new paradigms in microtubule-mediated endocrine signaling in … · treatment of crpcs. recent...

Review

New Paradigms in Microtubule-Mediated EndocrineSignaling in Prostate Cancer

Sucharita J. Mistry and William K. Oh

AbstractMetastatic prostate cancer has limited therapeutic options and has remained a major clinical challenge.

Historically, prostate cancer has been widely recognized as a chemotherapy-resistant disease. However,

clinical studieswith anti-microtubule agents over the past decade have shown important efficacy in improving

survival in patients with advanced disease. The favorable outcomes with microtubule-targeted agents have

thus rekindled interest in such therapies for the clinical management of prostate cancer. Microtubules are

dynamic polymers of tubulin molecules that play diverse roles within the cell. The dynamic property of

microtubules is responsible for forming the bipolar mitotic apparatus, the mitotic spindle, that functions to

precisely segregate the chromosomes during cell division. Thus, owing to the pivotal role that they play in the

orchestration ofmitotic events,microtubules provide excellent targets for anti-cancer therapy. Recent evidence

also suggests that microtubules play a crucial role in the regulation of endocrine signaling pathways.

Interestingly, microtubule-targeted agents such as taxanes not only inhibit cell division but also impair

endocrine receptor signaling in prostate cancer. Herein, we provide an overview of the current status of

microtubule-targeted therapies that are used in the treatment of prostate cancer anddiscuss novelmechanisms

by which such therapies modulate endocrine signaling in prostate cancer. We also address the emerging roles

of microtubule regulatory proteins in prostate carcinogenesis that could serve as attractive targets for prostate

cancer therapy and might also serve as predictive biomarkers to identify patients who may benefit from

endocrine and/or chemotherapy. This may have important implications in designing mechanism-based and

targeted-therapeutic strategies for prostate cancer. Mol Cancer Ther; 12(5); 555–66. �2013 AACR.

IntroductionProstate cancer is themost frequently diagnosedmalig-

nancy and the second leading cause of cancer mortality inmen inWestern countries (1). As prostate cancer growth isdrivenby circulating androgens (mainly testosterone), themainstay of first-line treatments for metastatic prostatecancer is suppression of gonadal androgens. Androgendeprivation therapy, either by medical or surgical inter-ventions, is initially effective at blocking tumor growth(2). However, these therapies eventually fail, leading to adrug-resistant androgen-independent stage, which isinvariably fatal (2). Unfortunately, androgen deprivationat best can only achieve a "hormone-reduced" rather thana "hormone-free" state. It is believed that residual levels ofintratumoral androgens following androgen deprivation

therapy are sufficient to activate the androgen receptor(AR) and drive prostate tumor growth (3). This trend ofprostate cancer progression has led to the recent renamingof this stage of the disease as "castration-resistant" insteadof "hormone-refractory" disease.Although androgendep-rivation by castration produces dramatic and rapiddeclines in prostate-specific antigen (PSA), bone pain,and urinary tract obstruction, limited options are avail-able for patients whose disease progresses despite a cas-trate testosterone level. Bonemetastasis occurs in amajor-ity of patients with advanced disease and such patientswith bone lesions cannot be cured (4). Thus, the clinicalmanagement of patients with castration-resistant prostatecancer (CRPC) has remained a major challenge.

Microtubules,which are one of the integral componentsof the cytoskeleton, have proven to be an effective ther-apeutic target for CRPCs. They are intrinsically dynamicprotein polymers made up of alternating subunits of a-and b-tubulin (5, 6). The formation of microtubulesinvolves a highly dynamic process of polymerization anddepolymerization of a- and b–tubulin heterodimers (5, 6).This dynamic property is crucial for the assembly of themitotic spindle and precise segregation of the chromo-somes during cell division (5, 6). Disruption of microtu-bule dynamics, either by inhibiting polymerization or bypreventing depolymerization of tubulin, interferes withthe regulation of the mitotic spindle that results in cell-

Authors' Affiliation: Division of Hematology-Medical Oncology, TheTisch Cancer Institute, Icahn School of Medicine at Mount Sinai, NewYork, New York

Corresponding Authors: Sucharita J. Mistry, Division of Hemato-logy-Oncology, Icahn School of Medicine at Mount Sinai, NewYork, NY 10029. Phone: 212-241-5281; Fax: 212-241-4096; E-mail:[email protected]; and William K. Oh, Division of Hemato-logy-Oncology, Icahn School of Medicine at Mount Sinai, NewYork. Phone: 212-659-5549; Fax: 212-659-8539; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-12-0871

�2013 American Association for Cancer Research.

MolecularCancer

Therapeutics

www.aacrjournals.org 555

on February 16, 2020. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst May 1, 2013; DOI: 10.1158/1535-7163.MCT-12-0871

http://mct.aacrjournals.org/

cycle arrest and eventually cell death (5, 6). Thus, owing totheir indispensability in mitosis and cell division, micro-tubules provide excellent targets for anti-cancer therapy.In addition to their role in spindle regulation, microtu-bules also play an important role in endocrine signalingpathways. In this review,we describe the current status ofmicrotubule-targeted therapies that are used in the treat-ment of prostate cancer and discuss novel mechanisms bywhich such therapies modulate endocrine signaling inprostate cancer. We also address the emerging roles ofmicrotubule regulatory proteins in prostate carcinogene-sis thatmight serve as attractive targets for prostate cancertherapy andmight also serve as prognostic and predictivetools to identify patients who would benefit from endo-crine and/or chemotherapy. This may have importantimplications in designing mechanism-based and tar-geted-therapeutic strategies for prostate cancer.

