ActivationofAndrogenReceptor,Lipogenesis,andOxidative ...by AIA-360 Immunoassay Analyzer (Tosoh...

Signaling and Regulation

Activation of AndrogenReceptor, Lipogenesis, andOxidativeStress Converged by SREBP-1 Is Responsible for RegulatingGrowth and Progression of Prostate Cancer Cells

Wen-Chin Huang1, Xiangyan Li1, Jian Liu2, Jentai Lin3, and Leland W.K. Chung1

AbstractWe previously reported that sterol regulatory element-binding protein-1 (SREBP-1) is involved in the

transcriptional regulation of androgen receptor (AR) and formation of fatty acid through altered expression offatty acid synthase (FASN). In this article, we provide a new finding that SREBP-1 induced oxidative stress inprostate cancer cells through increased production of reactive oxygen species (ROS) and expression of NADPHoxidase 5 (Nox5). We have shown that (i) expression of SREBP-1 protein is positively associated with the clinicalGleason grades in human prostate cancer; (ii) genetic overexpression or knockdown of SREBP-1 in prostate cancercells resulted in corresponding increased or decreased AR, FASN and Nox5 expression, fatty acid and lipid dropletaccumulation, and ROS generation; and (iii) SREBP-1 induces and promotes the growth, migration, invasion, andcastration-resistant progression of prostate cancer cells in vitro and in vivo. Our data show a novel molecularmechanism by which SREBP-1 promotes prostate cancer growth and progression through alterations in theconcerted intracellular metabolic and signaling networks involving AR, lipogenesis, and ROS in prostate cancercells. Mol Cancer Res; 10(1); 133–42. �2011 AACR.

Introduction

Cancer progression is the underlying cause of mortalityandmorbidity in prostate cancer patients. Lethal progressionof prostate cancer from androgen-dependent status to andro-gen-refractory (or castration resistant) status involves mul-tiplemechanisms inwhich androgens and androgen receptor(AR)-mediated signaling play key roles (1–3). Blockade ofandrogen action and the AR signaling axis is currently themain treatment for prostate cancer and its progression.Several reports have shown that androgen biosynthesis andAR signaling in prostate cancer cells are intimately affectedby lipogenesis (4–6). Lipid raft membrane-related intercel-lular signaling pathways have been shown to induce ARactivity (7–9). High-fat diets were shown to promote pros-tate cancer cell growth and aggressiveness (10, 11), and drugsthat interfere with fatty acid and cholesterol metabolism and

absorption were shown to reduce prostate cancer growth,angiogenesis, and progression (12–14). Identifying theunderlying molecular mechanisms linking lipogenesis andAR signaling could facilitate further development of prom-ising therapeutic approaches for human prostate cancer.Sterol regulatory element-binding protein-1 (SREBP-1) is

a critical transcription factor for lipogenesis (15–17). Threemajor isoforms of SREBP have been identified, such asSREBP-1a, SREBP-1c, and SREBP-2. SREBP proteins(125 kDa) are anchored to the endoplasmic reticulum (ER)membrane. Through proteolytic cleavage (18), the activatedamino-terminus (68 kDa) of SREBP translocates into thenucleus to bind SRE (sterol regulatory element) cis-actingelements and trigger gene expression. SRE is found in thepromoter regions of genes encoding enzymes for fatty acid,lipid, and cholesterol biosynthesis, including fatty acidsynthase (FASN), HMGCoA synthase (19, 20) and farnesyldiphosphate synthase (21). SREBP-1 and one of its targetgenes, FASN, reported to be a metabolic oncogene (22),have been shown to be involved in prostate cancer malignantprogression (6, 23). Our recent results showed that SREBP-1 regulated AR promoter activity and expression and cellviability in prostate cancer cells (5). The data concurred withthe observation of upregulation of SREBP-1 expression inhuman prostate cancer tissues during castration-resistantprogression (23). These experimental and clinical datasuggest that SREBP-1 may play an important role in theregulation of prostate cancer cell growth and progression tolethal castration-resistant disease.In this study, we revealed a new molecular mechanism by

which SREBP-1 promotes prostate cancer growth, survival,

Authors' Affiliations: 1Uro-Oncology Research Program, Department ofMedicine, Cedars-Sinai Medical Center; 2Department of Molecular andMedical Pharmacology, University of California, Los Angeles, California;and 3Graduate Institute of Clinical Medicine, National Cheng-Kung Uni-versity, Tainan, Taiwan

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Wen-Chin Huang, Uro-Oncology ResearchProgram, Cedars-Sinai Medical Center, 8750 Beverly Blvd., Atrium103, Los Angeles, CA 90048. Phone: 310-423-7378; Fax: 310-423-8543; E-mail: [email protected]; and Leland W.K. Chung,E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-11-0206

�2011 American Association for Cancer Research.

