rticle history:eceived 29 December 2014eceived in revised form 1st February 2015ccepted 4 February 2015
eywords:rostate cancerndrogen receptoradiotherapy
a b s t r a c t
Androgen deprivation therapy is widely used in combination with radiotherapy for the treatment ofprostate cancer. The knowledge of the biology of the androgen axis could help the radiation oncologistto combine both modalities in an efficient way. Moreover, new drugs have recently been approved andtheir role in combination with radiation needs pre-clinical and clinical studies. This review summarizedthe main data on the biology of androgen receptor and the potential implications for the physician.Mechanisms of interactions between androgen deprivation therapy and radiotherapy are also presentedand discussed.
ots clés :ancer de la prostateécepteur aux androgènesadiothérapie
r é s u m é
La suppression androgénique est de plus en plus utilisée en association avec la radiothérapie dans le can-cer de prostate. La connaissance de la biologie des androgènes peut aider l’oncologue radiothérapeutedans le maniement des associations d’hormonothérapie et de radiothérapie. En outre, de nouveauxmédicaments ont été récemment mis sur le marché dans le cadre du cancer de prostate résistant à
la castration. Leur emploi potentiel avec la radiothérapie doit être évalué dans le cadre d’études pré-cliniques et cliniques. Cette revue résume les connaissances acquises sur le récepteur aux androgèneset leurs implications potentielles pour le clinicien. Les mécanismes d’interaction entre la suppressionandrogénique et la radiothérapie sont également résumés.
Combination of androgen deprivation therapy and radiotherapys now the standard of care for prostate cancers patients of theigh-risk group and some of the intermediate group. If the clinicalenefit of this association is now clearly demonstrated, the biologi-al interactions between the hormonal treatment and radiotherapyre not fully understood. Moreover, new hormonal agents as abi-
aterone and enzalutamide have been approved for the treatmentf castration-resistant prostate cancers. Their use in earlier phases
of the disease, particularly with radiotherapy, is more and morediscussed.
The aim of this review is to summarize the present knowledge ofthe physiology of the androgen receptor and to discuss the potentialinteractions between old and new hormonal drugs with radiation.
2. Prostate cancer and androgens
2.1. Prostate cancer biology
Prostate is a gland that synthesizes components of the seminal
fluid, including proteases as prostate-specific antigen (PSA). In thenormal adult prostate, the androgen receptor is expressed in allluminal cells and in some epithelial basal cells as well in stromalcells.
Prostate cancer develops from epithelial cells, but it is not clearf it arises from basal or luminal cells [1]. However, the role oftromal cells is essential to promote growth of prostate cancer,hrough the secretion of various growth factors. More than 50%f prostate cancers harbour a gene fusion between the promoterf transmembrane protease, serine 2 (TMPRSS2) and portions ofhe coding regions of E-twenty-six (ETS) transcription factors [2],
ainly erythroblast transformation-specific gene (ERG). TMPRSS2s an androgen responsive gene (see below). The ETS family mem-ers are transcription factors, regulating cell proliferation, celligration, cell cycle control and apoptosis [3]. Genomic profiling
evealed that mutations in the androgen receptor signalling path-ay is much more altered than the others pathways [4].
.2. Androgen dependency of prostate cancer
Androgen stimulation is fundamental for prostate cancerrowth. Androgen deprivation therapy has demonstrated morehan 60 years ago a dramatic effect on cancer progression [5].ndrogen deprivation therapy is obtained by surgical castrationr by the use of luteinizing hormone-releasing hormone (LH-RH)gonists or antagonists. Androgen deprivation therapy is supposedo reduce the serum testosterone level under 0.5 ng/mL. It is stillebated whether a level lower than 0.2 ng/ml improves the clinicalesponse rate and its duration. Combination of LH-RH agonists andnti-androgens is called complete androgen blockade: its useful-ess was not demonstrated in the treatment of metastatic prostateancer. The androgen receptor could induce directly cancer growth,or example via the TMPRSS2-ETS fusion factor. But it can also crossalks with numerous growth factors (transforming growth factor�TGF�], vascular endothelial growth factor [VEGF], insulin-likerowth factor [IGF], epithelial growth factor [EGF] and fibroblastrowth factor [FGF]). For instance, TGF� cooperates with andro-en receptor signalling to promote stromal cell growth. Androgeneprivation therapy remains the principal treatment of relapsefter radiotherapy [6].