Microtubule-targeted chemotherapeutics in prostatecancer

Chemotherapy was once thought to play a clinicallyinsignificant role inmetastatic CRPCs. Although a varietyof cytotoxic agents have been investigated for the treat-ment of CRPCs, most agents showed only marginal effi-cacy and did not extend survival. One of the reasons whyprostate cancer cells may be so difficult to eradicate is thatmost chemotherapeutic agents are thought to be active inproliferating cellswith a short doubling time. The classicalview of the clinical success of chemotherapy is generallybased on the notion that mitotic microtubules are highlydynamic and their rapid dynamics makes the cells moresusceptible to cytotoxic agents leading to mitotic arrest inactively dividing cancer cells, eventually leading to apo-ptosis. In contrast, prostate cancer may have relativelylong doubling timewith only a small fraction of the tumorcells actively proliferating (7). Despite the slow growthkinetics of prostate cancer cells, microtubule-targetedagents (MTA) have shown considerable value in thetreatment of CRPCs. Recent advances in our understand-ing of the biology of the microtubule machinery have ledto a paradigm shift in the way we think how MTAs killcancer cells. Although microtubules that make up themitotic spindle have remained one of the key targets ofMTAs in rapidly dividing cells, microtubules are asimportant in nondividing cells as they are in the mitoticspindle of dividing cells. Thus, it is not surprising thatMTAs are effective against slower growing prostatetumors, where they might also interfere with interphasemicrotubule functions such as intracellular transport andsignaling. This idea is supported by recent studies show-ing the involvement of the dynamic interphase microtu-bules in the regulation of transcription factors such as p53and hypoxia-inducible factor (HIF)-1a (8, 9), implying arole for microtubules in transcription factor trafficking.

Taxanes in prostate cancerTaxanes are a powerful class of chemotherapeutic

agents that have shown significant clinical activity in a

wide range of solid tumor malignancies including meta-static CRPCs. Taxanes function primarily by interferingwith thedynamics of themitotic spindle causing cell-cyclearrest and apoptosis. They are routinely used in manycancers in the neoadjuvant, adjuvant, and metastatic set-ting alone and in combination with drugs with differentmechanism(s) of action and nonoverlapping toxicity pro-files. In this section, we will discuss some of the taxane-based drugs that have helped to improve the outlook ofpatients with CRPCs.

Docetaxel. Docetaxel chemotherapy represented akey breakthrough in the treatment of CRPCs based on aseries of clinical trials (10–12). Results of 2 pivotal phase IIItrials from independent teams formed the basis for doc-etaxel-based therapy as the first-line chemotherapyagainst CRPCs. Both trials randomized docetaxel versusmitoxantrone, an agent that was shown to improve qual-ity of life but failed to show any survival benefit. First, inthe TAX 327 trial, a total of 1,006menwere randomized toreceive either docetaxel or mitoxantrone along with oralprednisone in both arms (12, 13). Treatment with doce-taxel on a 21-day cycle was associated with an improve-ment inmedian overall survival (19.2 vs. 16.3months, P¼0.004; refs. 12, 13). Second, Southwest Oncology Group(SWOG) 9916 trial randomized 674 patients to receiveeither docetaxel and estramustine or mitoxantrone andprednisone (11). As in TAX 327, the SWOG 9916 trial alsoshowed a survival advantage associated with docetaxeltherapy (17.5 vs. 15.6 months, P ¼ 0.02; ref. 11). Thesestudies led to the U.S. Food and Drug Administration(FDA) approval of docetaxel in 2004 and laid the foun-dation for additional studies of docetaxel for high-riskdisease and novel docetaxel-based combinations forCRPCs. More recently, a phase I/IIa study was designedto examine the safety and efficacy of docetaxel in combi-nation with radium-223 chloride for patients with CRPCswith bonemetastases and this trial is ongoing (14). Table 1summarizes some of the significant positive phase IIItaxane-based clinical trials in CRPCs that are alreadyapproved and those under investigation.

Cabazitaxel. Menwith docetaxel resistance had, untilrecently, few options for treatment. However, in the past 2years, there has been progress made in the treatment ofthis disease with the FDA approval of at least 3 drugs(sipuleucel-T, abiraterone, and cabazitaxel) with differentmechanism of actions. Among these 3 drugs, cabazitaxel(Jevtana) is a novel taxoid compound that is a potentmicrotubule stabilizer like docetaxel and is an importantnew addition to the chemotherapeutic arsenal for patientswhose disease progresses during or after docetaxel treat-ment (15). Unlike paclitaxel and docetaxel, whose effec-tivenesswas limited due to their high substrate affinity forthe P-glycoprotein (P-gp) drug efflux pump (16), cabazi-taxel was engineered as a dimethyloxy derivative ofdocetaxel that offers 2 major advantages over its prede-cessor. First, the addition of the extra methyl groupeliminates the P-gp affinity that is characteristic of doc-etaxel, enabling cabazitaxel to be effective against

Mistry and Oh

Mol Cancer Ther; 12(5) May 2013 Molecular Cancer Therapeutics556




docetaxel-refractory prostate cancer. However, the dataon the effectiveness of cabazitaxel due to lack of P-gp haveonly been reported in preclinical models in prostate can-cer cell lines. The correlationbetweenP-gp expression anddocetaxel resistance remains to be investigated clinicallyin patients with prostate cancer. Thus, the poor affinity ofcabazitaxels for P-gp may or may not be its principalmechanism of action for its enhanced efficacy. Second,the addition of the extra methyl group on cabazitaxel isalso thought to enhance its ability to cross the blood–brainbarrier, a feature that is uncommon among chemothera-peutic agents. Interestingly, cabazitaxel was shown to beactive in both docetaxel-sensitive tumors and those thatfailed to respond to chemotherapy and was superior topaclitaxel and docetaxel in penetration of the blood–brainbarrier in phase I clinical trials (17).

