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Estrogen receptor activated apoptosis in benignhyperplasia and cancer of the prostate is androgenindependent and TNF mediatedStephen J. McPhersona,1, Shirin Hussaina,1, Preetika Balanathana, Shelley L. Hedwardsa, Birunthi Niranjana,Michael Granta, Upeksha P. Chandrasiria, Roxanne Toivanena, YuzhuoWangb,c, Renea A. Taylora, and Gail P. Risbridgera,2

aProstate and Breast Cancer Research Group, Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria 3800, Australia;bVancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada V6T 1Z4; and cDepartment of CancerEndocrinology, British Columbia Cancer Agency, Vancouver, BC, Canada V5Z 1L3

Edited* by Jan-ke Gustafsson, Karolinska Institutet, Huddinge, Sweden, and approved January 4, 2010 (received for review May 20, 2009)

Prostate cancer (PCa) and benign prostatic hyperplasia (BPH) areandrogen-dependent diseases commonly treatedby inhibiting andro-gen action. However, androgen ablation or castration fail to targetandrogen-independent cells implicated in disease etiology and recur-rence. Mechanistically different to castration, this study showsbeneficial proapoptotic actions of estrogen receptor (ER) in BPHand PCa. ER agonist induces apoptosis in prostatic stromal, luminaland castrate-resistant basal epithelial cells of estrogen-deficient aro-mataseknock-outmice. This occurs via extrinsic (caspase-8) pathways,without reducing serum hormones, and perturbs the regenerativecapacity of the epithelium. TNF knock-out mice fail to respond toER agonist, demonstrating the requirement for TNF signaling. Inhuman tissues, ER agonist induces apoptosis in stroma and epithe-lium of xenografted BPH specimens, including in the CD133+ enrichedputativestem/progenitor cells isolatedfromBPH-1cells invitro. InPCa,ER causes apoptosis in Gleason Grade 7 xenografted tissues andandrogen-independent cells lines (PC3 and DU145) via caspase-8.These data provide evidence of the beneficial effects of ER agoniston epithelium and stroma of BPH, as well as androgen-independenttumor cells implicated in recurrent disease. Our data are indicative ofthe therapeutic potential of ER agonist for treatment of PCa and/orBPH with or without androgen withdrawal.

castration | steroid receptors | selective estrogen receptor modulators

Benign prostatic hyperplasia (BPH) and prostate cancer (PCa)are the most common benign and malignant diseases in agingmen (1, 2).BPHarises in the transition zoneor peri-urethral glandswhere stromal and epithelial nodules develop, whereas PCa arisesin the peripheral zone of the prostate gland where epithelial cellsundergo malignant transformation. These androgen-dependentdiseases are treated by inhibiting androgens or their action. In PCa,androgen ablation fails to target castrate-resistant or androgen-independent cell types, implicated in disease etiology and recur-rence. Androgen blockade in men with PCa is effective initiallybecause it causes apoptotic regression in the bulk of the tumor,although significant side effects include hypogonadism, gyneco-mastia, anemia, and metabolic syndrome, for which further treat-ments are required. Nevertheless, relapse frequently occurs, assubpopulations of cells are either castrate-resistant or adapt toandrogen-deplete conditions, resulting in incurable castrate-resistant PCa (3). For BPH, anti-hormonal treatments are asso-ciated with the same side effects and often fail to permanentlyreduce prostatic volume or to ease lower urinary tract symptoms(4). Thus, new therapies for PCa or BPH are required that are aseffective as androgen withdrawal but also target castrate-resistantcells implicated in disease recurrence.Although estrogens were previously used for PCa therapy, their

efficacy was based on indirect suppression of androgen levels; theyalso resulted in adverse side effects such as cardiovascular andthromboembolic events (5). It is now known that estrogens actingvia ER mediate aberrant epithelial cell proliferation, prostatic

inflammation, and malignancy (69), and ER antagonists such asToremifine are in clinical trial for PCa prevention/progression (10).In contrast, effects of estrogen mediated by ER are beneficial; weand others previously reported anti-proliferative activity of ERagonist in the prostate, independent of systemic androgens (and notinvolving the suppression of serum testosterone) but requiringintraprostatic stromalepithelial cell signaling (6, 1113).The aimof this studywas to investigate the therapeutic potential

of an ER agonist of proven selectivity (1417), specificallyinvestigating its proapoptotic mechanism of action compared withcastration. This compound (8-VE2) has proven selectivity andwas previously used to dissect the physiological roles of ER andER in vivo in bone, cardiovascular, and metabolic studies (1418). To circumvent the use of a specific ER knock-out mousemodel because of reported variation in prostatic phenotypes fromdifferent colonies (19), we used aromatase knock-out (ArKO)mice that lack endogenous estrogen ligands but express functionalERs (20), thus obviating any confounding action of ER activationby endogenous ligands. Using these mice, we compared the cel-lular targets andmechanismof action of ER agonist to castration.We further verified our findings by comparing castration and ERagonist using human prostatic specimens and cell lines to test thetherapeutic potential of ER agonists in PCa andBPH.Our resultsprovide independent, unequivocal proof of the concept initiallyproposed by Gustafsson et al. that ER is anti-proliferative andproapoptotic in the prostate (13), and demonstrate a mechanismof ER action that is androgen-independent and mediated byTNF, targeting castrate-resistant epithelial cells.

