Review

Consequences of the Convergence of MultipleAlternate Pathways on the Estrogen Receptor in

the Treatment of Metastatic Breast CancerStefan Glück

AbstractEndocrine therapy is the usual first-line therapy for patients with hormone receptor-positive metastatic breast cancer.However, resistance to hormone therapies frequently occurs during the course of treatment. Growing understandingof the signal cascade of estrogen receptors and the signaling pathways that interact with estrogen receptors hasrevealed the complex role of these receptors in cell growth and proliferation, and on the mechanism in development ofresistance. These insights have led to the development of targeted therapies that may prove to be effective options forthe treatment of breast cancer and may overcome hormone therapy resistance. This article reviews current under-standing of the cellular receptor signaling pathways that interact with estrogen receptors. It also reviews data fromrecent ongoing clinical trials that examine the effects of targeted therapies, which might interfere with estrogen re-ceptor pathways and might reduce or reverse resistance to traditional, sequential, single-agent endocrine therapy.

Clinical Breast Cancer, Vol. -, No. -, --- ª 2016 The Author. Published by Elsevier Inc. This is an open access article underthe CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: Cyclin-dependent kinases, Endocrine therapy, Hormone receptor-positive, PI3K/Akt/mTOR pathway, Programmedcell death inhibitor

IntroductionBreast cancer is the most common malignancy and the second

leading cause of cancer-related death among women in the UnitedStates according to 2016 estimates.1 Approximately 6% of cases areadvanced or metastatic breast cancer (MBC) at diagnosis, and, forthose diagnosed at early stages, recurrence to distant sites occurs in20% to 30%.2-4 Of patients with breast cancer, approximately 75%have hormone receptor-positive (HRþ) tumors.5

Therapies for MBC are aimed at palliation of symptoms withimprovement or conservation of quality of life and, potentially, pro-longation of survival; unfortunately, prolonged survival is observed inonly a small percentage of patients.6,7 Both hormone receptor status(eg, estrogen receptor [ER] and progesterone receptor [PgR]) andhuman epidermal growth factor 2 (HER2) status are important pre-dictive markers for treatment efficacy.7,8 Patients with HRþ MBC arecandidates for initial endocrine therapy (ET).9 In premenopausal

Department of Medicine, Division of Hematology Oncology, Sylvester ComprehensiveCancer Center, Miami, FL

Submitted: Jun 17, 2016; Revised: Aug 1, 2016; Accepted: Aug 14, 2016

Address for correspondence: Stefan Glück, MD, PhD, FRCPC, Sylvester Professor ofMedicine, Department of Medicine, Division of Hematology Oncology, SylvesterComprehensive Cancer Center, Miami, FL 33136E-mail contact: sgluck1@mac.com

1526-8209/ª 2016 The Author. Published by Elsevier Inc. This is an open access articleunder the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).http://dx.doi.org/10.1016/j.clbc.2016.08.004

women with MBC, various endocrine treatments are employed,including ovarian suppression or oophorectomy, selective ER mod-ulators such as tamoxifen, aromatase inhibitors (AIs) in conjunctionwith ovarian suppression, and the selective ER downregulator, ful-vestrant, all of which have different mechanisms of action. Thesemechanisms have been reviewed extensively, demonstrating thediversity of approaches that ultimately antagonize the growth-promoting effects of estrogen on breast cancer cells. These mecha-nistic differences are also linked to dissimilarities in resistancemechanisms and thus influence treatment selection, particularly in thecontext of sequencing and the use of combination regimens.10-17 Inthe setting of postmenopausal HRþ MBC, ET has a better tolerabilityprofile and longer time to progression (TTP) than chemotherapy.18,19

In clinical trials of AIs employed for first- or second-line treatment ofpostmenopausal patients with MBC, AIs have greater efficacy thaneither tamoxifen or megestrol acetate.20

Resistance to ET in HRþ MBC is common, and given sufficienttime, most patients are faced with disease progression.21,22 Themechanisms underlying disease progression and the development ofresistance to endocrine treatment are complex and not fully un-derstood. Nevertheless, over the past several years, insights intoseveral pathways of resistance have grown and have led to increasedunderstanding of the clinical value of sequential lines of therapy andco-targeting strategies. While receiving ET, up to 20% of patients

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Alternate Pathways on Estrogen Receptor in Metastatic Breast Cancer

experience a loss of ERs with concomitant loss of response to es-trogen.23 Amplified expression of HER2 may reduce ER levels andserve as an alternative pathway for tumor cell survival.23,24 Cellularsignaling of growth-factor receptors interacts with ER nuclear andnonnuclear pathways and may contribute to resistance throughposttranslational modifications of ER and/or coregulators.25-27

Mutations and/or aberrant forms of ER and their coregulatorsmay also be a primary cause or contribute to the development ofresistance to ET.21,22,25

The aim of this review is to describe emerging information oncellular pathways that interact with ER and impact cell proliferationand development of resistance to ET. We will discuss the biologicrationale for combining ET with agents that target these pathwaysand review the current status of clinical trials that are investigatingthe effects of combination treatments (Table 1). We also illustratethe cellular receptors and intracellular signaling pathways thatinteract with ER (Figure 1).

Estrogen-Receptor SignalingER functions through 2 separate but interrelated pathways: nu-

clear (genomic) and nonnuclear (nongenomic) pathways. The nu-clear pathway mediates the effects of ER on genomic activity,thereby altering expression of many genes involved in physiologiccell function and in abnormal cell proliferation, resulting intumorigenesis.28 The ER nuclear pathway is activated by estrogenbinding, receptor dimerization and translocation to the nucleus,interaction with coregulator proteins (both coactivators and co-repressors), and through estrogen response elements modulation ofgene transcription.29 ER coregulators have been implicated in thepathogenesis of breast cancer. Most notable is amplified in breastcancer 1, which, as its name implies, is overexpressed in breastcancers. Moreover, amplified in breast cancer 1 overexpression mayplay a role in resistance to ET.30 The nonnuclear pathway of ERfunction (originating in cellular cytoplasm) is mediated by estrogen-bound ER acting outside the cell nucleus through interaction withmultiple cellular signaling pathways, tyrosine kinases, andmembrane-bound growth factor-receptor pathways, includingHER2, insulin-like growth factor-1 receptor (IGF1R), and fibro-blast growth factor receptor (FGFR).31,32 Multiple levels of inter-action or crosstalk between ER and growth factor and tyrosinekinase pathways may contribute to ER actions. These interactionsinclude modulation of ER activity, upregulation of competingpathways and development of resistance to ET, and alternative orescape pathways for cellular proliferation and tumorigenesis.