.3. Androgens synthesis
Testosterone is the principal circulating androgen and is syn-hesized by the testis for the major portion. Less than 3% of theirculating testosterone is bioavailable, most of it being boundo different proteins, sex hormone-binding protein (SHBG) andlbumin, essentially. The remaining androgens in the bloodstream5–10%) are dehydroepiandrosterone (DHEA), androstenediol andndrostenedione: they are produced by adrenals or by peripheralonversion of testosterone. Androgens production is regulated byhe hypothalamic-pituitary-gonadal axis. LH-RH is secreted by theypothalamus in pulses, thus stimulating luteinizing hormone (LH)ecretion, which acts on Leydig cells in the testis to induce androgenroduction. Testosterone acts on hypothalamus through a negativeeedback to prevent LHRH release.
Steroids synthesis begins with mobilization by the steroido-enic acute regulatory protein (StAR) and cleavage by CYP11A1f cholesterol in pregnenolone (Fig. 1). Subsequent metabolismo mineralocorticoids, glucocorticoids, androgens or oestrogensepends of the enzymatic equipment of the tissue. Androgens areroduced from the conversion of pregnenolone-like steroids by theYP17, a cytochrome P450 enzyme. CYP17 catalyzes hydroxylasend lyase reactions: the hydroxylase activity is similar for pre-nenolone and progesterone but the lyase activity is much moreotent for 17-OH-pregenolone than 17-OH-progesterone. Hence,
he synthesis of androgens is mainly performed via DHEA. Poly-
orphisms of the CYP17 gene have been associated with a higherisk of prostate cancer and a worst prognosis of castration-resistantrostate cancers [7,8].
rapie 19 (2015) 220–227 221
DHEA and androstenedione, synthesised in adrenal glands, aretransformed in testosterone in the Leydig cells of the testis. Testos-terone is released in the bloodstream and enters into the targetcell via passive diffusion through the plasma membrane, butactive transport by organic anion-transport polypeptides have beendescribed [9]. Testosterone is converted into target tissues (prostateand a limited number of other tissues) in 5�-dihydro-testosteroneby 5-alpha reductase (SRD5A1 or 2). There are two types of 5-alphareductase, depending on the target tissue, the type 2 being morespecific for prostatic epithelial cells.
3. The androgen receptor
The androgen receptor is a nuclear transcription factor and is amember of the steroid receptor superfamily. The androgen recep-tor gene is located on the X-chromosome at position Xq11-12 andcontains eight exons that encode a protein of ≈919 amino acids.Two isoforms of androgen receptor have been described: the pre-dominant isoform B (110 kDa) and the less dominant isoform A(80 kDa).
Like other members of the nuclear receptor superfamily, theandrogen receptor is composed of four domains (Fig. 2) [10]:
• the transcriptional activation domain (exon 1: N-terminaldomain): this domain is necessary and sufficient for transcrip-tion activity. It harbours a variable number of highly repetitiveDNA sequences, such as CAG, coding for polyglutamin. The num-ber of CAG repeats is correlated to the transcriptional activity ofthe androgen receptor, the shorter CAG repeat length resulting ina higher transcriptional activity. Racial groups with a high preva-lence of prostate cancer had also more frequently a short CAGrepeats, suggesting that early tumorigenesis is dependent on amore active androgen receptor. The N-terminal domain containsalso coregulators’ interaction domains;
• the DNA-binding domain (exon 2 and 3) with two zinc-fingerregions;
• a flexible hinge region separates the DNA-binding domain fromthe androgen-binding domain: it contains the nuclear localiza-tion signal, allowing the androgen receptor to interact with thecytoskeleton and to migrate to the nucleus, after binding ofimportin-� [11];
• the ligand-binding domain at the C-terminal end (exons 4-8),containing an androgen-binding pocket.
The activation function AF-1 and AF-2 domains are requiredfor optimal transactivation. AF-1 undergoes folding when con-tacted by transcription factor TFIIF after binding of the AR on DNA,enabling coregulators recruitment. AF2 is a coactivator-bindingsite, unmasked after ligand binding.