With these observations, cabazitaxel progressed fromphase I trial directly to a randomized open-label phase IIITROPIC trial inwhich 755 patientswith docetaxel-treatedmetastatic CRPCs were treated with prednisone andmitoxantrone or cabazitaxel/prednisone (18). Themedianoverall survival was 15.1 months in the cabazitaxel groupand 12.7 months in the mitoxantrone group, a 30% reduc-tion in the risk of death (HR, ¼ 0.70; P < 0.001; Table 1).Thus, cabazitaxel was the first chemotherapeutic agent toshow survival benefit in men with docetaxel-pretreateddisease. On this basis, cabazitaxel received FDA regula-tory approval in June 2010 for the treatment of patientswith metastatic CRPCs who had previously been treatedwith docetaxel. Although mitoxantrone was previouslyused for the treatment of docetaxel-refractory disease dueto its palliative effects, cabazitaxel nowoffers anewoptionfor patients with CRPCswhose disease progresses duringor after docetaxel treatment and has replaced mitoxan-trone as the chemotherapeutic treatment of choice afterdocetaxel treatment. Further assessment of drug safetyand quality-of-life benefits imparted by cabazitaxel incombination with different agents is ongoing (19).

The clinical success of the taxanes had led to a search forother drugs that enhance microtubule polymerization.Epothilones (ixabepilone and patupilone) are a new classof microtubule stabilizers that have been investigated inphase II clinical trials in patients with CRPCs and seem tohave promising antitumor effects but significant toxicityas well (20, 21). A randomized phase II trial evaluatedixabepilone monotherapy or ixabepilone in combinationwith estramustine phosphate (EMP) in patients with pro-gressive castrate metastatic prostate cancer (20). PSAdecline of at least 50% was noted more frequently in thecombination group than themonotherapy group (69% vs.48%). The rate of tumor regression was also higher in thecombination group than themonotherapy group (48% vs.32%). Despite the encouraging clinical activity, the mostcommon toxic effects in both the groups included signif-icant grade III and IV neutropenia and peripheral neu-ropathy (20, 21). Future use of epothilones in the treatmentof CRPCswill need optimization of the dosing schedule tolimit toxicities. Further studies will also be needed to

Tab

le1.

Sum

maryof

sign

ifica

ntpos

itive

pha

seIIIclinicaltrialsus

ingtaxa

ne-bas

edtherap

iesor

endoc

rinetherap

iesinCRPCsthat

areap

prove

dan

dthos

eun

der

inve

stigation

Clin

ical

trial

Treatmen

tarm

1Treatmen

tarm

2Priortrea

tmen

tMed

ianOS

HR

Referen

ces

Approve

dtaxa

ne-bas

edtrea

tmen

tsTA

X32

7Doc

etax

elwith

predn

ison

eMito

xantrone

with

predniso

neChe

mothe

rapy-na

ïve

19.2

vs.1

6.3mon

ths

0.78

24%

RDR

12,1

3SWOG

9916

Doc

etax

elwith

estram

ustin

eMito

xantrone

with

predniso

neChe

mothe

rapy-na

ïve

17.5

vs.1

5.6mon

ths

0.80

20%

RDR

11TR

OPIC

Cab

azita

xelw

ithpredn

ison

eMito

xantrone

with

predniso

neDoc

etax

el-treated

15.1

vs.1

2.7mon

ths

0.70

30%

RDR

18Approve

dEnd

ocrin

eTh

erap

ies

COU-301

Abira

terone

acetatewith

pred

niso

nePlace

bowith

pred

niso

neDoc

etax

el-treated

14.8

vs.1

0.9mon

ths

0.65

35%

RDR

37AFF

IRM

Enz

alutam

ide

Place

bo

Doc

etax

el-treated

18.4

vs.1

3.6mon

ths

0.63

37%

RDR

39Ta

xane

-bas

edtrea

tmen

tsun

der

inve

stigation

Pha

seI

Cab

azita

xelw

ithcisp

latin

Place

bo

Doc

etax

el-treated

Ong

oing

—19

Pha

seI/IIa

Doc

etax

elwith

radium-223

Doc

etax

elalon

eChe

mothe

rapy-na

ïve

Ong

oing

—14

Abbreviations

:RDR,red

uctio

nin

dea

thrate;H

R,h

azardratio

.

Microtubule-Mediated Endocrine Signaling

www.aacrjournals.org Mol Cancer Ther; 12(5) May 2013 557




compare these agents head-to-head with cabazitaxel in apost-docetaxel setting.

Effects of MTAs on androgen receptor signaling inprostate cancer

The androgens and the androgen receptors play acritically important role in prostate cancer developmentand progression (22). Classically, the androgens exerttheir biologic effects by binding to the androgen receptor,which is a member of the nuclear receptor superfamilythat acts as a ligand-dependent transcription factor (22).The binding of androgen to its receptor causes a confor-mational change in the receptor, followedbydimerizationand nuclear translocation of the hormone–receptor com-plex. Once translocated, the ligand–receptor complexbinds to specific androgen response elements (ARE)upstream of the target genes along with coactivator pro-teins, leading to transcription activation of androgenreceptor target genes (22). PSA, a clinicalmarker routinelyused for prostate cancer, is one of the key targets that isdirectly regulatedby the androgen receptor (23).Adeclinein serumPSA levels is often associatedwith tumor regres-sion following androgen deprivation therapy. Despitecastrate levels of testosterone, nearly all advanced pros-tate cancers develop evidence of recurrence characterizedby an increase in PSA and radiographic progression. Thissuggests that tumor progression is associated with inap-propriately restored androgen receptor function despitesustained androgen ablation (3, 22). The reactivation ofandrogen receptor in recurrent tumors is hypothesized tooccur through multiple mechanisms including androgenreceptor amplification, active androgen receptor signalingdespite low levels of androgen, androgen receptor muta-tion, and androgen receptor splice variants (3, 22, 24, 25).More recently, castration resistance in human prostatecancerwas shown to be conferred by frequently occurringalternatively spliced androgen receptor variants such asARv567es, AR3, and ARv7, which lack the ligand-bindingdomain (24, 25). However, these variants confer ligand-independent androgen receptor transactivation and pro-duce a constitutively active androgen receptor followingcastration that plays an important role in prostate cancerprogression (24, 25). ARv567eswas themost common of the3 variants identified in 59% of patients with CRPCs inresponse to androgen deprivation therapy and to abira-terone (25). Thus, owing to the crucial role of androgenreceptors in prostate cancer, inhibition of androgen recep-tor activity is a major therapeutic goal in themanagementof CRPCs. Recent developments of novel agents targetingthe androgen receptor signaling pathway have shownsignificant clinical activity in patients with CRPCs (26),thus corroborating the importance of androgen receptorsas a therapeutic target in CRPCs.