Estrogen can modify the activity of growth factor pathways byincreasing levels of growth factors such as transforming growth factor-a and IGF1R and, alternatively, by altering the expression ofepidermal growth factor receptors (EGFR).27,32,33 Conversely, acti-vation of growth factor-receptor signaling pathways may down-regulate expression of ER, resulting in decreased estrogen effects.34,35

This crosstalk, which can up- or downregulate competing pathwaysbetween ER and growth factor-receptor signaling, is recognized as amajor mechanism of ET resistance, and implies that ER-positive(ERþ)/HER2-positive (HER2þ) breast cancer should be treatedwith a combination of ET and HER2 inhibitors or antagonists.36

In addition to ER andHER2 status, assessment of PgR status is alsotypically used to characterize MBC and shape treatment decisions. In

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normal breast tissue, ER and PgR appear to be expressed in differentepithelial populations and regulate distinct pathways; estrogen isinvolved in extracellular signaling and progesterone is associated withcell growth.8 This independent activity is altered in breast cancer cellswhere ER and PgR expression become correlated and converge onpathways that promote tumor growth and metastasis, and both PgRand ER become regulated by estrogen.8 Although PgR in ERþ

patients withMBChas been used as a predictor of response to ET, it iscontroversial and not as well-accepted as the role of ER.37

Data indicate that ERþ/PgR-negative (PgR�) breast tumors arenot as responsive to selective ER modulator therapy as ERþ/PgR-positive (PgRþ) tumors. One proposed mechanism for this anties-trogen resistance involves aberrant growth factor signaling (a markerfor loss of PgR), but other unknown contributory mechanisms arealso likely to play a role. Further research is needed to identify suchmechanisms and thereby refine therapeutic strategies.38

In a recent study of MBC, PgR was found to associate with ER inthe presence of agonist ligands, and the ER-PgR ligand-activatedcomplex acted to modify chromatin-binding events and gene tran-scription, leading to an antitumorigenic effect.39 Stimulation withprogesterone in an estrogen-rich context induced interactions be-tween known ER cofactors and PgR, but did not alter the associationof those cofactors with ER, yielding an ER-PgR binding complex thatpromoted cell death, apoptosis, and differentiation pathways.39 Thisfinding, coupled with the association between PgR gene (PgR) copynumber loss and poorer clinical outcome possibly owing to a reduc-tion in ER-PgR binding complex formation, suggests a more intricaterelationship beyond PgR as a simple marker for ER pathway functionin the MBC setting.39 Although this contrasts with hormonereplacement studies in which synthetic progesterone (medrox-yprogesterone) was associated with an increased breast cancer risk, itwas found that natural progesterone did not have the same effect,which suggests different mechanisms of action for synthetic ago-nists.40,41 Interestingly, semi-synthetic progestin, megestrol acetate,provided clinical benefit to ERþ patients withMBCwho experienceddisease progression after estrogen suppression with a nonsteroidalAI.42Molecular subtyping of tumorsmay offer additional insight intotreatment of early-stage and locally advanced breast cancer. In a studythat evaluated molecular subtype and diagnostic classification of the154 patients classified as Luminal type B (high risk), a group that istypically sensitive to chemotherapy, 145were ERþ and 99were PgRþ

tumors.43 Among the Luminal B patients, pathologic completeresponse was 85%, suggesting a possible association between ER/PgRstatus and the benefit of chemotherapy.43 Clearly the complex rela-tionship between ER and PgR depends on many factors, includinggenomic alteration, interaction of cofactors at a transcriptional level,relative levels of estrogen and progesterone in the tumor cell envi-ronment, and possibly variations in conformational changes related tothe form of the agonist.

Growth-Factor ReceptorsGrowth-factor receptors, particularly receptor tyrosine kinases,

play an integral role in growth promotion, cellular proliferation, andtumorigenesis. Growth-factor pathways may act as ER-independentdrivers of tumor growth and survival, contributing to resistance toall types of ET. HER2, IGF1R, and FGFR have been implicated inresistance.26,44,45

Table 1 Clinical Trials Investigating the Effects of Combination Treatments in Breast Cancer (Available at https://clinicaltrials.gov/)

NCT Identifier (Acronym)Study Agents Phase Study Design Selected Clinical Outcomes

Start Date/EstimatedCompletion

Growth-factor receptor TKinhibitors

NCT00634634Sorafenib/letrozole

I/II � Open-label, single-arm� Postmenopausal, HRþ advanced/MBC

Phase IPrimary: RPTDPhase IIPrimary: CBRPhase I/IISecondary: TTP, OS, safety

August 2008/December 2015

NCT00752986 (ZACFAST)Vandetanib/fulvestrantPlacebo/fulvestrant

II � Randomized, double-blind� Placebo-controlled� Postmenopausal/advanced BC

Primary: Event-free survivalSecondary: TTP, PFS, OS, safety

December 2008/September 2013

NCT01528345Dovitinib/fulvestrantPlacebo/fulvestrant

II � Randomized, double-blind� Placebo-controlled� Postmenopausal/HRþ/HER2� advanced/MBC

Primary: PFSSecondary: ORR, DOR, OS, safety, ECOG performance

April 2012/October 2015

NCT02053636 (FINESSE)Lucitanib

II � Open-label, single-arm� HRþ MBC

Primary: ORR December 2013/July 2016

NCT00305825Bevacizumab/letrozole

II � Open-label, single-arm� Postmenopausal/HRþ

� Unresectable advanced/MBC

Primary: SafetySecondary: ORR, CBR

August 2004/November 2017

NCT00390455Fulvestrant/lapatinibFulvestrant/placebo

III � Randomized, double-blind� Placebo-controlled� Postmenopausal/HRþ advanced/MBC

Primary: PFSSecondary: OS, ORR, DOR, OS, safety, QOL

September 2006/July 2014

NCT01160211AI/trastuzumab/lapatinibAI/trastuzumab AI/lapatinib

III � Randomized, open-label, multiple-arm� Postmenopausal/HRþ/HER2þ, advanced/MBC; prior trastuzumab and ET