Ligand-free androgen receptor is sequestered in the cytoplasmbound to heat-shock proteins (HSP 70, 90 and p23), which protectit from degradation. After binding of the ligand, a conformationalchange of the androgen receptor occurs, which allows dissociationof heat-shock proteins.
Testosterone and dihydrotestosterone can bind to the andro-gen receptor, but the latter forms a more stable complex andis 3–10 times more potent. Binding of the ligand initiates anintramolecular interaction between the N-terminal domain andligand-binding domain, termed N/C interaction [12]. The andro-gen receptor becomes phosphorylated and translocates into thenucleus (Fig. 3). The androgen receptor is phosphorylated at
several sites by different kinases, including mitogen-activated pro-tein kinase (MAPK), protein kinase C (PKC) and protein kinaseB (AKT/PKB). They interact with the androgen receptor, usuallyin a ligand-dependent manner. Phosphorylation stabilizes the
222 L. Quero et al. / Cancer/Radiothérapie 19 (2015) 220–227
Fig. 1. Synthesis of steroids. In blue: classical pathway – cholesterol is transformed in pregnenolone, then to dehydroepiandrosterone (DHEA) by cytochrome P450 enzymeCYP17A1. DHEA is converted to androstenedione through the action of 3�-hydroxysteroid dehydrogenase (3�HSD) enzymes. 3�HSD1 is present in peripheral tissues such asprostate, skin or breast and is ten-fold more potent than 3�HSD2 expressed in adrenal, testes and ovaries. Androstenedione is converted in testosterone by 3�HSD3 (testis)or AKCR1C3 (peripheral tissues). Then 5�-dihydro-testosterone (5�-DHT) is produced by 5�-reductase 1 and 2. In green: Backdoor pathway–17-OH-progesterone are actedon by 5�-reductase and aldo-keto reductase 1C (AKR1C) prior to the lyase activity of CYP17A to obtain androsterone, which is converted to androstanediol. An oxidatives etc.) isa 5�-at
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tep, which could be made by different enzymes (17BHSD 6 or 10, RODH4, RDH5,
ndrostenedione pathway) – androstenedione is converted first by 5�-reductase toestosterone as a required intermediate.
igand/receptor complex and is required for translocation to theucleus [13]. The androgen receptor dephosphorylation by the pro-ein phosphatase 2A can lead to loss of its transcriptional activity14].
The androgen receptor forms homodimers (and also het-rodimers with others proteins) and binds to specific DNA regions,ermed androgen-responsive elements, in association with a seriesf transcriptional coregulators. These androgen-responsive ele-ents are located in the promoter regions of target genes and
erve to activate or repress transcription of specific androgen-
egulated genes. About 146 androgen-regulated genes have beenescribed [15]. In the mature gland, androgens promote cell divi-ion and proliferation of epithelial cells, modulate programmed celleath, regulate several aspects of prostate cellular metabolism and
ig. 2. Structure of the androgen receptor and its gene. NTD: N-terminal domain (transctivation function.
required to convert androstanediol to 5�-DHT. In pink: alternative pathway (5�-ndrostenedione and then by AKR1C3 (or 17�HSD) to 5�-DHT, thus circumventing
control the production of specific proteins such as PSA. For example,activation of androgen-responsive elements results in an enhancedexpression of cyclin-dependent kinases 2 and 4, down regulationof the cell cycle inhibitor p16, thereby enhancing cell proliferation.An up-regulation of p21, an inhibitor of apoptosis, is also observed.
4. Coregulators of the androgen receptor
By definition, these are proteins that increase or inhibit thetranscriptional activity of the androgen receptor [16]. Over 170
coregulators of androgen receptor functions have been identified,either activators or repressors [17].
Some co-activators, as c-jun or steroid receptor co-activator-2(src2), bind to the N-terminal domain of the androgen receptor and
L. Quero et al. / Cancer/Radiothérapie 19 (2015) 220–227 223
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ig. 3. Androgens and androgen receptor signalling in prostate cancer. Dihydrotrom heat-shock protein (HSP). The androgen receptor translocates to the nucleus,oactivators (COacT), androgen-regulated genes (ARG).
romote its homodimerization. SRC-2 is overexpressed or amplifiedn 11% of prostate cancers [4]. SRC-2 expression is repressed byndrogens and thus, androgen deprivation therapy increases theRC-2 level.