Interestingly, several lines of evidence suggest thattaxane-based chemotherapy can inhibit androgen recep-tor signaling in prostate cancer via its effects onmicrotubules. Figure 1 is a schematic illustration of someof the emerging mechanistic scenarios of the effects of

taxanes onmicrotubules and androgen receptor signalingin prostate cancer. Kuroda and colleagues recentlyshowed downregulation of androgen receptor and PSAexpression in different prostate cancer cell lines followingexposure to docetaxel in vitro (27). Conversely, overex-pression of androgen receptor in prostate cancer cellsresulted in partial abrogation of cytotoxic effects of doc-etaxel (27). Thus, docetaxel mediates inhibitory effects onthe expression of androgen receptors and PSA (Fig. 1).This suggests that the PSA responses observed withdocetaxel treatment may represent a combination of cyto-toxicity (decrease in tumor cell mass) via its effects onmicrotubule assembly as well as possible effects on PSAmetabolism. Another study by Gan and colleaguesshowed that taxanes (paclitaxel and docetaxel) can inhibitandrogen receptor activity in PTEN-positive CRPC cellsby inducing nuclear accumulation of the FOXO1 protein,which is a known androgen receptor–suppressive nuclearfactor (Fig. 1). FOXO1 accumulation was shown toincrease the interaction between FOXO1 and the andro-gen receptors in the nucleus (28). Thus, the observedFOXO1-mediated androgen receptor inhibitory effect ofpaclitaxel in CRPC cells may play an important role intaxane-mediated inhibition of CRPC growth. Moreover,as the tumor suppressor gene, PTEN, which is also animportant upstream regulator of FOXO1 is frequentlymutated or deleted in advanced CRPCs (29), it was pro-posed that deregulation of the PTEN/FOXO1 pathwaymay lead to the development of taxane resistance inprostate cancer. Furthermore, immunohistochemicalanalysis using tissue microarrays (TMA) of human pros-tate specimens from docetaxel-treated patients with pros-tate cancer showed a significant depletion of nuclearandrogen receptors, paralleled by an increase in the cyto-solic androgen receptors and a decrease in PSA immu-noreactivity in the individual tissue arrays in response toMTAs (30). Taken together, these studies suggest thatchemotherapy with docetaxel not only inhibits cell divi-sion but also impairs androgen receptor signaling byinterfering with androgen receptor translocation.

More recent studies investigated the contribution of themicrotubules on androgen-mediated signaling and thesubsequent inhibition of androgen receptor activity fol-lowing taxane treatment (30–32). Prostate cancer cellstreated with paclitaxel followed by the addition of thedihydrotestosterone analogue R1881 had shown signifi-cant microtubule bundling associated with a concomitantdecrease in androgen receptor nuclear accumulation (31).The profound cytoplasmic sequestration of androgenreceptor following paclitaxel treatment suggests a rolefor microtubules in the shuttling of the receptor from thecytoplasm to the nucleus (31). Furthermore, cells withacquired tubulin mutations, which are known to preventtaxane-induced microtubule stabilization, failed to trans-locate androgen receptors to the nucleus as expected,confirming a role for microtubules in androgen receptortrafficking (31). More interestingly, the same group alsoassessed whether taxanes could interfere with nuclear

Mistry and Oh





translocation of the androgen receptors in human speci-mens in vivo. Isolation of circulating tumor cells (CTC)from the blood of patients with CRPCs can enable molec-ular characterization of disease progression without theneed of invasive biopsy. Interestingly, perturbation of themicrotubule–androgen receptor axis in CTCs isolatedfrom patients with CRPCs receiving taxane therapy (pac-litaxel or docetaxel) showed a close correlation with clin-ical response. For instance, CTCs isolated from a patientwho was refractory to paclitaxel treatment exhibited nor-mal organization of the microtubule cytoskeleton withandrogen receptors present in both the nucleus and thecytoplasm (31). In contrast, patients who responded wellto docetaxel chemotherapy showed bundled microtu-bules and the androgen receptors was exclusively in thecytoplasm with intense androgen receptor perinuclearstaining with a concomitant decline in PSA followingchemotherapy (31). Further analysis of 18 additionalsamples obtained during clinical progression frompatients who had more than 25% increase in PSA, 13(72%) showed nuclear androgen receptor localizationwhereas among the 17 responders (those having at leasta 30% reduction in PSA) or with stable disease, 12 (70.6%)showed cytoplasmic androgen receptor localization (31).Theseobservations showasignificant correlationbetweenandrogen receptor sequestration and clinical response totaxane chemotherapy (Fig. 1). This suggests that the

activity of taxanes in CRPCs is mediated at least in partby inhibiting androgen receptor nuclear transport andsignaling via microtubules and that monitoring androgenreceptor subcellular localization in the CTCs of patientswithCRPCmight aid in the prediction of clinical responseto taxane chemotherapy and possibly combination ther-apy as well.