Primary: OSSecondary: PFS, ORR, CBR, OS, safety, QOL, TTP, DOR

May 2011/December 2017

PI3K/Akt/mTOR inhibitors

NCT01248494BEZ235/letrozoleBKM120/letrozoleBKM120 (intermittent)/letrozole

Ib � Open-label, multiple-arm� Postmenopausal, HRþ MBC

Primary: MTD BEZ235 or BKM120Secondary: PFS, ORR

November 2010/October 2013

NCT01344031MK-2206/anastrozoleMK-2206/fulvestrantMK-2206/anastrozole/fulvestrant

I � Open-label, multiple-arm� Postmenopausal, HRþ advanced/MBC

Primary: MTD MK-2206, RPTD for all agentsSecondary: Safety, CBR

April 2011/October 2013

NCT01339442BKM120/fulvestrant

I � Open-label, multiple-arm� Postmenopausal, HRþ advanced/MBC

Primary: MTD BKM120 safetySecondary: ORR, PK

November 2011/December 2014

NCT01791478Letrozole/BYL719

Ib � Open-label, single-arm� Postmenopausal HRþ MBC

Primary: MTD BYL719Secondary: CBR, PFS, ORR, Safety

April 2013/March 2016

NCT01797120Fulvestrant/everolimusFulvestrant/placebo

II � Randomized, double-blind, placebo-controlled� Postmenopausal HRþ MBC, AI refractory

Primary: PFSSecondary: Safety, ORR, TTP, OS

May 2013/November 2015

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Table 1 Continued

NCT Identifier (Acronym)Study Agents Phase Study Design Selected Clinical Outcomes

Start Date/EstimatedCompletion

NCT02049957Exemestane/MLN0128Fulvestrant/MLN0128

IIb/II � Open-label, multiple-arm� Postmenopausal HRþ/HER2�, advanced/MBC, everolimus refractory

Primary: Safety, CBRSecondary: ORR, tumor size, PK-PD, PFS, OS

February 2014/June 2016

NCT01992952 (FAKTION)Fulvestrant/AZD5363Fulvestrant/placebo

Ib/II � Randomized, double-blind, placebo-controlled� Postmenopausal HRþ, advanced/MBC

Phase Ib Primary: MTD AZD5363Phase II Primary: PFSPhase Ib/II Secondary: Safety, ORR, OS

May 2014/August 2016

NCT01698918 (BOLERO-4)Letrozole/everolimus (1st line)Exemestane/everolimus (2nd line)

II � Open-label, single-arm� Postmenopausal HRþ/HER2�, advanced/MBC

Primary: PFS after 1st lineSecondary: ORR, OS, CBR, safety, PFS after 2nd line

March 2013/October 2016

NCT01296555GDC0032 onlyLetrozole/GDC0032Fulvestrant/GDC0032

I/II � Open-label, multiple-arm� Phase I advanced/metastatic solid tumors� Phase II: Postmenopausal/HRþ advanced/MBC

Phase I Primary: SafetyPhase II: Fulvestrant/GDC0032Secondary: ORR, DOR, PFSPhase II Primary: CBR, ORRSecondary: DOR, PFS, OS

March 2011/July 2017

NCT01437566Fulvestrant/GDC0941Fulvestrant/GDC0980Fulvestrant/placebo

II � Randomized, double-blind, placebo-controlled� Postmenopausal HRþ, MBC, AI refractory

Primary: PFS, safetySecondary: ORR, CBR, DOR, PK

October 2011/November 2015

NCT02035813 (DETECT IV)Everolimus/standard ET

II � Open-label, single-arm� Postmenopausal HRþ/HER2�, advanced/MBC

Primary: PFSSecondary: ORR, DCR, OS

January 2014/December 2019

NCT01610284 (BELLE-2)Fulvestrant/BKM120Fulvestrant/placebo

III � Randomized, double-blind, placebo-controlled� Postmenopausal HRþ, MBC, AI refractory

Primary: PFSSecondary: OS, ORR, CBR, safety, PK

August 2012/March 2017

NCT01633060 (BELLE-3)Fulvestrant/BKM120Fulvestrant/placebo

III � Randomized, double-blind, placebo-controlled� Postmenopausal HRþ/HER2�, advanced/MBC, AI treated, progressedon/after mTOR

Primary: PFSSecondary: OS, ORR, CBR, safety, PK

October 2012/March 2017

Src inhibitors

NCT00696072Letrozole/dasatinibLetrozole only

II � Open-label, multiple-arm� Postmenopausal HRþ/HER2�, advanced/MBC

Primary: CBRSecondary: ORR, PFS, TTF, safety

August 2008/February 2015

CDK 4/6 Inhibitors

NCT01857193ExemestaneEverolimus/LEE011 (palbociclib)Exemestane/everolimusExemestane/LEE011 (palbociclib)

Ib/II � Open-label, multiple arm� Postmenopausal, HRþ/HER2� advanced/MBC

Phase I Primary: SafetyPhase II Primary: PFSPhase Ib/II Secondary: Safety, PK, ORR, DOR, OS, DCR

September 2013/July 2016

NCT00721409Letrozole/palbociclibLetrozole only

I/II � Open-label, multiple-arm� Postmenopausal HRþ/HER2�, advanced/MBC

Phase I Primary: SafetyPhase I Secondary: PK, QTc, antitumor activityPhase II Primary: PFSPhase II Secondary: Antitumor activity, OS, safety

September 2008/July 2018

Alternate

Pathw

ayson

Estrogen

Receptor

inMetastatic

Breast

Cancer

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Table 1 Continued

NCT Identifier (Acronym)Study Agents Phase Study Design Selected Clinical Outcomes

Start Date/EstimatedCompletion

NCT02088684LEE011/BKM120/Fulvestrant LEE011/BYL719/Fulvestrant LEE011/fulvestrant

IIb/II � Open-label, multiple-arm� Postmenopausal HRþ/HER2�, advanced/MBC

Phase Ib Primary: Dose-limiting toxicityPhase II Primary: PFSPhase Ib/II Secondary: Safety, PK, ORR, DOR, PFS (Phase Ionly), OS (Phase II only)

May 2014/February 2019

NCT01723774Postmenopausal: Anastrozole/palbociclibPremenopausal: Goserelin/palbociclib

II � Neoadjuvant, open-label, single-arm� Pre/postmenopausal HRþ/HER2�, Stage 2/3 BC

Primary: Complete cell cycle arrest (Ki67<2.7%)Secondary: PEPi 0 score, response- clinical, radiological,Ki67 level, pCR rate, PK, safety

