: archive for functional genomics data sets—update. Nucleic Acids Res. 41, D991–D995 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Edgar R., Domrachev M., Lash A. E. (2002) Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 30, 207–210 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Rusthoven C. G., Carlson J. A., Waxweiler T. V., Yeh N., Raben D., Flaig T. W., Kavanagh B. D. (2014) The prognostic significance of Gleason scores in metastatic prostate cancer. Urol. Oncol. 32, 707–713 [DOI] [PubMed] [Google Scholar]
  • 42.Karagianni N., Ly M. C., Psarras S., Chlichlia K., Schirrmacher V., Gounari F., Khazaie K. (2005) Novel adenomatous polyposis coli gene promoter is located 40 kb upstream of the initiating methionine. Genomics 85, 231–237 [DOI] [PubMed] [Google Scholar]
  • 43.Peng X., Maruo T., Cao Y., Punj V., Mehta R., Das Gupta T. K., Christov K. (2004) A novel RARbeta isoform directed by a distinct promoter P3 and mediated by retinoic acid in breast cancer cells. Cancer Res. 64, 8911–8918 [DOI] [PubMed] [Google Scholar]
  • 44.Nie D., Krishnamoorthy S., Jin R., Tang K., Chen Y., Qiao Y., Zacharek A., Guo Y., Milanini J., Pages G., Honn K. V. (2006) Mechanisms regulating tumor angiogenesis by 12-lipoxygenase in prostate cancer cells. J. Biol. Chem. 281, 18601–18609 [DOI] [PubMed] [Google Scholar]
  • 45.Nie D., Nemeth J., Qiao Y., Zacharek A., Li L., Hanna K., Tang K., Hillman G. G., Cher M. L., Grignon D. J., Honn K. V. (2003) Increased metastatic potential in human prostate carcinoma cells by overexpression of arachidonate 12-lipoxygenase. Clin. Exp. Metastasis 20, 657–663 [DOI] [PubMed] [Google Scholar]
  • 46.Steele V. E., Holmes C. A., Hawk E. T., Kopelovich L., Lubet R. A., Crowell J. A., Sigman C. C., Kelloff G. J. (1999) Lipoxygenase inhibitors as potential cancer chemopreventives. Cancer Epidemiol. Biomarkers Prev. 8, 467–483 [PubMed] [Google Scholar]
  • 47.Tang D. G., Chen Y. Q., Honn K. V. (1996) Arachidonate lipoxygenases as essential regulators of cell survival and apoptosis. Proc. Natl. Acad. Sci. USA 93, 5241–5246 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Arenberger P., Kemény L., Ruzicka T. (1993) Characterization of high-affinity 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) binding sites on normal human keratinocytes. Epithelial Cell Biol. 2, 1–6 [PubMed] [Google Scholar]
  • 49.Arenberger P., Raap A., Armah B., Kemény L., Ruzicka T. (1993) The lipoxygenase inhibitor 2-phenylmethyl-1-naphthol (DuP 654) is a 12(S)-hydroxyeicosatetraenoic acid receptor antagonist in the human epidermal cell line SCL-II. Skin Pharmacol. 6, 148–151 [DOI] [PubMed] [Google Scholar]
  • 50.Herbertsson H., Hammarström S. (1992) High-affinity binding sites for 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) in carcinoma cells. FEBS Lett. 298, 249–252 [DOI] [PubMed] [Google Scholar]
  • 51.Zingoni A., Rocchi M., Storlazzi C. T., Bernardini G., Santoni A., Napolitano M. (1997) Isolation and chromosomal localization of GPR31, a human gene encoding a putative G protein-coupled receptor. Genomics 42, 519–523 [DOI] [PubMed] [Google Scholar]