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on June 30, 2021. © 2012 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst November 7, 2011; DOI: 10.1158/1541-7786.MCR-11-0206

http://mcr.aacrjournals.org/

and lethal progression. We genetically manipulated SREBP-1 using either an expression vector or a sequence-specificshort hairpin RNA (shRNA) approach to show, respectively,increased or decreased AR expression, cell proliferation,migration, and invasion in prostate cancer cells. Throughthe induction of FASN expression, SREBP-1 induced fattyacid and lipid droplet formation and accumulation inprostate cancer cells. Furthermore, SREBP-1 increased reac-tive oxygen species (ROS) levels via increased NADPHoxidase 5 (Nox5) expression in prostate cancer cells. ROShas been shown to induce signal transduction, survival, andprogression of cancer cells (24, 25). In mouse xenograftmodels, SREBP-1 promoted human prostate tumor growthand supported the development of a castration-resistantprogression phenotype through the induction of AR, FASN,and Nox5 expression. SREBP-1 and its downstream AR/lipogenesis/ROS signaling axis, therefore, provide noveltherapeutic targets for prostate cancer treatment.

Materials and Methods

Prostate cancer cell lines, cell culture, reagents, andmaterialsHuman prostate cancer cell lines LNCaP and C4-2B (26)

were cultured in T-medium (Invitrogen) supplemented with5% FBS. SREBP-1 expression vector and shRNA wereobtained from OriGene Technologies, Inc. and Santa CruzBiotechnology, respectively. Oil Red O and diphenyleneio-donium (DPI) were purchased from Sigma-Aldrich. Ahuman prostate carcinoma tissue microarray (TMA) wasobtained from IMGENEX.

Western blot analysisCell lysates were prepared from prostate cancer cells as

previously described (27). Nuclear extracts were prepared byaNucBuster Protein Extraction kit (Novagen).Western blotanalysis was done by NOVEX system (Invitrogen). Primaryantibodies against human SREBP-1, FASN, AR, Nox5(Santa Cruz Biotechnology), catalase (Abcam), Akt, andphospho-Akt (p-Akt, Ser 473; Cell Signaling Technology)were utilized.

Fatty acid content quantification and Oil Red O stainingThe long chain fatty acid contents were determined by a

Fatty Acid Quantification Kit (MBL International Corpo-ration). Lipid droplet formationwas assayed by anOil RedOstaining method (28). Oil Red O staining images wereexamined and recorded by a phase contrast microscope. Forquantification, Oil Red O retained in cells were extracted by100% isopropanol and measured optical absorbance at 500nm normalized by total cell numbers.

Cell proliferation, in vitromigration, and invasion assaysFor cell proliferation assay, prostate cancer cells (1 � 105

cells per well) were seeded on 6-well plates for 3-day incuba-tion. Cells were harvested and cell numbers were counted byhemocytometer. The Boyden chamber method was utilizedto examine in vitro cell migration and invasion of prostate

cancer cells. Briefly, the undersides of the upper Boydenchambers were precoated with collagen I (2.5 mg/cm2, formigration assay) or growth factor–depleted Matrigel matrix(1:4 dilution, for invasion assay). Cells (5 � 104 cells) wereseeded inside theprecoatedupper chambers.After incubationat 37�C for 12 to 24 hours (migration), or 24 to 48 hours(invasion), the numbers of migrated or invading cells weremeasured by the crystal violet staining method (29).

Intracellular ROS determinationSuperoxide or hydrogen peroxide was assayed by dihy-

droethidium (DHE) or 5-(and-6)-chloromethyl-20,70-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA; Invitrogen). DHE was oxidized to red fluores-cent ethidium by superoxide, and CM-H2DCFDA wasoxidized to green fluorescent dichlorofluorescein (DCF) byhydrogen peroxide. Cells were treated with 10mmol/LDHEor 5 mmol/L CM-H2DCFDA for 30 minutes, respectively,at 37�C. Subsequently, treated prostate cancer cells werewashed with PBS and cultured in T-medium for 30minutes.The mean fluorescence intensity was determined by flowcytometry FACS Calibur (BD Bioscience) as relative ROS(superoxide or hydrogen peroxide) compared with controls.