Most coregulators are recruited to gene-promoter regions afterinding of androgen receptor dimers on androgen-responsive ele-ents [12]. They function to facilitate or suppress transcription.arious members of the steroids family can recognize the samendrogen-responsive element. So, the specific response of a tissues dependent on the coactivators or repressors. Some coactiva-ors, including SRC-1 and cyclic adenosine monophosphate (cAMP)esponse element-binding protein can induce the remodelling ofhromatin by acetylating histones, opening the nucleosomal struc-ure of the gene and allowing the initiation of transcription by RNAolymerase II.
. Mechanisms of castration resistance
In advanced cancer treated with castration, a clinical and bio-hemical response is observed in the majority of patients, butventually, a resistance state will emerge after a variable delay. Cas-ration resistance is defined as a progression of the disease, eitheriological or clinical, with a testosterone level less than 0.5 ng/mL.lthough the exact mechanisms of castration resistance are notell understood, available data support the physical presence and
ctivity of the androgen receptor. Castration-resistant prostate can-er is resistant to androgen deprivation therapy but remains oftenndrogen receptor-dependent [18]. This is evidenced by the usualise in serum PSA levels at the time of prostate cancer progression.
.1. Mutation, amplification or overexpression of the androgeneceptor
In a relatively large proportion of patients with castration-
esistant prostate cancers, the androgen receptor is expressed,verexpressed, mutated or amplified. Mutations of the receptorave been described, particularly in the ligand-binding domain.pecificity of the ligand-binding domain could be modify, allowing
erone (DHT) binds to the androgen receptor (AR) and promotes its dissociationrizes and binds to androgen-response elements (ARE), activating, with the help of
receptor activation by a low concentration of androgens [19] or byweak endogenous androgens. The androgen receptor could respondto non-androgen steroidal hormones as progesterone and estradiol[20]. Even anti-androgens such as flutamide or bicalutamide couldbecome agonists of the androgen receptor [21]. However, thesemutations are detected in 10 to 20% of cancer specimens and areresponsible of the androgen escape in only a few patients [22,23].
Androgen receptor gene amplification is very common duringthe castration-resistant state [24], leading eventually to its overex-pression, making tumours cells extremely sensitive to low levels ofcirculating androgens. Combined androgen blockade is potentiallya good way to overcome this androgen receptor amplification [25].
5.2. Alternative splicing of the androgen receptor messenger RNA
Many genes are transcribed with alternative exon usage, andthis is the case for the androgen receptor gene. It has been demon-strated that alternative splicing of the androgen receptor mRNAcould culminate in a receptor that is capable of translocation andDNA binding without ligand binding [26]. Expression of these vari-ants is correlated with castration resistance [27]. AR-V7 is one of thebest-characterized splice variants, present in castration-resistantprostate cancers patients [28]: it lacks the ligand-binding domainand is constitutively active, independently of any androgens. Othersplice variants are required to dimerize with full-length recep-tors for nuclear translocation and transcriptional activity, and soremains dependent of androgens [29].
The transcript ARv567es, in which exons 5, 6, and 7 are deleted,appears to be less dependent of full-length receptors for transcrip-tional activity. On the other hand, AR-V7 and Arv567es facilitatefull-length receptors nuclear localization in the absence of andro-gen [30]. It has been recently shown that the presence of thisvariant is correlated to abiraterone and enzalutamide resistance
[31]. However, there is at this moment no evidence that thesesplice variants are present in localized diseases and in hormone-naive prostate cancers. For locally advanced diseases, when acombination of androgen deprivation therapy and new
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nti-androgens is considered, looking for this characteristicould be recommended.
.3. Alterations of coactivators and others intracellular pathways
Modifications of coactivators has also been described: theyould increase the androgen receptor transcriptional activity in theresence of low ligand concentration or by altering the ligand speci-city of the receptor, allowing antiandrogens and estrogens to act asgonists [32]. ARA70 is often overexpressed in castration-resistantrostate cancers and facilitates the activation of the androgeneceptor by non-androgenic molecules such as 17�-estradiol (E2)33]. Interaction of SRC-1 with the androgen receptor results in itso the same magnitude as that obtained by dihydrotestosterone34]. SRC-1 and -2 expression is increased in more than 60% ofastration-resistant prostate cancers. In conclusion, it is clear thatverexpression of androgen receptor coactivators contribute torostate cancer progression.