ResultsER Agonist Increases Apoptosis and Reduces Proliferation inProstatic Stroma and Epithelia. Treatment with ER agonist for 6weeks abrogated prostatic hyperplasia and hypertrophy of ArKOmice (21) because of reduced cellular proliferation; more acutely,a time course study showed that ER-induced apoptosis wasmaximal at 37 days, compared with the effect of an ER agonistthat causes inflammation (Fig. S1 A and B). Figure 1 showsproapoptotic effects of ER agonist in ArKO or wt mice within

Author contributions: S.J.M., S.H., andG.P.R.designed research; S.J.M., S.H., P.B., S.L.H., B.N.,M.G., and U.P.C. performed research; S.J.M., S.H., R.T., and Y.W. contributed new reagents/analytic tools; S.J.M., S.H., P.B., S.L.H.,M.G.,U.P.C., andR.A.T. analyzeddata; andS.J.M., S.H.,R.A.T., and G.P.R. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

*This Direct Submission article had a prearranged editor.1S.J.M. and S.H. contributed equally to this work.2To whom correspondence should be addressed. E-mail: gail.risbridger@med.monash.edu.au.

This article contains supporting information online at www.pnas.org/cgi/content/full/0905524107/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0905524107 PNAS | February 16, 2010 | vol. 107 | no. 7 | 31233128

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http://www.pnas.org/cgi/data/0905524107/DCSupplemental/Supplemental_PDF#nameddest=sfig01mailto:gail.risbridger@med.monash.edu.aumailto:gail.risbridger@med.monash.edu.auhttp://www.pnas.org/cgi/content/full/0905524107/DCSupplementalhttp://www.pnas.org/cgi/content/full/0905524107/DCSupplementalwww.pnas.org/cgi/doi/10.1073/pnas.0905524107

3 days, compared with those in intact vehicle-treated, castrate, orER agonisttreated mice. Contemporary stereology and mor-phometric analyses show that ER agonist significantly increasedepithelial and/or stromal apoptosis vs. vehicle controls in ArKO(Fig. 1A) and wt mice (Fig. 1B). Castration significantly increasedepithelial but not stromal apoptosis, whereas ER agonisttreatedtissues showed levels of apoptosis similar to controls in all cellularcompartments (Fig. 1A andB). Further subdivision into epithelialluminal and basal cells based on location and CKH immunor-eactivity (basal cells are CKH-positive) showed that luminal epi-thelial cell apoptosis was significantly increased by both castrationand ER agonist, but only ER agonist caused apoptosis of basalcells (Fig. 1A and B and Fig. S1C). In ArKOmice, ER agonist orcastration (but not ER agonist) significantly reduced epithelial(but not stromal) cell proliferation (quantified by PCNA staining)compared with controls; proliferation was reduced in luminal andbasal epithelia (Fig. S2A). Similar results were observed in wild-type (wt) mice in which epithelial (but not stromal) cell pro-liferation was lowered by castration and ER agonist (Fig. S2B).Altogether, these data showed that ER agonist uniquely causedapoptosis in the castrate-resistant basal cell layer, reducing cellproliferation and increasing apoptosis in the luminal epithelial andstromal cells of hyperplastic and normal mouse prostate.

Epithelial Regeneration After ER Agonist Results in Cystic Atrophyand Depletion of p63+ Basal Cells.Basal cells maintain the structuralintegrity of the prostatic epithelium (22) and are necessary fortissue regeneration occurring over repeated cycles of androgendeprivation and replacement. Following ER agonist-inducedapoptosis in basal cells, we examined whether ER agonisttreatment disrupted epithelial regenerative capacity. Twenty-onedays posttreatment, ER agonist-treated tissues showed regions

of cystic atrophy with expansion of the fluid-filled lumen (Fig.S3A) not seen in control or castrate-recovery tissues as evidenceof perturbed glandular secretion. Prostates from castrated ani-mals treated with androgens or intact control animal tissuesshowed normal morphology. The apparent frequency of p63+cells within atrophic regions of ERagonist treated tissues wasre