Mouse xenograft experimentsAll the mouse experiments were approved and carried out

in accordance with institutional guidelines. Four-week-oldathymic nu/nu male mice (Charles River) were inoculatedsubcutaneously with control Neo or overexpressing SREBP-1 LNCaP (H2) with 1 � 106 cells per mouse. The tumorburdens were monitored by tumor volume [V ¼ 4/3p X(d/2)2�D/2, in which d is theminor tumor axis andD is themajor tumor axis] weekly. To determine the effects ofsurgical castration on the growth of LNCaP tumors, nu/numale mice were subcutaneously inoculated with 3 � 106Neo or H2 cells per mouse. After 6 weeks of tumor growth,micewere either surgically castrated or shamoperated. Bloodspecimens were harvested and serum PSA was determinedby AIA-360 Immunoassay Analyzer (Tosoh Bioscience)weekly. At the end of the animal experiments, mice wereeuthanized and prostate tumor tissues were harvested, fixedin 10% formalin, dehydrated in ethanol, embedded inparaffin, and sectioned for histomorphologic and immuno-histochemical (IHC) analyses (5).

Statistical analysisStatistical analyses were done as described previously (30).

Student's t test and 2-tailed distribution were applied in theanalysis of statistical significance. Statistical analysis ofhuman prostate carcinoma TMA results was done usingFisher's exact test.

Results

Overexpression of SREBP-1 is associated with aggressivepathologic features in human prostate cancerTo study the clinical significance of a lipogenic transcrip-

tion factor, SREBP-1, in prostate cancer progression, we

Huang et al.

Mol Cancer Res; 10(1) January 2012 Molecular Cancer Research134




determined the expression of SREBP-1 protein in humanprostate carcinoma TMA. We assayed SREBP-1 expressionusing a clinical prostate cancer progression set representativeof tumors at different stages of the disease from normal/benign to localized cancer with different Gleason grades(Fig. 1 and Table 1). SREBP-1 showed only 20% (3/15)positive expression in normal/benign prostate tissues, where-as expression of SREBP-1 protein increased with higherGleason grades of disease [from 50% (grade 3) to 71%(grade 5); Table 1]. Interestingly, nuclear SREBP-1 wasdetected prevalently in grade 4 and 5 prostate cancers(Fig. 1C and D). Statistical analysis revealed that overallSREBP-1 expression levels were strongly correlated withGleason grades (P ¼ 0.003). These results suggested that

expression of SREBP-1 protein is closely linked with thedevelopment of aggressive pathologic features in humanprostate cancer. SREBP-1 may be a potential prognosticbiomarker for human prostate cancer.

SREBP-1 induces expression of AR and FASN andincreases formation of fatty acid and lipid droplets inprostate cancer cellsWe previously showed that SREBP-1 regulated AR tran-

scriptional expression by binding the 50-flanking AR pro-moter region in prostate cancer cells (5). To further inves-tigate the biological functions of SREBP-1 in prostatecancer, we established LNCaP cells stably overexpressingSREBP-1 under the control of a universal CMV promoter(5) because LNCaP cells showed lower intrinsic SREBP-1[both precursor SREBP-1 (125 kDa) and mature nuclearSREBP-1 (68 kDa)] than aggressive C4-2B cells (Fig. 2A;ref. 26). After antibiotic screening, we selected the 2 higheststably overexpressing both precursor and nuclear SREBP-1LNCaP clones, H1 and H2 (Fig. 2B). Consistent withprevious observations, SREBP-1 induced expression of AR(5) and FASN (31) in H1 and H2 cells (Fig. 2B). FASN isone of the SREBP-1 target proteins and has been shown as ametabolic oncogene in prostate cancer (32). Because FASNis a key enzyme for de novo synthesis of fatty acid and lipids,we subsequently examined the levels of long chain fatty acidand lipid droplets in cells. As shown in Fig. 2C, fatty acidcontents were significantly increased in H1 and H2 com-pared with untransfected LNCaP and control Neo (emptyexpression vector transfected) cells. Also, lipid droplets werehighly accumulated in both H1 and H2 compared with

Figure 1. Overexpression of SREBP-1 isassociated with aggressive pathologic featuresin human prostate cancer. A, human prostatecarcinoma TMA was used to determineexpression of SREBP-1. A, prostate normal/benign tissues; B, Gleason grade 3; C, grade 4;and D, grade 5 of prostate cancer tissues.SREBP-1 protein showed lower expression inhuman normal/benign tissues, an increasingexpression with higher Gleason grade. Inparticular, SREBP-1 was highly detected innuclei in grade 4 and 5 prostate cancer.Scale bar ¼ 275 mm.