June 2013/August 2015

NCT02040857Palbociclib/standard ET

II � Open-label, single-arm� Pre/postmenopausal HRþ/HER2�, Stage 2/3 BC

Primary: Treatment DC rate January 2014/June 2019

NCT01942135 (PALOMA-3)Fulvestrant/palbociclibFulvestrant/placebo

III � Randomized, double-blind, placebo-controlled� Postmenopausal HRþ/HER2�, MBC, ET refractory

Primary: PFSSecondary: OS, ORR, DOR, CBR, QOL, TTD

September 2013/January 2017

NCT01958021 (MONALEESA-2)Letrozole/LEE011Letrozole/placebo

III � Randomized, double-blind, placebo-controlled� Postmenopausal HRþ/HER2�, advanced/MBC, therapy-naive

Primary: PFSSecondary: OS, ORR, CBR, safety, QOL, QTc

December 2013/January 2017

PD-1 inhibitors

NCT02129556 (PANACEA)MK-3475/trastuzumab

I/II � Open-label, single-arm� HER2þ, advanced/MBC, trastuzumab refractory

Primary: Dose-limiting toxicity, RPTDSecondary: Safety, CBR, DOR, TTP, PFS, OS

July 2014/July 2023

Proteasome inhibitors

NCT01142401Fulvestrant/bortezomibFulvestrant only

II � Randomized, double-blind� Postmenopausal HRþ, advanced/MBC

Primary: PFSSecondary: CBR, OS, safety

May 2010/June 2014

Epigenetic modifiers

NCT02374099Oral azacitidine/fulvestrantFulvestrant only

II � Randomized, open-label� Postmenopausal ERþ, HER2� MBC who progressed on an AI

Primary: PFSSecondary: ORR, CBR, OS, DOR, safety

March 2015/February 2018

NCT00676663 (ENCORE301)Exemestane/entinostatExemestane only

II � Randomized, double-blind� Postmenopausal ERþ, locally recurrent or MBC who progressed on an AI

Primary: PFSSecondary: ORR, CBR, safety

May 2008/October 2012

NCT01349959Azacitidine/entinostat

II � Randomized, open-label� TNBC or HRþ/HER2�, advanced/MBC

Primary: Confirmed response rate by RECISTSecondary: CBR, OS, PFS, Safety

April 2011/March 2014

NCT01194908Decitabine/panobinostat/tamoxifen

I/II � Randomized, open-label� TNBC, MBC or locally advanced

Primary: MTDSecondary: Safety

July 2010/January 2014

Abbreviations: AI ¼ aromatase inhibitor; BC ¼ breast cancer; CBR ¼ clinical benefit rate; DC ¼ discontinuation rate; DOR ¼ duration of response; ECOG ¼ Eastern Cooperative Oncology Group; ET ¼ endocrine therapy; HER2þ/HER2� ¼ human epidermal growth factorreceptor-2 positive/negative; HRþ ¼ hormone receptor-positive; MBC ¼ metastatic breast cancer; MTD ¼ maximum tolerated dose; mTOR ¼ mammalian target of rapamycin inhibitor; ORR ¼ overall response rate; OS ¼ overall survival; pCR ¼ pathologic complete response;PD ¼ pharmacodynamics; PD-1 ¼ programmed cell death protein 1; PFS ¼ progression-free survival; PK ¼ pharmacokinetics; QOL ¼ quality of life; QTc ¼ corrected QT interval; RPTD ¼ recommended phase II dose; TK ¼ tyrosine kinase; TNBC ¼ triple negative breastcancer; TTD ¼ time to deterioration; TTF ¼ time to treatment failure; TTP ¼ time to progression.

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In breast cancer, HER2, a member of the EGFR family, is animportant independent pathway for cell proliferation.46 HER2 isconstitutively in an active conformation that allows dimerization withitself (homodimerization) or other members of the EGFR family(heterodimerization).WhenHER2 is overexpressed, as it is inw15%to 20% of all breast cancers, it mediates cell growth and survivalthrough activation of its downstream mediators, PI3K/Akt/mTORand Ras/Raf/MEK/MAP kinase.47 Overexpression of HER2(HER2þ) is an independent prognostic (and predictive for sensitivityto its antagonists/inhibitors) factor indicating a more aggressive formof cancer with a higher recurrence rate and higher mortality.46,48

Approximately 10% of patients with HRþ MBC are also HER2þ

and, conversely, approximately 50%ofHER2þ breast cancers are alsoHRþ.WomenwithHRþ andHER2þ diseasemay not respond to ETas well as women withHRþ andHER2-negative (HER2�)MBC.7,49

Reversing resistance to antiestrogen therapy in MBC is an importanttactic to avoid and delay the use of cytotoxic compounds. Themammalian target of rapamycin (mTOR) has been associated within vitro reversal of drug resistance, including tamoxifen resistance.50

A number of compounds that interfere with HER2 are eitherapproved for therapy ofHER2þ breast cancer or are under investigationas treatments for HER2þ MBC and HRþ HER2þ MBC. Trastuzu-mab is a humanized monoclonal antibody directed against the extra-cellular domain-2 of HER2, which prevents ligand-independentsignaling.51,52 Trastuzumab has been widely studied for treatment ofHER2þ breast cancer and is approved by the US Food and DrugAdministration (FDA) for first-line treatment of HER2þMBC as wellas a component of adjuvant systemic therapy for HER2þ breast can-cer.53 Two large phase III trials have demonstrated the benefit oftrastuzumab treatment with ET in patients with HRþ and HER2þ

MBC. In the Trastuzumab and Anastrozole Directed AgainstER-Positive HER2-Positive Mammary Carcinoma (TAnDEM) trial,median progression-free survival (PFS) was significantly improved inpatients with HRþ and HER2þ MBC who received anastrozole andtrastuzumab (n ¼ 103) compared with anastrozole (n ¼ 104) alone(4.8 vs. 2.4 months, respectively; P ¼ .0016).54 In the Efficacy ofletrozole in combination with trastuzumab compared to letrozolemonotherapy (eLEcTRA) trial, median time to progression wasnumerically longer in HRþ and HER2þ MBC patients receivingletrozole and trastuzumab (n¼26) versus letrozole (n¼ 31) alone (14.1vs. 3.3 months, respectively; P¼ .23). The failure to achieve statisticalsignificance was attributed to the small sample of patients evaluated.55