On the basis of the evidence that prostate cancer pro-gression remains dependent on androgen receptor acti-vation,with tumor cells generating their ownhormones denovo (26), novel hormonal agents such as abirateroneacetate (AA) and enzalutamide (formerly calledMDV3100) have recently been developed. Abirateroneacetate is anoral inhibitor ofCYP17 that reduces androgenproduction by inhibiting the steroid synthesis pathway inthe testes, adrenals, and in the tumor itself (33). In contrastto abiraterone acetate that abrogates testosterone, enza-lutamide is a novel androgen receptor antagonist thatprevents translocation of the androgen receptor and inhi-bits DNA-binding and androgen-regulated expression(34). As taxanes impact androgen receptor signaling, it isimportant to determine whether taxanes are as active inpatients with CRPCs following treatment with abirater-one acetate or enzalutamide or whether these agentswould negatively impact taxane benefit. Following pro-mising phase 1 and II clinical trials (35, 36), abirateroneacetate was shown to significantly improve overall

Nuclear accumulation of FOXO1 interferes

with AR activity via its interaction with AR

Inhibition of nuclear translocation of AR

Target geneARE

PSA

AR Expression

ApoptosisMitotic

arrest

Microtubule stabilization

Taxanes

AR

AR

A

A

FOX01

On/off

© 2013 American Association for Cancer Research

Figure 1. Emerging mechanistic scenarios of the effects of taxane-based chemotherapy on microtubules and androgen receptor signaling. Taxanes promotethe assembly of stable microtubule bundles that cannot disassemble, thus inducing mitotic arrest and apoptosis. Taxanes also downregulate androgenreceptor expression and cause PSA decline. Microtubules have been proposed to facilitate the nuclear translocation of the androgen receptor andenhance downstream androgen receptor transcriptional activity in prostate cancer cells. Taxanes block this pathway and interfere with the androgen receptoractivity, in part, through microtubule stabilization and inhibition of androgen receptor nuclear transport and signaling. Taxane-induced inhibition ofandrogen receptor activity may also be mediated by the accumulation of FOXO1 proteins in the nucleus and increased interaction between FOXO1 andthe androgen receptor. A, androgen.






survival (OS) in men with metastatic CRPCs in the post-docetaxel setting in phase III COU-AA-301 trial andCOU-AA-302 trial in chemonaive patients (37). The median OSwas 14.8months in the abiraterone acetate armversus 10.9months in the placebo arm with a 35% reduction in therisk for death (HR, 0.65; P < 0.001) in the COU-AA-301trial (Table 1). A more recent study evaluated theresponse to abiraterone acetate in the postchemother-apy setting in patients with CRPCs whose disease pro-gressed early on docetaxel (38). This study suggests thatpatients who are refractory to docetaxel do not respondto abiraterone acetate. In addition, patients who wererefractory to abiraterone acetate did not respond todocetaxel either, raising the potential for cross-resis-tance between these 2 agents (38). More recently, 2 largerandomized phase III trials were initiated to evaluatethe efficacy of enzalutamide in patients with CRPCs inthe pre-docetaxel (PREVAIL trial) and post-docetaxel(AFFIRM trial) settings. The AFFIRM trial reported aprolongation in OS in patients with CRPCs who pro-gressed following docetaxel-based chemotherapy (39).The estimated median OS was 18.4 months for enzalu-tamide-treated patients compared with 13.6 months forplacebo-treated men reducing the risk of death by 37%(HR, 0.631; P < 0.0001; Table 1). However, comparativedata on the efficacy of enzalutamide in a post-docetaxelversus pre-docetaxel setting has not been reported asyet. Enzalutamide was recently approved by the FDAand is marketed as Xtandi for the treatment of meta-static CRPCs.

Effects of estrogen therapy on microtubules inprostate cancer

While the role of androgens in the regulation of normaland malignant growth of the prostate has been well-established, increasing evidence suggests that estrogensalso significantly contribute to normal prostatic functionsas well as to the genesis of prostate cancer. Paradoxically,however, estrogen therapy has previously been a main-stay treatment for advanced prostate cancer (40). Thetherapeutic effect of estrogen is primarily mediated bysuppression of the hypothalamic–pituitary–gonadal axisand by direct effects on the Leydig cells in testis, whichtogether lead to decreased serum levels of testosteroneand produces castration-like effects (41). Several decadesago, diethylstilbestrol, a synthetic estrogen, was used asthe first standard therapy for prostate cancer treatment(41). Subsequently, diethylstilbestrol diphosphate(DESdP), a nontoxic pro-drug form of diethylstilbestrolwas also shown to have palliative effects in advancedpatients with prostate cancer (42). Administration ofestrogenic analogues, diethylstilbestrol and DESdP,showed reproducibly lower levels of serum testosteroneto the concentration range observed in castrated patients(41, 42). In addition to their anti-androgenic effects, estro-gens also exert direct growth-inhibitory effects by dis-rupting microtubule formation. Diethylstilbestrol wasshown to have concentration-dependent effects on tubu-

lin polymerization, which was associated with the induc-tion of mitotic arrest and subsequent apoptosis in andro-gen-independent prostate cancer cell lines in vitro (43, 44).Although estrogen therapy was effective for advancedprostate cancer, unfortunately, itwas associatedwithhighrisk of serious cardiovascular complications, which hadstrongly limited its clinical use, at least in theUnited States(40, 45). Since then, several new estrogenic compoundshave been evaluated for their potential in the next gen-eration of prostate cancer therapies.

The therapeutic benefits of estrogen therapy wereapparent in combination studies with estramustine.Estramustine is a structural analogue of 17b-estradiol,with unusual pharmacologic properties that are distinctfrom its estrogenic activities. In addition to its hormonalaction, estramustine also acts as a microtubule disruptorthat can mediate cytotoxic effects through microtubuledisassembly and antimitotic activity (46). Clinical trialswith the microtubule inhibitors, estramustine and vin-blastine, were shown to induce antitumor response inpatients with CRPCs (47). In addition, 2 randomized trialsuggested that estramustine increases the efficacy ofdocetaxel (48, 49). Results of a multicenter randomizedphase II trial of docetaxel (arm A) or docetaxel plus oralEMP (arm B) showed more than 50% PSA decline in 40%of the patients with CRPCs in arm A and in 75% of thosein arm B (48). The median time to PSA progression wassignificantly increased to 30 weeks in arm B comparedwith 20 weeks in arm A (48). Subsequent studies of theimpact of the combination of docetaxel-based chemother-apy with EMP on the quality of life showed significantdecrease in pain with fewer symptoms in patients withCRPCs who were more likely to achieve a biochemicalresponse (49, 50). Overall, however, the side effects ofestramustine continue to limit its routine use in theUnited States.