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C D

Table 1. Expression of SREBP-1 in humanprostate carcinoma TMA

SREBP-1 expression,n (%)

Clinicopathologiccharacteristics Positive Negative

Gleason gradeNormal/benign (n ¼ 15) 3 (20) 12 (80)Grade 3 (n ¼ 14) 7 (50) 7 (50)Grade 4 (n ¼ 27) 19 (70) 8 (30)Grade 5 (n ¼ 14) 10 (71) 4 (29)NOTE: n indicates the numbers of samples.

SREBP-1 Promotes Prostate Cancer Growth and Progression

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control cells (Fig. 2D). The results suggested that SREBP-1induced expression of AR and FASN and increased accu-mulation of fatty acid and lipid droplets in prostate cancercells.

SREBP-1 regulates cell proliferation, migration, andinvasion in prostate cancer cellsNext, we determined the biological role of SREBP-1 in

regulating cell proliferation, migration, and invasion inprostate cancer cells. Overexpressing SREBP-1 H1 andH2 cells showed increased cell proliferation compared withuntransfected LNCaP and control Neo cells during a 3-dayincubation (Fig. 3A). One of the hallmarks of progressiveand metastatic cells is their ability to invade surroundingtissues and migrate efficiently. The assays of in vitro migra-tion and invasion were conducted by the Boyden chambermethod. H1 and H2 showed significantly increased migra-tory and invasive capabilities compared with the controlgroups (Fig. 3B). The results supported the conclusion thatSREBP-1 promoted cell proliferation, migration, and inva-sion, the hallmarks of progressive cancer cells. Conversely,knockdown of SREBP-1 using a sequence specific shRNAwith lentiviral delivery to C4-2B cells as known with high

intrinsic SREBP-1 expression (Fig. 2A), showed decreasedSREBP-1, AR, and FASN expression (Fig. 3C). SREBP-1shRNA also significantly inhibited cell proliferation (Fig.3D), in vitro migration (23.8% � 10.0% inhibition, P ¼0.04) and invasion (31.8% � 10.3% inhibition, P ¼ 0.01)in aggressive C4-2B cells. These data, in aggregate, revealedthat SREBP-1 plays an important role in regulation of cellproliferation, migration, and invasion in prostate cancercells.

SREBP-1 induces cell proliferation and progressionthrough increased Nox5 expression and intracellularROS levels in prostate cancer cellsROS and Nox (a ROS generator) have been reported to

regulate cell proliferation, progression, metastasis, and radi-ation resistance of prostate cancer cells (33–35). Our cDNAmicroarray data revealed that Nox5 was upregulated inoverexpressing SREBP-1 prostate cancer cells comparedwith control cells (unpublished data). To further determinewhether SREBP-1 induces prostate cancer cell proliferationand progression through activation of Nox5 and ROS, wefirst examined expression of Nox5 in control and SREBP-1stably overexpressing H1 and H2 cells. Consistent with

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Figure 2. SREBP-1 induces expression of AR and FASN and accumulation of fat in prostate cancer cells. A, expression of intrinsic SREBP-1 protein in LNCaPand C4-2B cells was analyzed by Western blot. LNCaP cells showed lower expression of precursor (125 kDa) and mature SREBP-1 (68 kDa) thanC4-2B cells. b-Actin was used as an internal loading control. B, expression of FASN and AR was increased in the 2 highest overexpressing SREBP-1 LNCaPclones, H1 and H2, compared with untransfected LNCaP and control Neo. C, SREBP-1 increased fatty acid contents in H1 and H2. The relative fattyacid content (%) was assigned as 100% in LNCaP. ��, P < 0.005, significant differences from Neo. Data represent the mean� SD of 2 independent triplicateexperiments. D, SREBP-1 greatly increased lipid droplet accumulation in H1 and H2. ��, P < 0.005, significant differences from Neo. In the left panel,arrowheads indicate lipid droplets in prostate cancer cells. Scale bars ¼ 40 mm.