Activation of the androgen receptor could be a consequence ofctivating the PI3K/AKT/mTOR pathway via IGF-1, KGF or EGF [35].he androgen receptor activation can result also by binding of longon-coding RNAs [36].
.4. Modifications of the androgen synthesis pathways
It has been demonstrated that significant amount of dihy-rotestosterone have been detected in castration-resistantumours [37]. Numerous studies suggest that synthesis of andro-ens occur de novo in castration-resistant prostate cancers. Genesncoding enzymes involved in androgen metabolism (and partic-larly CYP17A1) are up regulating in castration-resistant prostateancer cells [38]. Testosterone and dihydrotestosterone are usuallyroduced from cholesterol via the classical pathway in adrenallands and testis. However, in castration-resistant prostate cancerells, different pathways (backdoor and alternative pathways) arectivated to produce dihydrotestosterone (see Fig. 1) [37,39–41].ntracrine conversion of adrenal weak androgens such as DHEA, 11-H- androstenedione or deoxycorticosterone in steroids activating
he androgen receptor has been also identified [42]. In prostate,HEA is first converted to androstenedione by 3�-hydroxysteroidehydrogenase (3�HSD1). Following castration, adrenal androgenndrostenedione serum and tissue levels are unchanged and aignificant amount of androstenedione is observed in castration-esistant prostate cancer cells [43]. The alternative pathway seemso be predominant in these cells [41]. Androstenedione is alsoonverted in 11-OH-androstenedione by CYP11B1,2 then reducedn 11-OH-5�-dihydrotestosterone by 5�-reductase: this moleculeould activate the wild type androgen receptor [44]. All thesenzymes are potential targets for new drugs.
To summarize, four different molecular states of prostate canceran be defined [45]:
endocrine androgen-dependent and androgen receptor-dependent: prostate cancer growth is driven by serum androgensproduced by the testis;intracrine androgen-dependent and androgen receptor-dependent: androgens are produced within prostate cancercells;androgen (ligand)-independent and androgen receptor-dependent: this is the case for androgen receptor variants;androgen-independent and androgen receptor-independent.
. New hormonal drugs
In castration-resistant prostate cancers, growth of cancer cellss often always dependent of the androgen receptor. X-ray
rapie 19 (2015) 220–227
crystallography and computer modelling allow the understandingof the biochemical interaction between drugs and the androgenreceptor at the atomic level. This helps to design new drugs toovercome castration resistance.
Residual levels of androgens can be found in cancer cells duein part to an intratumoral synthesis. Ketoconazole inhibits the 17-hydroxylase activities of CYP17A1: at high dose, it has been shownto obtain PSA response but no survival benefit could be demon-strated [46]. Abiraterone acetate (Zytiga®) is an oral irreversibleinhibitor of CYP17A1, one of the major enzymes involved in andro-gen synthesis (Fig. 3) [47]. CYP17A1 has both 17a-hydroxylaseand 17,20-lyase activities. However, this enzyme is also requiredfor synthesis of glucocorticoids. Low dose dexamethasone isrequired to prevent reflexive increase of adrenocorticotropichormone (ACTH) and mineralosteroid effects, as hypertension[48]. Abiraterone acetate decreased the production of androgens(androstenedione, DHEA, testosterone 5�-dihydrotestosterone) bythe adrenal glands, prostate and tumour cells [49].
Clinical trials showed that abiraterone acetate induced a dra-matic response in many patients with a castration-resistantprostate cancers [50]. In a retrospective study, abiraterone acetatewas shown to be superior to ketoconazole in terms of PSAresponse and survival [51]. Randomized trials have demonstratedan improvement in survival in castration-resistant prostate cancerseither after or before docetaxel [52,53].
TAK-700 (orteronel) is an another CYP17A1 inhibitor,more potent to inhibit CYP17,20 lyase function than CYP17-�hydroxylase. It does not require an association with prednisoneto prevent mineralocorticoid effects. However, recently presentedphase III studies could not demonstrated an improvement insurvival in castration-resistant prostate cancers and this drug willbe probably not approved by the US Food and Drug Administration(FDA) and the European Medicines Agency (EMA).