2-Methoxyestradiol (2-ME) is another natural metab-olite of 17b-estradiol hormone that interferes with tubu-lin polymerization and has been shown to exert anti-tumor and anti-angiogenic effects in prostate cancercells lines in vitro and in xenograft models in vivo (51,52). In the last decade, 2-ME had gained increasedattention due to its marked antitumor properties andpossible cardiovascular benefits, particularly in patientswith taxane-refractory, metastatic CRPCs (53). Howev-er, in a recent phase II single-arm open-label study, 2-ME did not show improved clinical outcomes in menwith taxane-refractory CRPCs (54). Although 2-ME didnot appear to have significant clinical activity as amonotherapy, there is still considerable interest in theclinical evaluation of novel anti-angiogenic agents forprostate cancer. It may be critically important to stratifypatients whomay best benefit from such agents. Furtherstudies are needed to evaluate the efficacy of 2-MEin combination with conventional MTAs. This mayprovide patients with more effective options for che-motherapy in the treatment of advanced metastaticdisease.

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Emerging roles ofmicrotubule regulatory proteins inprostate cancerMicrotubules are regulated by a variety of cellular

proteins thatmodulate their functions and dynamic prop-erties. The intracellular dynamic behavior ofmicrotubulesis regulated by a balance in the activities of 2major classesof microtubule-regulatory proteins, the microtubule-sta-bilizing proteins, andmicrotubule-destabilizing proteins.Because of the success of anti-microtubule agents liketaxanes in the clinical management of CRPCs, there hasbeen considerable interest generated to identify novelapproaches to interfere with the dynamics of the micro-tubules system. In this section, we discuss some of theimportant cellular regulators of microtubule dynamicsthat are attractive targets for prostate cancer therapyand might also serve as prognostic and predictive bio-markers to identify patients responsive to endocrine ther-apy and/or chemotherapy.Stathmin. Stathmin (also known as Oncoprotein 18,

p18, Lap 18) is the founding member of a family ofmicrotubule-destabilizing proteins that regulates theassembly and disassembly of the mitotic spindle throughchanges in its cell-cycle–specific phosphorylation(6, 55, 56). The initial evidence for the role of stathmin inthe regulation of mitosis came from genetic studies thatshowed that manipulation of stathmin expression inter-fereswith the progression of cells throughmitosis (56, 57).These studies were subsequently followed by the identi-ficationof stathmin as amajor regulator of thedynamics ofpolymerization and depolymerization of the microtu-bules that make up the mitotic spindle (58). Stathminpromotes microtubule depolymerization by 2 distinctmechanisms (6). First, stathmin promotes a catastrophe-promoting microtubule-depolymerizing activity thatincreases the catastrophe rate at both the plus and theminus ends of the microtubules. The second is a tubulin-sequestering activity that inhibits microtubule polymer-ization by sequestering tubulin heterodimers and thusdepleting the pool of free tubulin heterodimers availablefor polymerization. Both of these activities of stathminplay a critically important role in the regulation of mitoticand interphase microtubules.High levels of stathmin expression have been found in a

wide variety of human malignancies including prostatecancer (6, 59). Interestingly, when biopsy specimens fromhuman prostate tumors were stained with an anti-stath-min antibody, immunoreactivity was seen in poorly dif-ferentiated tumors but not in hyperplastic prostate orhighly differentiated tumors (59). Thus, it was proposedthat the level of stathmin expression may serve as animportant prognostic marker for prostate cancer. A morerecent retrospective study of 240 radical prostatectomycases included representative cancer and benign tissuefrom each prostatectomy specimen in TMAs that werestained with different antibodies including anti-stathminantibody (60). By univariate analysis, staining with anti-stathmin antibody showed statistically significant out-come predictability (60). Positive staining with anti-stath-

min antibody was found to be statistically significant inpredicting biochemical failure (60), identifying stathminas a reliable molecular marker of prognosis in prostatecancer.

In preclinical studies that aimed to target stathminexpression, inhibition of stathmin expression by adeno-virus-mediated gene transfer of anti-stathmin ribozymewas shown to mediate significant antiproliferative andantitumorigenic effects in prostate cancer cells in vitro (61),suggesting that stathmin provides an attractive target forprostate cancer therapy. Subsequent evaluation of thetherapeutic interactions between stathmin inhibition anddifferent chemotherapeutic agents in prostate cancershowed a synergy, when stathmin inhibition was com-bined with either a microtubule-interfering drug such aspaclitaxel or a topoisomerase inhibitor such as etoposide(62). Furthermore, triple combinations of anti-stathmintherapy with low-dose, noninhibitory concentrations ofpaclitaxel and etoposide resulted in more profound anti-proliferative and antitumorigenic effects in prostate can-cer (62). These studies suggest that stathmin inhibitionsensitizes prostate cancer cells to chemotherapy. The exactmolecular mechanism responsible for the observed syn-ergistic interaction between MTAs and topoisomerases isnot clear. Numerous lines of evidence have shown that adeficiency in stathmin decreases the rate of catastrophesand sequestration of tubulin molecules, thereby shiftingthe equilibrium between the polymerized and unpoly-merized tubulin in favor of polymerized tubulin(55, 58, 63). Paclitaxel, on the other hand, stabilizes micro-tubules by binding to polymerized tubulin (64). Interest-ingly, unlike paclitaxel, etoposide induces G2 arrest as aresult from the rapid inhibition of the activity of p34cdc2, aprotein kinase that is critical for the transition from the G2

phase into mitosis in eukaryotic cells of the cell cycle (65).Hence, when prostate cancer cells in which stathmin isinhibited are exposed to taxol and etoposide, cells willhave difficulty entering mitosis as a result of etoposide-induced G2 arrest, will have difficulty depolymerizingtheir spindles as a result of stathmin deficiency, and willhave difficulty exiting mitosis, as the polymerized micro-tubules will be further stabilized by taxol binding (62).This may explain, at least in part, the observed synergybetween stathmin inhibition and exposure to paclitaxeland toposisomerases. As paclitaxel and etoposide havebeen frequently used in the treatment of prostate cancer,combination of these agents with anti-stathmin therapymay provide a superior form of combination therapy thatcould avoid toxicities associated with the use of chemo-therapeutic agents at their maximally tolerated doses.