Another FDA-approved agent, lapatinib, is a dual EGFR/ErbB2reversible tyrosine kinase inhibitor that competes with ATP for itsbinding site on the tyrosine kinase domain. It blocks PI3K/Akt/mTOR and Ras/Raf/MEK/MAP kinase pathways downstream ofHER2.56 Lapatinib is FDA-approved for systemic treatment ofHER2þ advanced or MBC with systemic chemotherapy as second-line treatment or in first-line combination with letrozole for HRþ

and HER2þMBC.57 In the EGF30008 trial, addition of lapatinib toletrozole significantly reduced the risk of disease progression versusletrozole plus placebo; median PFS was 8.2 versus 3.0 months,respectively, (P¼ .019) in patients with ERþ andHER2þ early breastcancer. The clinical benefit rate was significantly greater for lapatinib-letrozole versus letrozole-placebo (48% vs. 29%, respectively;P¼ .003).58,59 These findings led to FDA approval of first-line MBCtherapy in ERþ and HER2þ disease. Phase III trials of lapatinib for

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postmenopausal advanced HRþ MBC are also underway. Forexample, one study is examining lapatinib plus fulvestrant comparedwith lapatinib and placebo (NCT00390455). Another study iscomparing 3 arms: lapatinib plus trastuzumab plus an AI, trastuzu-mab plus an AI, and lapatinib plus an AI (NCT01160211).

Combined targeting of HER2 with lapatinib and trastuzumab mayimprove outcomes compared with single-agent targeting. Dual inhibi-tion with these agents may also provide a regimen with greater tolera-bility compared with alternative treatments that involve the use ofcytotoxic agents.60 A phase III trial demonstrated that lapatinib plustrastuzumab significantly improvedPFS andoverall survival (OS)versuslapatinib monotherapy in patients with HER2þ MBC whose diseasehad progressed during prior trastuzumab therapy.61 The ALTERNA-TIVE (Safety and efficacy of lapatinib [L], trastuzumab [T], or both incombination with an aromatase inhibitor [AI]) study is an ongoingphase III, randomized, open-label, multicenter trial in HRþ HERþ

MBC examining the safety and efficacy of lapatinib plus trastuzumabplus AI versus trastuzumab plus AI.62 The rationale for this trial is toevaluate dual anti-HER2 therapywith anAI compared with single anti-HER2 therapy and an AI because HER2þ MBC is associated with apoor prognosis and increased resistance to ET in patients who areHRþ.

Pertuzumab is a monoclonal antibody to the extracellular domain4 of HER2 that blocks heterodimerization between HER2 andHER3 as well as other HER family members, thereby inhibitingsignal transduction via MAPK and PI3K pathways, which can leadto cell growth arrest and apoptosis.63 In a phase II trial, pertuzumabplus trastuzumab showed some improvements in patients withHER2þ MBC with progression on trastuzumab.64 A phase III trialof pertuzumab plus trastuzumab plus cytotoxic chemotherapy withdocetaxel as first-line treatment in HER2þ MBC demonstratedimproved PFS compared with trastuzumab plus docetaxel alone,and resulted in the FDA approval of this combination.65

IGF1R is a tyrosine kinase growth factor receptor that has beenshown to promote the growth of breast cancer cells.66 Similar to theHER2/EGFR family of receptors, growth-promoting effects ofIGF1R are mediated by PI3K and Ras/Raf/Mek/MAPkinase intra-cellular signaling. Inhibition of IGF1R function blocks the growth-promoting effects of IGF1 on breast cancer.67,68 Early preclinicalstudies were promising; however, trials with the monoclonal anti-bodies figitumumab, AVE1642, ganitumab, and dalotuzumab tar-geting IGF1R with ET in HRþ MBC have not shown advantage overET alone.66 A potential reason for this lack of benefit may be relatedto findings that breast cancer cells, which are resistant to tamoxifen,are also refractory to IGF1R antibody treatment.69 Inhibition ofIGF1R function at different stages of HRþ MBC or in combinationwith inhibitors of other pathways may prove more effective.

Growth factor receptor pathways such asHER2, IGF1R, andFGFR,which are independent of ER signaling, can subvert ET and lead totumor cell growth and proliferation in MBC. Combination strategies,which include monoclonal antibodies that block growth factor re-ceptors, coupled with tyrosine kinase inhibitors or other agents that actto disrupt signaling of multiple downstream effector molecules may bean effective approach to optimize treatment for the patient withMBC.

Intracellular Signaling CascadesPI3K/Akt/mTOR Pathway. The PI3K signaling pathway plays a

vital role in cell proliferation, differentiation, and cell survival.70 The

Figure 1 Cellular Signaling Pathways in Breast Cancer and Resistance to Endocrine Therapy. Estrogen Activates Cytoplasmic andNuclear ER. Cytoplasmic ER Binds to Growth Factor Signaling Components Such as PI3K, Activating Key Signaling MoleculesSuch as Akt and RAS, and Downstream Molecules Such as mTOR, Raf, and MAPK, Which Promote Cell Proliferation andSurvival. In Addition, Many of These Signal-Transduction Molecules Can Phosphorylate and Activate ER and its Co-Regulators to Enhance Nuclear Genomic ER-Mediated Responses. Src Can Also Interact With ER and Mediate NongenomicER Activation of Signaling Pathways and Gene Transcription. Src May Be a Key Signaling Molecule in the Development ofResistance to ET. ER Degradation by the 26S Proteasome is Depicted by the Schematic Model. Src Activation and ERProteolysis Support a Model Whereby Crosstalk Between Ligand-Bound ER and Src Drives ER Transcriptional Activity andTargets ER for Ubiquitin-Dependent Proteolysis. Src Activation May Promote Not Only Proliferation, but Also Estrogen-Activated ER Loss Through Proteolysis. Cyclin E1-CDK2 and Cyclin E2-CDK2 Are Activated During Estrogen-MediatedProliferation. Estrogen, via ER, Upregulates Cyclin D1. Cyclin D1 Upregulates E2F Transcription Factors, Leading to ActiveCyclin E2-CDK2 Complexes. Mitogenic Signals Converge at the Level of Cyclin D1 Upregulation and CDK4/6 Association,Localization, and Kinase Activity

Abbreviations: AI ¼ Aromatase inhibitor; CDK ¼ Cyclin-dependent kinases; EGFR¼ epidermal growth factor receptor; ER ¼ estrogen receptor; ERE ¼ estrogen response elements; FGFR ¼ fibroblastgrowth factor receptor; HER2 ¼ human epidermal growth factor; IGF ¼ insulin-like growth factor; MAPK ¼ mitogen-activated protein kinases; mTOR ¼ mammalian target of rapamycin inhibitor; p ¼phosphorylation sites; PI3K ¼ phosphatidylinositol 3-kinase; RTK ¼ receptor tyrosine kinase; Ub ¼ ubiquitin; TF ¼ transcription factor.

