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Ligand, Receptor, and Cell Type–Dependent Regulation of ABCA1 and ABCG1 Mrna in Prostate Cancer Epithelial Cells

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Ligand, Receptor, and Cell Type–Dependent Regulation of ABCA1 and ABCG1 Mrna in Prostate Cancer Epithelial Cells Published OnlineFirst June 16, 2009; DOI: 10.1158/1535-7163.MCT-09-0020 Published Online First on June 16, 2009 as 10.1158/1535-7163.MCT-09-0020 OF1 Ligand, receptor, and cell type–dependent regulation of ABCA1 and ABCG1 mRNA in prostate cancer epithelial cells Steven E. Trasino,1 Young S. Kim,2 that ABCA1 gene expression is differentially regulated by and Thomas T.Y. Wang1 synthetic and natural LXR ligands, possibly involving kinase mediated signal transduction. [Mol Cancer Ther 2009;8(7): 1 Diet, Genomics, and Immunology Laboratory, Beltsville Human OF1–12] Nutrition Research Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland and 2Nutritional Sciences Research Group, Division of Cancer Introduction Prevention, National Cancer Institute, NIH, Bethesda, Maryland Advanced and metastatic prostate cancer (PCa) remains the most prominent cancer type and the second leading cause of Abstract all cancer deaths among males in the United States (1). Recent evidence suggests that the liver X receptor (LXR) Treatment of recurrent or advanced PCa with hormone ab- is a potential anticancer target in prostate carcinoma. lation therapy typically results in disease regression with There is little characterization, however, of which of the eventual recurrence of PCa with a more aggressive and two LXR isoforms, LXRα or LXRβ, regulates the LXR- untreatable phenotype (2). In the absence of any effective responsive genes ATP-binding cassette subfamily members long-term therapy and with such an overwhelming social A1 (ABCA1)andG1(ABCG1) in transformed prostatic ep- burden, characterization of novel targets for effective che- ithelial cells. In this study, small interfering RNA (siRNA) moprevention or treatment of PCa has been a priority for α β cancer researchers. was used to determine whether LXR or LXR is involved α β in regulating ABCA1 and ABCG1 mRNA expression in Liver X receptors (LXR , LXR ) are nuclear receptor tran- LNCaP and PC-3 cells. Treatment of both cell lines with scription factors that have been recently identified as puta- tive chemotherapeutic targets in models of PCa (3). The the synthetic LXR ligand T0901317 and oxysterols: 25- α β hydroxycholesterol (25HC) and 24(S), 25-epoxycholesterol natural ligands for LXR and LXR are oxysterols, which (24,25EC), resulted in more than a 10-fold increase of are oxygenated cholesterol intermediates such as 25-hydro- ABCA1 and ABCG1 mRNA expression. Transfection of xycholesterol (25HC) and 24(S),25-epoxycholesterol LNCaP cells with siRNA against either LXRβ or LXRα failed (24,25EC) that are derived from both cholesterol catabolism to inhibit T0901317 and 25HC-mediated increase of and synthesis, respectively (4, 5). A highly specific synthetic ABCA1 mRNA. siRNA silencing of LXRβ did, however, in- LXR ligand (T0901317) has also been developed (6). Studies hibit ABCA1 mRNA expression in 24,25EC-treated LNCaP have shown that oxysterols and T0901317 can inhibit cells. In contrast, LXRβ siRNA inhibited T0901317, 25HC, growth of prostate carcinoma cells in culture and repress and 24,25EC induction of ABCA1 mRNA in PC-3 cells and progression of androgen-dependent tumor xenografts to a ABCG1 mRNA in both LNCaP and PC-3 cells. Additional more aggressive androgen-independent phenotype (7). experiments revealed that T0901317 and 25HC induction More recently, it has also been reported that T0901317 can ABCA1 behave as an antiandrogen agent in models of PCa (8). of mRNA expression was significantly inhibited by α β the p38 stress kinase antagonist SB202190 and PKA inhib- LXR and LXR isotypes have been primarily studied for itor H89. Our study is the first to show that LXRβ,butnot their critical role in limiting accumulation of free cholesterol LXRα, is the major regulatory isoform of ABCG1 mRNA in peripheral tissue and macrophages through regulation of expression in LNCaP and PC-3 cells. Our study also reveals reverse cholesterol transporters ATP-binding cassette, sub- family A, member 1 (ABCA1) and subfamily member G1 (ABCG1; refs. 9, 10). LXRα is primarily expressed in liver, adipose, and enterocytes where LXRβ is expressed ubiqui- Received 1/8/09; revised 3/30/09; accepted 4/13/09; published tously (11). Although both LXRα and LXRβ are expressed in OnlineFirst 6/16/09. the human adult prostate (12), it is unclear whether the ob- Grant support: U.S. appropriated funds to USDA project number 1235-51530-052-00 (T.T.Y. Wang and S.E. Trasino), and the National served anticancer effect of oxysterols and LXR ligands in Cancer Institute (Y.S. Kim). PCa models is related to regulation of cholesterol efflux or The costs ofpublication ofthis article were defrayedin part by the if LXR and its ligands take part in yet undetermined signal- payment ofpage charges. This article must thereforebe hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to ing pathways that are prostate specific. The latter could be indicate this fact. true as it has been shown that LXRs are uniquely involved Requests for reprints: Thomas T.Y. Wang, Diet, Genomics, & Immunology in the innate immune response of macrophages and CD4- Laboratory, Beltsville Human Nutrition Research Center, Agricultural positive lymphocytes (13). Research Service, U.S. Department ofAgriculture, Building 307C, Room 132, Beltsville, MD 20705. Phone: 301-504-8459; Fax: 301-504-9062. There is still considerable debate whether selective activa- E-mail: [email protected] tion of LXRα or LXRβ has a differential effect on cholesterol Copyright © 2009 American Association for Cancer Research. homeostasis or whether they exist as functionally redundant doi:10.1158/1535-7163.MCT-09-0020 paralogs (14, 15). Studies using LXRα/β null mice suggest Mol Cancer Ther 2009;8(7). July 2009 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst June 16, 2009; DOI: 10.1158/1535-7163.MCT-09-0020 OF2 ABCA1 and ABCG1 mRNA in LNCaP and PC-3 Cells that the regulation of genes in liver and peripheral tissue in- cells per well and grown for 24 h in culture medium, then volved in cholesterol homeostasis is primarily under the treated with various concentrations (0.1–10 μmol/L) of control of LXRα, and activation of LXRβ can partially rescue 25HC, 24,25EC, T0901317, or vehicle (DMSO 0.05% v/v). LXRα null animals from gross peripheral cholesterol accu- After termination of experiments, total RNA was isolated mulation (16). However, outside of its role in cholesterol ef- using the TRIzol reagent (Invitrogen), and reverse tran- flux, the broader biological functions of LXRβ are emerging, scribed to complementary DNA using StrataScript First yet remain unclear (17). Strand complementary DNA Synthesis kit (Stratagene; Unlike ABCG1 mRNA expression, which seems to be ex- ref. 21). Real-time PCR was carried out using a TaqMan clusively under the transcriptional control of LXRα, ABCA1 Universal PCR Master Mix on an ABI Prism 7000 Se- mRNA in a number of cell types is regulated through sig- quence Detection System (Applied Biosystems). The Taq- naling mechanisms independent of both LXR isotypes and Man probes and primers were purchased from Applied its role in cholesterol transport (18). Despite this, measuring Biosystems using inventoried TaqMan gene expression as- ABCA1 mRNA changes is often used as a surrogate marker says: LXRα, (assay ID: Hs00167445), LXRβ (assay ID: for in vitro and in vivo LXR activation (19–21). Hs00180254), ABCG1 (assay ID: Hs00167476), ABCA1 (as- In light of the heterogeneity between LXRα and LXRβ say ID: Hs02576345), CYP3A4 (assay ID: Hs01546612), and signaling in other cell types, and the observed anti-PCa CYP3A5 (assay ID: Hs00241417), MDR1 (assay ID: properties of LXR ligands, we sought to identify the rele- Hs00184500). Human glyceraldehyde-3-phosphate dehy- vant LXR isotypes and ligand behavior of T0901317, drogenase (assay ID: Hs99999905) was used as an endog- 25HC, and 24,25EC in the regulation of ABCA1 and ABCG1 enous control for all gene expression except for basal mRNA in two models of transformed prostatic epithelial mRNA quantitation in Fig. 1, where 18S rRNA (assay cells—LNCaP and PC-3. In this report, we show that LXRβ, ID: Hs99999901) was used as an additional control. The but not LXRα, is the primary regulator of ABCA1 and amplification parameters used were as follows: 50°C for ABCG1 mRNA expression in these in vitro cell line models. 2 min, 95°C for 10 min, followed by 46 cycles of amplifi- We also show in LNCaP cells that ABCA1 mRNA expres- cation at 95°C for 15 s and 60°C for 1 min. Quantitation of sion comes under LXRβ regulation only when cells are ex- mRNA fold changes were derived using the comparative −ΔΔCt posed to the endogenously synthesized oxysterol 24,25EC. CT (2 ) cycle (ΔCt) method (22). Lastly, our data suggests that synthetic LXR ligand Quantitation of Basal mRNA levels within LNCaP and T0901317 and the natural LXR ligand 25HC can regulate PC-3 Cells ABCA1 mRNA expression through LXRβ-independent me- We first assigned the largest ΔCt value of one gene as the chanisms involving the kinase-mediated pathway. control and normalized the lower ΔCt value of the second gene to determine a comparative ΔΔCt value. LXRβ ΔCt values were normalized to LXRα ΔCt values (control), Materials and Methods and ABCG1 ΔCt values were normalized to ABCA1 ΔCt Chemicals and Reagents values (control). Dharmacon ON-TARGETplus SMARTpool siRNA Quantitation of Comparative Basal mRNA Levels reagents targeting LXRα (NM_005693) or LXRβ Between LNCaP and PC-3 Cells (NM_007121) were purchased from Thermo Fisher Scientific. We assigned the higher LNCaP cells ΔCt values of each HiPerFect Transfection Reagent was purchased from Qiagen. gene as control and normalized the lower ΔCt value of The selective inhibitors for p38α/β (SB203580), c-Jun corresponding genes in PC-3 cells to determine a compara- NH2-terminal kinase (JNK; SP600125), extracellular signal- tive ΔΔCt value.
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