Tau. Tau is one of the most extensively investigatedmicrotubule-associatedproteins that interactswithmicro-tubules of the cytoskeleton. Tau proteins are known tostabilizemicrotubules andpromote tubulin assembly intomicrotubules (66). Tau is found primarily in the neuronsof the central nervous system (CNS) as different isoformsthatmodulatemicrotubule stability via changes in its stateof phosphorylation (67). While the role of Tau in the






regulation of processes in the CNS is well-documented,tau may also have distinct roles in nonneuronal cells ofvarious tissue origins. Interestingly, numerous studiesshowed a close correlation between increased expressionof tau and increased drug resistance to antimitotic drugsin various cancers, including prostate cancer. Previousstudies showed that overexpression of tau in humanprostate cancer cell lines correlates with estramustineresistance (68). Comparative analysis of tau expressionin estramustine-sensitive and estramustine-resistantprostate cancer cell lines showed higher levels of tauexpression in the estramustine-resistant cell line than inestramustine-sensitive prostate cancer cell line (68). Theincrease in the level of tau in estramustine-resistant cellswas attributed to enhanced stability of microtubules as aresult of binding of tau to the microtubules. As mostMTAs, including estramustine, are known to interferewith microtubule dynamics, it is possible that alteredratios of tau and tubulinmay confer a resistant phenotypeby modulating the kinetic parameters of microtubulebehavior.

Recent studies indicate that the level of tau expressioncan also influence paclitaxel sensitivity (69). Low levelof tau expression was associated with increased sensi-tivity to paclitaxel (69). Tau can bind to b-tubulin onboth the inner and the outer surfaces of microtubulesand it can bind at the same site as paclitaxel on the innersurface of microtubules. Thus, it was proposed that taucompromises the efficacy of MTAs by competing withthe drugs for the microtubule-binding site. The clinicalsignificance of tau expression and taxane sensitivity hasbeen extensively investigated in patients with differentcancers. For instance, reduced expression of tau wasassociated with a favorable response to paclitaxel in

gastric cancer cases (70). Similarly, low expression of tauwas associated with higher rates of pathologic completeresponse (pCR) to preoperative paclitaxel chemothera-py in patients with breast cancer (BrCa; ref. 71). Asubsequent study investigated the prognostic and pre-dictive values of tau expression in estrogen receptor–positive primary breast cancer in 3 patient cohorts thatincluded patients who received no systemic adjuvanttherapy, patients who received adjuvant endocrine ther-apy but no adjuvant chemotherapy and patients whoreceived preoperative paclitaxel containing chemother-apy (72). This study reported an inverse correlationbetween endocrine sensitivity and chemosensitivity.High tau expression was significantly associated withno recurrence at 5 and 10 years (P ¼ 0.005 and P ¼ 0.05,respectively) in patients treated with tamoxifen, indi-cating a predictive value for endocrine therapy. On theother hand, tau expression was significantly lower inpatients who achieved pCR to paclitaxel containingchemotherapy (P < 0.001). Thus, this study suggestedthat high tau expression in estrogen receptor–positivebreast cancer could indicate an endocrine sensitivity butno chemosensitivity. In contrast, low tau expressioncould identify a subset of patients that have poor prog-nosis with endocrine therapy alone and may benefitfrom adjuvant taxane-containing chemotherapy. Thepredictive value of tau expression remains to be deter-mined in prostate cancer. Figure 2 illustrates the role ofmicrotubule-regulatory proteins and their link to hor-monal and chemotherapy.

Stathmin-Tau as biomarkers of chemosensitivity andendocrine sensitivity. While stathmin overexpressionhas been linked to reduced sensitivity to taxanes, inhibi-tion of stathmin results in chemosensitization of prostate

MT Polymer

Polymerization

MAP-Tau

Depolymerization

αβαβ heterodimer

Stathmin

MT destabilizing

factor

MT stabilizing

factor

High Tau

Endocrine-sensitive

Chemo insensitive

High stathmin

Chemo insensitive

Endocrine-insensitive

Low stathmin

Chemo sensitive

Endocrine-sensitive

Low Tau

Endocrine-insensitive

Chemo sensitive

© 2013 American Association for Cancer Research

Figure 2. Schematic illustrationof the role of microtubuleregulatory proteins and theirlink to hormonal andchemotherapy. The dynamicsof microtubule polymerizationand depolymerization isregulated by microtubule-stabilizing factors such asMAP–tau and microtubule-destabilizing factors such asstathmin. Stathmin and tauhave opposite butcomplementary roles in thedynamic regulation ofmicrotubules. The level ofexpression of stathmin and tauseems to play a criticallyimportant role in themodulation of chemosensitivityand endocrine sensitivity asindicated. MT, microtubule.