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cDNA microarry results, Nox5 protein increased in H1 andH2 compared with Neo and untransfected LNCaP cells(Fig. 4A). We also found that SREBP-1 increased p-Aktexpression (Fig. 4A), which is involved in prostate cancer cellproliferation, survival, and progression (36). Next, weassessed ROS (superoxide and hydrogen peroxide) levels incells. The levels of hydrogen peroxide were increased in H1and H2 cells (Fig. 4B, the right panel); the levels ofsuperoxide, however, were not significantly changed bySREBP-1 (Fig. 4B, the left panel). We also observed that2 superoxide-degraded enzymes, extracellular superoxidedismutase (SOD3) and mitochondrial SOD (SOD2), wereupregulated in H1 and H2 cells (data not shown). Inaddition, catalase, an enzyme responsible for hydrogenperoxide degradation, was decreased in H1 and H2 cells(Fig. 4A). To further investigate whether SREBP-1 inducesprostate cancer cell proliferation through activation of Nox5and ROS, a Nox inhibitor and ROS scavenger, DPI, wasused to treat these prostate cancer cells. As shown in Fig. 4C,cell proliferation in both Neo and H2 cells was affected byDPI in a concentration-dependent inhibition. H2 cells withhigh Nox5 and ROS levels exhibited a phenotype withincreased resistance to DPI-mediated suppression of cellgrowth (Fig. 4C). Interestingly, DPI inhibited AR expres-sion in prostate cancer cells (Fig. 4D). Hydrogen peroxidehas been shown to affect AR expression in LNCaP cells (37).DPI inhibited AR expression could be through ROS bydecreasing hydrogen peroxide in prostate cancer cells. These

data collectively indicated that SREBP-1 induced prostatecancer cell proliferation and progression through increasedNox5 expression and intracellular ROS levels.

SREBP-1 promotes prostate tumor growth andcastration resistance in subcutaneous xenograft mousemodelsBecause SREBP-1 expression is increased in advanced

forms of human prostate cancer (Fig. 1; ref. 23), we sought todetermine whether SREBP-1 confers growth advantages inhormone-naive mice and resistance to tumor shrinkage insurgically castrated mice. We found that SREBP-1 over-expressing H2 cells inoculated subcutaneously developed a100% incidence of tumor formation (8/8) in mice. ControlNeo cells only exhibited 50% incidence of tumor formation(4/8) during an 8-week observation period. LNCaP classi-cally showed less aggressive and less tumorigenic character-istics in mouse models (26). Furthermore, H2 tumorsexhibited a 14-fold increased growth rate over that of theNeo tumors, as assessed by tumor volumes (Neo: 8.8� 5.0mm3 and H2: 124.0 � 40.0 mm3) after 8 weeks of in vivogrowth (Fig. 5A). Consistent with previous Western blotresults, IHC data showed that H2 highly expressed SREBP-1 (mostly in nuclei), FASN (cytoplasm), Nox5 (cell mem-branes), and AR (mostly in nuclei) in comparison with Neotumors harvested from mouse subcutaneous spaces (Fig.5B). Next, we sought to determine whether SREBP-1 wouldmediate castration resistance in prostate tumor xenografts

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Figure 3. SREBP-1 promotes cell proliferation,migration and invasion in prostate cancer cells. A, H1andH2showed increased cell proliferation comparedwithLNCaP and control Neo during 3-day incubation. The relative cell proliferation (%) was assigned as 100% in LNCaP cells. ��, P < 0.005, significantdifferences from Neo. Data represent the mean � SD of 2 independent quadruplicate experiments. B, SREBP-1 significantly induced in vitro cell migrationand invasion in H1 and H2 compared with control groups. �, P < 0.05; ��, P < 0.005 significant differences from Neo. C, knocking down SREBP-1 by shRNAwith lentiviral delivery in high intrinsic SREBP-1 C4-2B cells showed decreased SREBP-1, AR, and FASN expression determined by Western blot. D,downregulationofSREBP-1byshRNAshoweddecreasedcell proliferationofC4-2B. The relative cell proliferation (%)wasassignedas100%inuntransfectedC4-2B cells (-). ��, P < 0.005 significant differences from nonspecific control shRNA-transfected C4-2B cells (Con).