Stefan Glück

cell cycle is often dysregulated in cancer cells, resulting in reducedgrowth-factor dependency and increased replicative potential.Approximately 8% of breast cancers have mutations in PI3K; gain-of-function mutations in the PI3K alpha catalytic subunit(PIK3CA) occur in approximately 30% of ERþ breast cancers andare a common genomic alteration in this subtype of breast can-cer.71,72 PI3K is activated by growth-factor receptor tyrosine ki-nases, HER2, EGFR, IGF1R, and FGFR, and through a series ofsteps, activation of PI3K stimulates the downstream mediators Aktand mTOR.73 The PI3K pathway interacts with ER directly andindirectly in that PI3K and Akt can phosphorylate and therebyactivate ER in the absence of estrogen.74,75 This interaction of thePI3K pathway with ER confers ET resistance.74,76 A phase IIclinical trial that studied the combination of ET with everolimus, aninhibitor of mTOR, tested the rationale that inhibition of the PI3Kpathway might amplify response or reverse resistance to ET. Inpatients with HRþ breast cancer, the combination of everolimus

and letrozole resulted in increased clinical response and suppressionof tumor cell proliferation compared with letrozole and placebo.77

The Tamoxifen Plus Everolimus (TAMRAD) study in patientswith HRþ HER2� MBC who had progressed on an AI found thatthe addition of everolimus to tamoxifen improved clinical benefitrate, TTP, and OS compared with tamoxifen alone.78 The phase IIICombination of everolimus with trastuzumab plus paclitaxel as first-line treatment for patients with HER2-positive advanced breastcancer (BOLERO-2) trial showed that treatment with everolimusplus exemestane demonstrated a median TTP of 10.6 monthscompared with 4.1 months with exemestane alone in post-menopausal HRþ MBC that recurred or progressed on previousET.79,80 However, in the phase III Placebo-controlled trial ofletrozole plus oral temsirolimus (HORIZON) trial, the addition ofthe mTOR inhibitor temsirolimus to letrozole did not lead toimprovement in PFS in AI-naive advanced breast cancer.81 Severaldifferences between these trials may contribute to the varied results.

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First, patient populations differed, with AI-resistant patients in theTAMRAD and BOLERO-2 trials, but AI-naive patients in theHORIZON trial. With both mTOR inhibitors subject to meta-bolism by the same CYP enzyme (CYP3A), population differencesin its expression might have resulted in variable metabolism acrossdiverse ethnic groups. Genotypic and phenotypic variation acrosspopulations may have also accounted for variable response. Finally,relative differences in drug effectiveness between everolimus andtemsirolimus cannot be ruled out.82 Ongoing studies of fulvestrantwith everolimus and standard ET with everolimus will provideadditional perspective on the potential combined benefit of mTORand ER inhibition (NCT01797120, NCT02035813).

Inhibitors of PI3K in preclinical and clinical trials for breastcancer include BKM120, LY294002, GDC0941, BYL719, andGDC0032.83 Additionally, combination PI3K/mTOR inhibitors,GDC0980 and BEZ235, are also being studied. In preclinicalstudies, GDC0980, GDC0032, and GDC0941, given in combi-nation, enhanced activity of fulvestrant in MCF-7 xenografts,resulting in tumor regression and tumor growth delay.84 A com-bination of tamoxifen, everolimus, and LY294002 producedincreased antitumor effects compared with tamoxifen alone ortamoxifen and everolimus in a breast cancer cell line.85 In addition,treating breast cancer cells with BEZ235 induced apoptosis whencombined with estrogen deprivation, suggesting potential activity inHRþ breast cancer.86 In an initial phase I/II study, patients withtrastuzumab-resistant locally advanced or metastatic HER2þ breastcancer were treated with BKM120 (buparlisib) and trastuzumab.Two patients (17%) had partial responses, and 7 (58%) had stabledisease (� 6 weeks) with a disease-control rate of 75%.87 Buparlisibin combination with letrozole has also been used to treat ERþ

HER2� MBC, and provided clinical benefit in both patients withPI3K-wild type and mutant tumors, but patients with coexistingPI3K and MAP3K mutations derived the greatest clinical benefitfrom this regimen.88 The Akt inhibitor MK2206 is also beingstudied in breast cancer. In MCF-7 cells, overexpression of consti-tutively active Akt1 induced resistance to anastrozole; addition ofthe Akt inhibitor MK2206 restored sensitivity to this AI.89

Taken together, the preclinical and clinical data suggest that ERand PI3K pathways counter-regulate each other with low PI3Kactivation resulting in high ER levels, and hyperactivation ofPI3K causing a reduction in ER levels.90 Further validation of thisnotion comes from studies showing that inhibition of HER2 with theantibody trastuzumab or the tyrosine kinase inhibitor lapatinibrestores or upregulates ER levels or transcriptional activity in breastcancer cells and patient tumors.91,92 Also, AIs or fulvestrant inhibitthe growth ofHER2þ tumors that have progressed on trastuzumab orlapatinib.92 Numerous ongoing phase I to III studies are examiningthe effects of AIs or fulvestrant in combination with PI3K andAkt inhibitors (NCT01610284, NCT01633060, NCT01791478,NCT02088684,NCT01248494,NCT01339442,NCT01437566,NCT01296555, and NCT01344031).

Src kinases, intracellular tyrosine kinases that contribute tocontrol of cell proliferation, have been implicated in oncogenicactivity in breast cancer, particularly HRþ breast cancer.93 Dasatinibis a small-molecule inhibitor of several Src family kinases approvedfor use in chronic myelogenous leukemia.94 A recent phase II trial ofdasatinib in women with MBC showed modest activity with disease

nical Breast Cancer Month 2016

control rates (ie, complete response, partial response, or stable dis-ease for at least 16 weeks) for 8.3% of patients with HER2þ MBCand 15.6% of those with HRþ MBC.95 Early results of a trial ofdasatinib in combination with fulvestrant as second-line treatmentof HRþ MBC in postmenopausal women did not find improvedefficacy with the addition of dasatinib.96

With the variety of agents available that target the PI3K/Akt/mTOR pathway, the challenge remains to identify which subset oftargets, and therefore, which combination of agents may be themost impactful to maximize clinical benefit and adapt to ETresistance in individual patients.