Mistry and Oh





cancer cells following paclitaxel treatment (62). The levelof stathmin expression has also been shown to correlatewith efficacy of endocrine therapy (73). Althoughmuch ofthese data have been generated in patients with breastcancer and not in prostate cancer, these observations arestill of considerable clinical interest as breast cancer andprostate cancer are closely related diseases that sharemany similarities in terms of the biologic and pathologicprocesses. Low stathmin staining intensity could predictfavorable outcome in patients on endocrine monotherapyand could identify a low-risk group for which 5 years ofendocrine therapy is sufficient (73). In contrast, highstathmin expression could possibly serve as a marker forendocrine resistance (73, 74). A recent study used a 2-biomarker model that examined stathmin expression inconjunction with tau to determine their prognostic val-ue in a large cohort of patients with primary breastcancer (75). When stratified by hormonal receptor sta-tus, low stathmin correlated with receptor positivityshowing improved survival whereas high stathminexpression predicted worse overall survival (HR, 1.48;P ¼ 0.06). Survival analysis showed 10-year survival of53.1% for patients with high stathmin expression versus67% for low expressers (P < 0.003). The ratio of tau tostathmin expression showed a positive correlation todisease-free survival (HR, 0.679; P ¼ 0.0053) with a 10-year survival rate of 65.4% for patients who had a highratio of tau to stathmin versus 52.5% 10-year survivalrate for those with a low ratio (P ¼ 0.0009). This studyrevealed that the ratio of tau to stathmin was an inde-pendent predictor of overall survival (HR, 0.609; P ¼0.008). Although these studies suggested that low stath-min and high tau are associated with increased micro-tubule stability and better prognosis in breast cancer, itdid not include a taxane-treated cohort to evaluate theprognostic value of either stathmin or tau/stathminratio in response to taxane therapy. While the connec-tion between stathmin-tau as biomarkers of chemosen-sitivity and endocrine sensitivity is interesting, some ofthese studies measured only one of the microtubule-regulatory proteins and not both. Therefore, these clin-ical observations may not be internally consistent.Nonetheless, the above clinical studies on stathmin andtau are intriguing in breast cancer, and it would be ofinterest to confirm the relevance of these observations inpatients with CRPCs. Further studies on stathmin andtau are needed to better assess the response to endocrinetherapy and chemotherapy.

ConclusionThe rapidly changing landscape of therapeutic options

during thepast 2 years hasmarked anewera in the clinicalmanagement of CRPCs. The classical paradigm of theclinical activity of taxanes through exclusive inhibitionof mitosis has dramatically changed with the discoverythat taxanes can induce interphasemicrotubule bundling,a cellular insult that can impair nuclear translocation of

the androgen receptor and thus inhibit transcription of theARE-containing target genes (30–32). These observationsreveal for the first time an unconventional link betweenthe microtubule and the endocrine signaling pathways,highlighting the therapeutic importance of microtubule-mediated androgen receptor signaling events that areimpaired downstream of drug-inducedmicrotubule bun-dling (30–32). These findings suggest that themicrotubuleand the endocrine signaling pathways interact with eachother and can strongly impact clinical outcome bymarkedly inhibiting prostate cancer cell growth. Whilemicrotubules can serve as dynamic highway tracks forandrogen receptor trafficking to enable rapid and targetednuclear delivery for androgen receptor transcriptionalactivity, perturbation of themicrotubule–androgen recep-tor axis may prove to be an important determinant oftaxane activity. The intersection of the androgen receptorand the microtubule functions may have important clin-ical implications in terms of providing unique opportu-nities to better understand the molecular basis of taxaneresistance in CRPCs and to identify patients who wouldbenefit from such a treatment. Furthermore, as the andro-gen receptor splice variants can contribute to CRPCs (24,25), it would be of interest to determine whether thealternatively spliced androgen receptor variants are alsomodulated by the microtubule signaling pathway in amanner similar to the wild-type androgen receptor andwhether they would respond to taxanes.

Recent advances in the molecular understanding of themicrotubule and the androgen receptor signaling path-ways have led to the development of not only novelMTAssuch as cabazitaxel but also hormonal agents such asabiraterone and enzalutamide, which have now beenplaced at the forefront in the therapeutic armamentariumfor the treatment of patients with CRPCs. Combination oftaxaneswith abiraterone or enzalutamide is attractive andmay have better clinical outcomes, as such a combinationwould inhibit the microtubule–androgen receptor axis by2 different but converging pathways. However, it wouldbe critically important to determine the optimal role andtiming of these agents, either given sequentially or incombination. Unfortunately, there is no validated genesignature that correlates with individual treatmentresponse in prostate cancer. The identification of novelbiomarkers might help to identify patients who wouldbenefit most from each treatment and to determine theappropriate drug touse in a given situation. In this regard,microtubule regulatory proteins, such as stathmin andtau, may provide powerful predictive and prognostictools to identify patients who would benefit from endo-crine therapy and/or taxane-containing chemotherapy.

Despite the recent landmark developments in the ther-apeutic options for patientswith CRPCs, it is still essentialto build on developing molecular-targeted therapies forprostate cancer. Compelling results from preclinical stud-ies on targeting microtubule regulatory proteins such asstathmin (61, 62) are intriguing and suggest that futuredevelopment of targeted therapies holds great promise.






The discovery of synergism between taxanes and anti-stathmin therapy in prostate cancer is particularly attrac-tive, as both agents target the microtubule pathway (62).Such molecular-targeted therapies may be designed incombination with taxanes at much lower doses than attheir maximum tolerated doses. This would avoid toxi-cities associated with the use of multiple chemotherapeu-tic agents at full therapeutic doses. With the growingdiscoveries of novel drugs under investigation and theidentification of novel molecular targets, application ofcombinatorial strategies might be the key to the futuredevelopment of effective regimens in CRPCs.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: S.J. Mistry, W.K. OhWriting of the manuscript: S.J. MistryReviewing of the manuscript: W.K. OhStudy supervision: W.K. Oh

Grant SupportW.K. Oh has received research funding fromGenentech and S.J. Mistry

is supported by the American Cancer Society Research Scholar Award.

Received September 7, 2012; revised December 21, 2012; acceptedJanuary 14, 2013; published OnlineFirst May 1, 2013.

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2013;12:555-566. Published OnlineFirst May 1, 2013.Mol Cancer Ther Sucharita J. Mistry and William K. Oh Prostate CancerNew Paradigms in Microtubule-Mediated Endocrine Signaling in

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