grown in mice. We first observed, as expected, that R1881induced in vitro cell proliferation in control Neo but notSREBP-1 expressing H2 cells (Supplementary Fig. S1).Strikingly, upon castration (week 6), subcutaneous H2tumor growth continued compared with Neo tumors (Fig.5C, top panel). Serum PSA levels of both Neo and H2tumor–bearingmice dropped in the first week postcastration(week 7). However, serum PSA levels of H2 mice signifi-cantly rebounded 4 weeks after castration (week 10) com-pared with Neo mice (Fig. 5C, bottom panel). These resultssuggested that SREBP-1 regulates prostate tumor occur-rence, growth, and even resistance to the effects of castrationin mice.

Discussion

Aberration of cellularmetabolisms has been reported to bestrongly linked with cancer. Cancer cells reprogram energyproduction through aerobic glycolysis followed by lactic acidfermentation in the presence of oxygen (the Warburg effect;ref. 38). Upregulation of de novo lipogenesis (fatty acid, lipid,and cholesterol biosynthesis) in cancer cells is associatedwithincreased needs for membranes and energy storage, andactivation of intracellular signaling pathways during uncon-trolled cell proliferation and division as well as cancerdevelopment and progression (6, 7, 9, 39, 40). Lipogenicactivation has also been shown to increase the biosynthesis ofandrogens in prostate cancer cells (4–6). Androgens and ARsignaling are involved in the regulation of prostate cancerdevelopment and lethal castration-resistant progression.

However, the mechanism of dysregulation of lipogenesis inprostate cancer cells and its contribution to prostate cancerdevelopment and progression remain unclear.We previouslyshowed that a key lipogenic transcription factor (15–17),SREBP-1, regulated AR promoter activity and transcrip-tional expression as well as cell viability in prostate cancercells (5). In this study, we further revealed a novel molecularmechanism by which SREBP-1 promotes prostate cancergrowth and progression through collaborative induction ofAR expression, lipogenesis and oxidative stress. The resultswere confirmed by genetic approaches in which SREBP-1overexpression or knockdown affected AR, FASN, andNox5 expression, fatty acid and lipid droplet accumulation,and ROS levels in prostate cancer cells. These biochemicalalterations were found to be closely associated with cellgrowth and behavioral changes, which can be monitoredby prostate cancer cell proliferation,migration, and invasion.The data in aggregate suggest that SREBP-1 not only is acrucial mediator for lipogenesis as previously described butalso induces AR a known survival factor and increasesoxidative stress through induction of Nox5, an importantROS generator, in prostate cancer cells.ROSandNoxhave been closely linked to the initiation and

progression of cancer (34, 35, 41, 42). ROS are produced incells when oxygen is metabolized, including superoxide,hydrogen peroxide, and hydroxyl radicals. Excessive ROSaccumulation in cells can cause cell injury by damaging vitalmolecules such as DNA, RNA, and proteins. Increasedintracellular ROS, however, often leads to the enhancedgrowth, survival, and progression of cancer cells (43–45).

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Figure 4. SREBP-1 induces cell proliferation and progression through alterations of Nox5 and ROS in prostate cancer cells. A, SREBP-1 induced Nox5 andp-Akt, and inhibited catalase expression in H1 andH2 cells determined byWestern blot. B, SREBP-1 increased the levels of hydrogen peroxide in H1 and H2.The levels of superoxidewere not significantly changed by SREBP-1. The relative DCF (%) andDHE (%)were assigned as 100% in LNCaP cells. ��,P < 0.005,significant differences from Neo. Data represent the mean � SD of 2 independent triplicate experiments. C, DPI, a Nox inhibitor and ROS scavenger,inhibited cell proliferation of Neo and H2 in a dose-dependent pattern (0–5 mmol/L) during a 2-day treatment. H2 cells showed increased resistance toDPI-mediated suppression of cell proliferation. The relative cell proliferation (%) was assigned as 100% for each cell without DPI treatment. ��, P < 0.005,significant differences from cells without DPI treatment. Data represent the mean � SD of 2 independent quadruplicate experiments. D, DPI also inhibitedAR expression in H2 cells.