Cell Cycle Control and ProliferationCellular pathways involved in cell proliferation are targets for new

drugs that interfere with development of resistance to ET and thattreat HRþ MBC. Cyclin-dependent kinases (CDKs) are a subgroupof serine/threonine kinases that play a key role in regulating cellcycle progression.97 The cell cycle G1 to S-phase transit iscontrolled by CDK4 and CDK6, which are activated upon bindingto D-type cyclins leading to expression of genes required for S-phaseentry.98 Several studies have identified alterations of cell cycle reg-ulators in human breast cancer and provide a rationale for the po-tential therapeutic role for CDK4/6 inhibition in breast tumors.Cyclin D1 is a direct transcriptional target of ER.99,100 Treatmentwith antiestrogens induces growth arrest of HRþ breast cancer cellsand decreases cyclin D1 expression.101 Conversely, microinjectionof antibodies into cyclin D1 inhibits estrogen-induced S-phase en-try.102,103 Endocrine resistance is associated with persistent cyclinD1 expression and Rb phosphorylation.104 Compared with othersubtypes of breast cancer, HRþ breast cancer is commonly associ-ated with hyperactivation of cyclin D1-CDK4/6.105

CDK4/6 inhibitors, including palbociclib, abemaciclib, and ribo-ciclib, are being investigated in both the preclinical and clinical settings;some are becoming available for treatment of HRþ MBC. In 2015,palbociclib, a highly selective inhibitor of CDK4/6 kinase, wasapproved by the FDA in combination with letrozole for the treatmentof postmenopausal women with ERþ HER2� advanced breast canceras initial endocrine-based therapy for their metastatic disease.106,107

Approval for this indication was accelerated based on PFS data.Continued approval for this indication may be contingent upon veri-fication and description of clinical benefit in a confirmatory trial. Inpreclinical studies, palbociclib inhibited growth of HRþ cancer cells,including those that are resistant to antiestrogen.105 Additionally, asynergistic antitumor effect was observed when palbociclib was com-bined with tamoxifen in both tamoxifen-sensitive and tamoxifen-resistant cell lines. Palbociclib in combination with letrozole versusletrozole alone (PALOMA-1), a randomized phase II study of the AIletrozole with or without palbociclib as first-line therapy for ERþ,HER2�MBC(n¼ 165), reported a striking improvement inPFS from7.5 to 26.2 months (hazard ratio [HR], 0.32; 95% confidence interval[CI], 0.19-0.56; P < .001) without additional safety concerns.108 Inthe final results analysis, the median PFS was 20.2 months withletrozole plus palbociclib compared with 10.2 months for letrozolealone (HR, 0.488; 95%CI, 0.319-0.748; 1-sided P¼ .0004), and thecombination demonstrated significant clinical benefit as first-linetreatment in this patient population.109 Several phase I, II, and IIIstudies of palbociclib in combination with fulvestrant and AIs (eg,

Stefan Glück

PALOMA-2 and PALOMA-3) in HRþ MBC are currently underwayor have recently concluded.110,111 Most recently, treatment with thecombination of palbociclib and fulvestrant in patients with advancedHRþ HER2� breast cancer who had relapsed or progressed duringprior endocrine therapy, received FDA approval based on the findingsof the phase III study, PALOMA-3.110 The study demonstrated thatthe combination of palbociclib and fulvestrant resulted in significantlylongermedian PFS (9.5months [95%CI, 9.2-11.0months] comparedwith 4.6months [95%CI, 3.5-5.6months]) for fulvestrant alone (HR,0.46; 95% CI, 0.36-0.59; P < .001).110

Other CDK inhibitors are in various stages of clinical develop-ment (NCT00721409, NCT01723774, NCT01942135,NCT02040857). Additionally, another CDK inhibitor, LEE001,ribociclib, is being used in addition to everolimus and exemestane inpatients with advanced HRþ, HER2� breast cancer who haveanastrozole- or letrozole-resistant disease (NCT01857193). Interimresults from the phase Ib/II study showed a 70.9% disease controlrate.112 This agent is also being examined in combination withfulvestrant and PI3K inhibitors (NCT02088684).

The CDK pathway regulates the cell cycle, with CDK4 and 6directly controlling the gap phase 1 of mitosis (G1). Dysregulation ofthese components may occur through multiple mechanismscontributing to tumor growth and metastatic potential, and is acommon feature in many cancers, including breast cancer. Therefore,therapeutic inhibition of CDK is a relevant treatment strategy, and anumber of agents are currently under investigation in clinical tri-als.98,105 Ongoing clinical trials that combine CDK inhibitors withinhibitors of other dysregulated pathways commonly found in breastcancer may improve outcomes and act to overcome ET resistance.

Immune Response ModulationAntitumor immunity in human cancer is often ineffective owing

to the tight regulation associated with the maintenance of immunehomeostasis. Previous attempts to develop effective immunother-apies for cancer have been unsuccessful because there are significantbarriers that have been difficult to overcome. However, blockade ofthe programmed death receptor (PD-1), which may play a role inthe tumor’s ability to bypass immune modulation and is overex-pressed in certain tumors such as melanoma, has been proveneffective for treatment.113-115 Additionally, a monoclonal antibodytargeting cytotoxic T-lymphocyte antigen-4 (CTLA-4), an immunecheckpoint molecule that downregulates pathways of T-cell activa-tion, has also been proven effective for treatment of melanoma.116

The place of PD-1 and CTLA-4 inhibition in the treatment ofHRþ MBC is not as clearly defined.