  • 52.Christensen G. B., Baffoe-Bonnie A. B., George A., Powell I., Bailey-Wilson J. E., Carpten J. D., Giles G. G., Hopper J. L., Severi G., English D. R., Foulkes W. D., Maehle L., Moller P., Eeles R., Easton D., Badzioch M. D., Whittemore A. S., Oakley-Girvan I., Hsieh C. L., Dimitrov L., Xu J., Stanford J. L., Johanneson B., Deutsch K., McIntosh L., Ostrander E. A., Wiley K. E., Isaacs S. D., Walsh P. C., Isaacs W. B., Thibodeau S. N., McDonnell S. K., Hebbring S., Schaid D. J., Lange E. M., Cooney K. A., Tammela T. L., Schleutker J., Paiss T., Maier C., Grönberg H., Wiklund F., Emanuelsson M., Farnham J. M., Cannon-Albright L. A., Camp N. J.; International Consortium for Prostate Cancer Genetics (2010) Genome-wide linkage analysis of 1,233 prostate cancer pedigrees from the International Consortium for Prostate Cancer Genetics using novel sumLINK and sumLOD analyses. Prostate 70, 735–744 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Kim J. H., Dhanasekaran S. M., Mehra R., Tomlins S. A., Gu W., Yu J., Kumar-Sinha C., Cao X., Dash A., Wang L., Ghosh D., Shedden K., Montie J. E., Rubin M. A., Pienta K. J., Shah R. B., Chinnaiyan A. M. (2007) Integrative analysis of genomic aberrations associated with prostate cancer progression. Cancer Res. 67, 8229–8239 [DOI] [PubMed] [Google Scholar]
  • 54.Coller H. A. (2014) The Runt-related transcription factor 1 in prostate cancer–associated fibroblasts. Proc. Natl. Acad. Sci. USA 111, 16238–16239 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Fitzgerald K. A., Evans J. C., McCarthy J., Guo J., Prencipe M., Kearney M., Watson W. R., O’Driscoll C. M. (2014) The role of transcription factors in prostate cancer and potential for future RNA interference therapy. Expert Opin. Ther. Targets 18, 633–649 [DOI] [PubMed] [Google Scholar]
  • 56.Gasi D., Trapman J. (2011) Androgen regulation of ETS gene fusion transcripts in prostate cancer. Methods Mol. Biol. 776, 335–348 [DOI] [PubMed] [Google Scholar]
  • 57.Liao Z., Nevalainen M. T. (2011) Targeting transcription factor Stat5a/b as a therapeutic strategy for prostate cancer. Am. J. Transl. Res. 3, 133–138 [PMC free article] [PubMed] [Google Scholar]
  • 58.Morgan R., Boxall A., Harrington K. J., Simpson G. R., Michael A., Pandha H. S. (2014) Targeting HOX transcription factors in prostate cancer. BMC Urol. 14, 17 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Sankpal U. T., Goodison S., Abdelrahim M., Basha R. (2011) Targeting Sp1 transcription factors in prostate cancer therapy. Med. Chem. 7, 518–525 [DOI] [PubMed] [Google Scholar]
  • 60.Tomlins S. A., Rhodes D. R., Perner S., Dhanasekaran S. M., Mehra R., Sun X. W., Varambally S., Cao X., Tchinda J., Kuefer R., Lee C., Montie J. E., Shah R. B., Pienta K. J., Rubin M. A., Chinnaiyan A. M. (2005) Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science 310, 644–648 [DOI] [PubMed] [Google Scholar]
  • 61.Wang Q., Li W., Liu X. S., Carroll J. S., Jänne O. A., Keeton E. K., Chinnaiyan A. M., Pienta K. J., Brown M. (2007) A hierarchical network of transcription factors governs androgen receptor–dependent prostate cancer growth. Mol. Cell 27, 380–392 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Pennacchio L. A., Ahituv N., Moses A. M., Prabhakar S., Nobrega M. A., Shoukry M., Minovitsky S., Dubchak I., Holt A., Lewis K. D., Plajzer-Frick I., Akiyama J., De Val S., Afzal V., Black B. L., Couronne O., Eisen M. B., Visel A., Rubin E. M. (2006) In vivo enhancer analysis of human conserved non-coding sequences. Nature 444, 499–502 [DOI] [PubMed] [Google Scholar]