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Hydrogen peroxide has been shown to induce cell transfor-mation of nontumorigenic urothelial cells (44) and increaseprostate tumorigenicity (42). Significant spheroid growthstimulation occurred when cancer cells were exposed tohydrogen peroxide (46). Our results confirmed that over-expressing SREBP-1 in prostate cancer cells increased ROS(hydrogen peroxide) levels, induced cell proliferation, migra-tion, and invasion in vitro, and promoted subcutaneoustumor growth inmice. Furthermore, SREBP-1 also increasedp-Akt protein expression (Fig. 4A). The data are consistentwith published results in which activation of an Akt survivalsignaling pathway can occur through ROS regulation inseveral cancer models (25, 47, 48). Together, our resultsreveal a previously unrecognized role of SREBP-1 promotingprostate cancer growth and survival and increasing prostatecancer cell migration and invasion through augmentedNox5

expression, intracellular ROS, and activation of AR and Aktsignaling, which ultimately could be responsible for theincreased aggressiveness and malignancy of prostate cancercells commonly associated with castration resistance.We propose that one of the key mechanisms by which

SREBP-1 promotes prostate cancer progression is throughthe induction of Nox5 expression, which enhances ROSlevels in cells. We identified a SREBP-1 binding site in the50-flanking Nox5 promoter region (data not shown), whichsuggests that SREBP-1 controls Nox5 expression via pro-moter transcriptional regulation. Studies using DPI, a Noxactivity inhibitor and ROS scavenger, provide additionalevidence that SREBP-1 increases ROS levels and promotesprostate cancer cell proliferation through Nox5. In addition,inhibition of Nox5 expression and decrease of ROS pro-duction by Nox5-specific antisense oligonucleotides caused

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Figure 5. SREBP-1 promotes human prostate tumor growth and castration resistance in mouse subcutaneous xenograft models. A, tumor growth wasassayed by tumor volume after subcutaneous inoculation of H2 and control Neo cells inmice. SREBP-1 significantly induced the growth of H2 comparedwithNeo tumors. ��, P < 0.005, significant differences from Neo tumors. B, IHC of subcutaneous Neo and H2 tumor specimens. H2 tumors highly expressedSREBP-1 (mostly in nuclei), FASN (cytoplasm), Nox5 (cell membranes), and AR (mostly in nuclei) proteins comparedwith Neo tumors. Scale bar¼ 100 mm.C,mouse castration study. Tumor volumesof subcutaneousH2 tumors continuously increasedaftermouse castration (week6) comparedwithNeo tumors (top).Serum PSA levels of both Neo and H2 tumor–bearing mice dropped the first week postcastration (week 7, bottom). However, PSA levels of H2 micesignificantly rebounded 4 weeks after castration (week 10) compared with the Neo group. �, P < 0.05, significant differences from Neo.






a reduction in the growth of prostate cancer cells (35). Thisarticle further supports our findings. Furthermore, DPI hasbeen shown to inhibit cell migration and invasion anddecrease matrix metalloproteinase (MMP)-2 and MMP-9expression and activity in PC3 prostate cancer cells (34).Interestingly, by decreasing Nox activity and ROS levels,DPI greatly inhibited AR expression in LNCaP cells (Fig.4D). It could be due to DPI decreasing intracellular hydro-gen peroxide levels (42) because hydrogen peroxide has beenshown to affect AR expression in LNCaP cells (37). Thesedata are consistent with the suggestion that SREBP-1increases ROS production through transcriptional regula-tion of Nox5 expression in prostate cancer cells. TargetingNox, ROS, and AR by DPI may be a promising therapeuticapproach for the treatment of lethal progression of humanprostate cancer.Very limited information is currently available on the