The extent of mutations and aberrant protein expression associ-ated with cancer cells may also dictate the potential for efficacy ofimmunotherapy.117 Foreign antigens expressed on the surface ofpathogens trigger the body’s immune system to target and eliminatethese invaders. However, tumor cells are often undetected by im-mune surveillance owing to identification of tumor cells as “self.”Although tumor cells may over express tumor-associated antigens,these same antigens are expressed at low levels on normal cells,leading to immune tolerance. In contrast, neoantigens that arise dueto mutations in a tumor cell, which are not expressed on any othernormal cells, represent prime targets for Tecell-mediated elimina-tion with no risk to normal tissues.117 This has led to the hypothesis

that tumors with high mutation rates have increased neoepitopes,and use of checkpoint blockade therapies (PD-1, PD-L1, andCTLA-4) can reactivate tumor-infiltrating T cells in these cases,leading to tumor regression.117

The heterogeneous nature of breast cancer has led to studies thatseek to identify biomarkers associated with drug response. Triple-negative breast cancer (TNBC) lacks the molecular targets formany treatment options and is associated with high genetic alter-ation rates. In particular, one study of 2111 patients with TNBC(ER�/PgR�/HER2�) found that 91% of patients showed dimin-ished androgen receptor expression and/or mutations in PI3KCA/PTEN/AKT1, or loss of PTEN.118 In addition, expression of PD-L1is more prevalent in TNBC, and this coupled with increased PD-L1-mediated T-cell reactivation suggests that PD-L1 inhibitors maybe effective in this patient population.117,119 In fact, a preliminarystudy that targeted PD-L1 showed promising objective clinical ac-tivity in TNBC patients (NCT01375842).119

Estrogen-Receptor Degradation and ProteasomeInhibitors (PIs)

Proteasomes are multicatalytic protease complexes in the cell,involved in the non-lysosomal recycling of intracellular proteins. Pro-teasomes play a critical role in regulation of cell division in both normaland cancer cells.120-123 In cancer cells, this homeostatic function isderegulated, leading to the hyperactivation of the proteasomes. PIsinterfere with the ubiquitin proteasome pathway and lead to theaccumulation of proteins engaged in cell cycle progression. They ulti-mately put a halt to cancer cell division and induce apoptosis. Cell cycleproteins are regulatory proteins whose temporal activity is tightlycontrolled and are thus important targets of proteasome degradation.Major proteins related to the cell cycle process are cyclins, CDKs, CDKinhibitors (CKIs), and some transcription factors. Cyclin proteins arefound to be highly upregulated in cases of aberrant cell division incancer cells, particularly cyclinsD andE.124,125 The precisemechanismof actionof PIs in cancer treatment is notwell-understood. In treatmentof breast cancer, 2 potential mechanisms have been described: the PI,bortezomib, may inhibit cell proliferation and may decrease levels ofER.126,127 Results from early preclinical and clinical trials have beenmixed, with 1 small study showing no effect of treatment with borte-zomib and another showing enhancedMCF-7 tumor suppression withbortezomib and trastuzumab.128 Data in MCF-7 breast cancer cellssuggest that fulvestrant’s antiestrogenic effects of induction of protea-somal degradation of ER may involve c-SRC, a cellular kinase proto-oncogene. In light of this potential interaction, a phase II study offulvestrant and bortezomib in women with locally advanced orMBC isunderway (NCT01142401).

Epigenetic ModifiersIn addition to genetic alterations such as the mutations and de-

letions that alter the composition of genomic DNA, epigeneticchanges including DNA methylation, histone modifications, andchromatin remodeling can reversibly alter gene expression and playa key role in tumorigenicity and metastatic potential of breast cancercells.129 Therapeutic interventions that target epigenetic modifica-tions have been used to partially or completely revert the alteredgene expression, and several epigenetic agents that are approved foruse in other settings have potential as breast cancer therapies.

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Several clinical trials of these epigenetic agents are planned orunderway in the breast cancer setting. Azacitidine and decitabine arehypomethylating agents that inhibit DNA methyltransferase,thereby halting tumor cell growth and division through epigeneticmanipulation.130,131 Both agents have been approved for use inmyelodysplastic syndrome and are currently being investigated forbreast cancer treatment.130,131 In combination with fulvestrant,azacitidine is being evaluated in a phase II trial in patients withERþ, HER2� MBC who have progressed on an AI(NCT02374099). In addition, a class I HDAC inhibitor, entino-stat, was assessed in a phase II trial (NCT00676663) with orwithout the AI exemestane in patients with ERþ breast cancer whohave progressed on an AI, and the combination was found to notonly improve PFS and OS, but restore sensitivity to hormonetherapy.132 Entinostat is currently being studied in combinationwith azacitidine in a phase II trial of patients with either TNBC orHRþ HER2� breast cancer (NCT01349959). Decitabine incombination with the HDAC inhibitor panobinostat (LBH589)was also being investigated in TNBC in an attempt to re-expressER; however, the study was closed due to slow accrual(NCT01194908).

ConclusionsER-targeted therapy is important for many women with breast

cancer, but resistance to therapy inevitably occurs. The ERsignaling pathway is a complex network of extensive crosstalk withgrowth-factor signaling pathways, cell cycle control pathways, andprotein degradation pathways. These pathways provide manyalternative targets for agents that may be useful in combinationwith ET to decrease resistance to treatment and to extend benefitto patients who do not achieve optimal benefit from ET alone. Aseffective treatments are identified, an important area of focus mustbe on the factors that determine optimal combination treatmentsin subtypes of breast cancer. Such factors comprise not only theresponsiveness of the cells of a particular subgroup of tumors, butalso the characteristics of different treatment combinations withrespect to the temporal profile of effects, relative tolerability ofdifferent combinations, and the compatibility of routes ofadministration. Several therapies that are discussed in this review,among them everolimus (mTOR inhibitor), palbociclib (CDK4/6inhibitor), and MK-3475 (PD-1 inhibitor), are already proving tobe effective treatments for MBC and useful in reversing or pre-venting the development of resistance to ET. Through inhibitionof central components of pathways for growth factor-promotedcellular proliferation, cell cycle-mediated cellular proliferation,and immunosuppressive programs that initiate and sustain tumorgrowth, these agents are effective options that may extend survivaland maintain quality of life for women with MBC who haverelapsed on endocrine therapies.

AcknowledgmentsMedical writing and editorial services were provided by SCI

Scientific Communications & Information, Parsippany, NJ, andThe Lockwood Group, Stamford, CT (funded by AstraZeneca LP).

Clinical Breast Cancer Month 2016

DisclosureThis article was supported by AstraZeneca LP. Dr Glück has

received grant support and consulting fees from Agendia, Celgene,Dara, Eisai, Genentech/Roche, GHI, Mylan, and Onyx/Amgen. Atthe time of the initiation of manuscript development, Dr Glück wasthe Sylvester Professor of Medicine, Department of Medicine, Di-vision of Hematology Oncology, Sylvester Comprehensive CancerCenter, Miami, FL. Dr Glück is a current employee and shareholderof Celgene Corporation, Summit, NJ.

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