Non-steroidal androgen receptor antagonists, such as flutamideand bicalutamide, have relatively low affinity for the recep-tor and could, in some cases, become agonists. They do notinhibit ligand-induced nuclear translocation but recruits nuclearco-repressors to block androgen-regulated gene transcription[54]. Enzalutamide (formerly MDV3100-Xtandi®) is eight timesmore potent than bicalutamide to inhibit the androgen receptor[55]; it inhibits not only the binding of testosterone and dihy-drotestosterone, but also receptor translocation and binding ofcoactivators [56]. Importantly, enzalutamide remains antagonistof the androgen receptor in overexpressing cells and mutant celllines, which convert bicalutamide to an agonist [55]. Enzalutamideincreased overall survival for patients with castration-resistantprostate cancers either after or before treatment with docetaxel[57,58].
ARN-509 is an androgen receptor antagonist, differing fromenzalutamide by only one atom [59]. It also prevents androgenreceptor translocation and DNA-binding. Clinical trials are ongo-ing. Galeterone (TOK-001) inhibits CYP17 at low concentration, butat higher doses, it could inhibit the androgen receptor and evenfacilitates its degradation [60].
All these androgen receptor antagonists are targeting theligand-binding domain, which is lost in splice variants. Some newinhibitors, as EPI-001, are directed against the N-terminal domainand are supposed to inhibit protein – protein interactions [61].Although abiraterone and enzalutamide provide a survival bene-fit, resistance almost always develops. Several new antiandrogensagents are being evaluated either in preclinical or clinical studies[62].
Finally, docetaxel, which inhibits the dissociation of micro-tubules, prevents nuclear translocation of the androgen receptor
and decreases its intracellular level. Part of its action could be medi-ated by hormonal inhibition [63,64].
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L. Quero et al. / Cancer/Ra
. Interactions between hormonal treatments andadiation
Androgen deprivation therapy is today the standard hormonalreatment of prostate cancer. In locally advanced stages, the com-ination of androgen deprivation therapy and radiotherapy hasemonstrated better survival rates than one modality used alone.ong-term androgen deprivation therapy is used for high-riskrostate cancers and short-term therapy for intermediate risk can-ers. In contrast with the number of clinical trials involved, only aew preclinical studies, either in vitro or in vivo, have evaluated the
echanisms of interaction between hormonal manipulations andadiations. This could be due to the lack of good preclinical modelsor localized disease.
Preclinical models implicated prostate cancer cell lines: some asAPC4 or LNCaP are androgen-sensitive, unlike PC3 or DU145 cellines which do not expressed the androgen receptor. LNCaP celline presents an androgen receptor mutation (T877A LBD), whichllows binding of progesterone or cortisol [65]. LAPC4 cells have aild type androgen receptor.
Androgen deprivation therapy by itself induced a major reduc-ion in epithelial cells mainly by apoptosis and decreased cellroliferation [66]. The LNCaP cell line expressed the androgeneceptor and was regularly used for in vitro studies of combi-ation of androgen deprivation therapy and radiation. Androgeneprivation therapy was achieved by culture in charcoal-strippederum-containing medium. After irradiation, an increase in thepoptosis rate was observed for LNCaP cultured in medium withoutndrogens compared to complete medium, but without any differ-nce in clonogenic cell survival, suggesting a shift in the modalityf cell death [67].
Hermann et al. evaluated the effect of goserelin, an LH-RH ago-ist, on two cell lines, LNCaP et PC3; only LnCaP expressed thendrogen receptor but both cell lines expressed LH-RH recep-ors, as 86% prostate cancers do [68,69]. No radiosensitization wasbserved in both cell lines despite inhibition of the EGF receptorathway and cell proliferation.
We have observed that combined treatment with bicalutamide,n antiandrogen, with radiation concomitantly or sequentially, had
protective effect over radiation alone in LNCaP cell line. There waso significant effect of bicalutamide on DU145 cell line, which doesot expressed the androgen receptor [70]. High levels of bicalu-amide had a cytostatic effect on LnCap blocking the cell cycle in1 phase; senescence was the main cell death pathway.