SREBP-1 expression profile of clinical prostate cancer. Oneearly study showed that SREBP-1 was elevated in humanprimary prostate tumors compared with benign prostatichypertrophy (23). In addition, dysregulated SREBP-1expression may be relevant to prostate cancer castration-resistant progression (23). Our results in a human prostatecarcinoma TMA further showed that overexpression ofSREBP-1 protein is significantly associated with aggressivepathologic features in human prostate cancer (Fig. 1and Table 1). Importantly, SREBP-1 was highly expressedin the nuclei of prostate tumor cells with higher Gleasongrades (Fig. 1). The precursor of SREBP-1 protein is an ERmembrane–bound form. Through a proteolytic process(18), the mature amino-terminal polypeptide is translocatedto the nuclei to activate the expression of lipogenesis relatedand other genes containing SREBP-1 binding sites in theirpromoter regions, such as FASN (31), AR (5), andNox5 (thepresent study). Furthermore, IHC results of mouse bearingsubcutaneous human prostate tumor xenografts showed thatSREBP-1 was highly expressed in the nuclei of H2 (i.e.,LNCaP–SREBP-1) tumor specimens collected from bothintact (Fig. 5B) and castrated (data not shown) mice, whichexhibited higher tumor incidences, burdens, and serum PSAlevels, when compared with control Neo tumor specimens.The clinical and animal data collectively indicate thatSREBP-1 expression and nuclear translocation play a criticalrole in the regulation of prostate cancer development andprogression to castration resistance. Further investigation ofthe regulatorymechanism of SREBP-1 nuclear translocationin prostate cancer cells might be of importance.The present studies show for the first time that (i) analysis

of a human prostate carcinoma TMA with varying grades ofdiseases revealed that SREBP-1 expression positively corre-lates with prostate cancer progression. Nuclear translocationof SREBP-1may also be closely associated with the degree ofprostate cancer malignancy. (ii) Genetic alterations ofSREBP-1 expression led to coordinated regulation of FASN,AR, and Nox5 expression in prostate cancer cells. (iii)Through the dual induction of FASN and Nox5 expression,SREBP-1 increased fat (fatty acid and lipid droplets) andROS (hydrogen peroxide) accumulation in prostate cancer

cells. (iv) SREBP-1 induced prostate cancer cell prolifera-tion, migration, and invasion in vitro and promoted prostatetumor growth and castration-resistant progression in vivo.Collectively, the molecular mechanism by which SREBP-1promotes prostate tumor growth and resistance to castrationand androgen responsiveness is through a concerted activa-tion of AR, lipogenesis, andROS signaling networks (Fig. 6).Data presented in this article are collected from the study ofestablished AR-positive human prostate cancer cell lines, andfurther investigation of this concept in AR-negative prostatecancer cells might be of importance. Also, additional studiesmay be warranted to define whether the concerted signalingwould function in tumor microenvironment, for example,tumor–stroma interaction. Taken together, we identifiedthat SREBP-1, a transcription factor known to regulate fatbiosynthesis and homeostasis, promotes and maintainsprostate cancer growth and progression by activating andreprogramming the AR/lipogenesis/ROS signaling axis.SREBP-1 and its ancillary regulatory signaling pathwaysmay, therefore, be novel promising therapeutic targets forthe prevention and treatment of lethal progression in humanprostate cancer.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank their colleagues for helpful discussions andMr.GaryMawyer forediting the manuscript and also thank Drs. Xiaojian Yang and Yueming Wang for thehelp of mouse study, Mr. Dror Berel (Cedars-Sinai Medical Center) for the assistanceof statistical analysis, and Dr. Gene Siegal (Anatomic Pathology, the University ofAlabama at Birmingham) for the help of score of human prostate carcinoma tissuemicroarray.

SREBP-1

AR

FASN

LipogenesisROS/oxidative stress

Prostate cancer growth, development, and progression

Nox5(a metabolic oncogene)

Figure 6. Proposed mechanism by which SREBP-1 promotes the growthand progression of prostate cancer by the activation of AR, lipogenesis,and ROS/oxidative stress. SREBP-1 induces AR transcriptionalexpression and activity in prostate cancer cells. SREBP-1 also activatesFASN expression and further induces lipogenesis. By induction of Nox5expression, SREBP-1 increases ROS levels and oxidative stress inprostate cancer cells. Through a concerted activation of AR, lipogenesisand ROS signaling networks, SREBP-1 promotes cell growth,development, and lethal progression of prostate cancer.

Huang et al.





Grant Support

This work was supported by grants from NIH grants 2P01 CA098912(L.W.K. Chung) and Department of Defense grants W81XWH-08-1-0321(W-C. Huang).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received May 6, 2011; revised September 12, 2011; accepted November 2, 2011;published OnlineFirst November 7, 2011.

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2012;10:133-142. Published OnlineFirst November 7, 2011.Mol Cancer Res Wen-Chin Huang, Xiangyan Li, Jian Liu, et al. Growth and Progression of Prostate Cancer CellsStress Converged by SREBP-1 Is Responsible for Regulating Activation of Androgen Receptor, Lipogenesis, and Oxidative

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