  • 63.Natoli G., Andrau J. C. (2012) Noncoding transcription at enhancers: general principles and functional models. Annu. Rev. Genet. 46, 1–19 [DOI] [PubMed] [Google Scholar]
  • 64.Guo A. M., Liu X., Al-Wahab Z., Maddippati K. R., Ali-Fehmi R., Scicli A. G., Munkarah A. R. (2011) Role of 12-lipoxygenase in regulation of ovarian cancer cell proliferation and survival. Cancer Chemother. Pharmacol. 68, 1273–1283 [DOI] [PubMed] [Google Scholar]
  • 65.Klampfl T., Bogner E., Bednar W., Mager L., Massudom D., Kalny I., Heinzle C., Berger W., Stättner S., Karner J., Klimpfinger M., Fürstenberger G., Krieg P., Marian B. (2012) Up-regulation of 12(S)-lipoxygenase induces a migratory phenotype in colorectal cancer cells. Exp. Cell Res. 318, 768–778 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Liu B., Marnett L. J., Chaudhary A., Ji C., Blair I. A., Johnson C. R., Diglio C. A., Honn K. V. (1994) Biosynthesis of 12(S)-hydroxyeicosatetraenoic acid by B16 amelanotic melanoma cells is a determinant of their metastatic potential. Lab. Invest. 70, 314–323 [PubMed] [Google Scholar]
  • 67.Piao Y. S., Du Y. C., Oshima H., Jin J. C., Nomura M., Yoshimoto T., Oshima M. (2008) Platelet-type 12-lipoxygenase accelerates tumor promotion of mouse epidermal cells through enhancement of cloning efficiency. Carcinogenesis 29, 440–447 [DOI] [PubMed] [Google Scholar]
  • 68.Cole B. K., Lieb D. C., Dobrian A. D., Nadler J. L. (2013) 12- and 15-lipoxygenases in adipose tissue inflammation. Prostaglandins Other Lipid Mediat. 104-105, 84–92 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Tajima Y., Ishikawa M., Maekawa K., Murayama M., Senoo Y., Nishimaki-Mogami T., Nakanishi H., Ikeda K., Arita M., Taguchi R., Okuno A., Mikawa R., Niida S., Takikawa O., Saito Y. (2013) Lipidomic analysis of brain tissues and plasma in a mouse model expressing mutated human amyloid precursor protein/tau for Alzheimer’s disease. Lipids Health Dis. 12, 68 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 70.Taylor-Fishwick D. A., Weaver J., Glenn L., Kuhn N., Rai G., Jadhav A., Simeonov A., Dudda A., Schmoll D., Holman T. R., Maloney D. J., Nadler J. L. (2015) Selective inhibition of 12-lipoxygenase protects islets and beta cells from inflammatory cytokine-mediated beta cell dysfunction. Diabetologia 58, 549–557 [DOI] [PubMed] [Google Scholar]
  • 71.Xu J., Zhang Y., Xiao Y., Ma S., Liu Q., Dang S., Jin M., Shi Y., Wan B., Zhang Y. (2013) Inhibition of 12/15-lipoxygenase by baicalein induces microglia PPARβ/δ: a potential therapeutic role for CNS autoimmune disease. Cell Death Dis. 4, e569 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 72.Tucker S. C., Honn K. V. (2013) Emerging targets in lipid-based therapy. Biochem. Pharmacol. 85, 673–688 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Data
supp_30_6_2360__index.html (941B, html)
supp_fj.201500076_Supplemental_Figure1.docx (4.7MB, docx)

Articles from The FASEB Journal are provided here courtesy of The Federation of American Societies for Experimental Biology

RESOURCES