In vivo, in different models of prostate cancer xenografts, iteems that a neoadjuvant androgen deprivation therapy is requiredo obtain and maximize the radiosensitizing effect. With thehionogi model, Zietman et al. showed that the TCD50 (radia-ion dose required to control 50% of the tumours) decreased whenndrogen deprivation therapy was added to radiation, particularlyf it was given 12 days before irradiation [71]. This effect was corre-ated to tumour volume regression, suggesting that the decreasef the number of clonogenic cells explained this supra-additiveffect. In another model, neoadjuvant androgen deprivation ther-py (3 days) increased the apoptotic rate compared to radiationlone or to androgen deprivation therapy given at the same timehan radiation [72]. However, this effect was not found in case of
ultiple fractions, suggesting that apoptosis is not the main typef radiation effect on prostate cancer cells [73]. In a model using3327-G dunning rat prostate tumour cells, neoadjuvant androgeneprivation therapy resulted in prolonged suppression of tumourrowth, compared to irradiation alone or adjuvant androgen depri-
ation therapy [74].
The number of clonogenic cells, rate of apoptosis but alsoypoxia have been discussed as possible factors of radiosensiti-ity. Hypoxia is frequent in prostate cancer tissues and maybe also
rapie 19 (2015) 220–227 225
in benign tissues [75,76]. The outcome is negatively influenced byhypoxia in patients treated by prostatectomy or radiotherapy [77].More recently, in a study of 247 patients, in which hypoxia wasmeasured before radiotherapy, a pO2 ≤ 10 mmHg was an indepen-dent of biochemical control and local control [78].
Inhibition of hypoxia-inducible factor (HIF) 1, the main mediatorof hypoxia, increases the radiosensitivity of PC3 cell line [79]. Look-ing at the relationship with the androgen axis, hypoxia increasesandrogen receptor activation in LNCap cells [80]; HIF1 could bea coactivator of the androgen receptor [81]. Androgen depriva-tion therapy decreases the hypoxia-induced expression of VEGFin androgen-dependent LNCap cell line but not in the androgen-independent PC3 cell line [82]. In Shionogi prostate tumour model,androgen deprivation therapy decreases the hypoxic fraction from30 to 2% [83]. Antiandrogens reduce the transcriptional activityof HIF1 [84]. In one study of 22 patients, hypoxia was measuredusing Eppendorf microelectrode technique, androgen deprivationtherapy increased the median pO2 values from 6.4 to 15 mmHg:part of the radiosensitizing effect of androgen deprivation ther-apy could be explained by this way [85]. On the other hand, ina study of 20 patients where dynamic magnetic resonance imag-ing (MRI) evaluated vascularisation, androgen deprivation therapyinduced a profound reduction in tumour blood flow and an increaseof hypoxia [86].
More recently, it has been demonstrated that ionizing radiationsactivated the androgen receptor. Moreover, the androgen recep-tor upregulated a number of DNA repair genes, particularly genesof the non-homologous pathway, because these genes contain theandrogen receptor binding sites in their enhancer sequences [87].In contrast, castration reduced the Ku67 level, a protein essentialfor non-homologous end-joining repair of double-strand breaks[88]. By this way, the androgen receptor promotes radioresis-tance and androgen deprivation increases DNA-damage and slowdouble-strand break repair [89]. New anti-androgen drugs (ARN509, enzalutamide) enhanced DNA damage and slow repair of DNAdouble-strand breaks.
Looking at these results, mechanisms of interaction betweenandrogen deprivation therapy and radiation are not fully eluci-dated. New drugs such as abiraterone and enzalutamide give newopportunities to increase the effect of radiation on prostate cancer.Preclinical and clinical studies of combination of these drugs withradiation are urgently needed.
8. Conclusion
The androgen receptor remains the main target of prostate can-cer either in locally or advanced disease, in castration dependantor resistant conditions. Androgen deprivation therapy is an impor-tant tool to improve the effect of radiotherapy. The knowledge ofandrogen receptor effects is important for the radiation oncologiststo manage at its best the combination of both modalities. New hor-monal drugs bring new opportunities to increase the potential ofradiotherapy.
Disclosure of interest
C.H. Occasional involvements: advisory services to Astellas,Janssen and Ipsen.
L.Q., F.R., P.B. have not supplied their declaration of conflict ofinterest.
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