The kinome associated with estrogen receptor-positive status in human breast cancer

Endocrine-RelatedCancer

ReviewM C Bruce et al. Kinome associated with

ER-positive status21 :5 R357–R370

The kinome associatedwith estrogenreceptor-positive status in humanbreast cancer

M Christine Bruce, Danielle McAllister and Leigh C Murphy

Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, University of Manitoba

and CancerCare Manitoba, 675 McDermot Avenue, Winnipeg, Manitoba, Canada R3E 0V9

http://erc.endocrinology-journals.org q 2014 Society for EndocrinologyDOI: 10.1530/ERC-14-0232 Printed in Great Britain

Published by Bioscientifica Ltd.

Correspondence

should be addressed

to L C Murphy

Email

Leigh.Murphy@

med.umanitoba.ca

Abstract

Estrogen receptor alpha (ERa) regulates and is regulated by kinases involved in several

functions associated with the hallmarks of cancer. The following literature review strongly

suggests that distinct kinomes exist for ERa-positive and -negative human breast cancers.

Importantly, consistent with the known heterogeneity of ERa-positive cancers, different

subgroups exist, which can be defined by different kinome signatures, which in turn are

correlated with clinical outcome. Strong evidence supports the interplay of kinase networks,

suggesting that targeting a single node may not be sufficient to inhibit the network.

Therefore, identifying the important hubs/nodes associated with each clinically relevant

kinome in ERC tumors could offer the ability to implement the best therapy options at

diagnosis, either endocrine therapy alone or together with other targeted therapies,

for improved overall outcome.

Key Words

" estrogen receptors

" breast cancer

" phosphorylation

" kinases

" endocrine therapy sensitivity

" biomarker

Endocrine-Related Cancer

(2014) 21, R357–R370

Introduction

The idea of personalized approaches to therapy in breast

cancer based on the molecular nature of the tumor can be

traced back to the late 1960s and early 1970s, when it was

discovered that some but not all human breast tumors

express estrogen receptors (ERs; Jensen et al. 1971). This

heralded the beginning of an era of targeted therapies for

breast cancer, identified the first biomarker used clinically

to predict the biological behavior of breast cancer, and

established the beginnings of understanding molecular

mechanisms by which the ovarian hormone, estrogen,

drives the growth and survival of the majority of human

breast cancers (Jensen & Jordan 2003), at least initially.

Inhibiting the activity of ERs with the antiestrogen

tamoxifen was the first targeted therapy in breast cancer.

Although the knowledge that female hormones were

involved in breast cancer and the hormonal/endocrine

therapy was used for breast cancer at least in the form of

ablation surgery such as ovariectomy dates back well over

a century, the identification of ERs in breast cancers

provided a molecular mechanism and rationale for the use

of hormonal therapies (Jensen & Jordan 2003). This led

directly to the development of the successful modern

endocrine therapies such as tamoxifen and other selective

estrogen receptor modulators (SERMs), which bind to the

ERs and induce conformational changes that modify and

in some cases inactivate the ERs (Jensen & Jordan 2003).

The newer endocrine therapies, the aromatase inhibitors

(AIs), which inhibit the aromatase enzyme, eliminate the

production of estrogen and therefore inhibit estrogen’s

proliferative action (Goss et al. 2011).

It was evident from the start that not all ERC breast

tumors were created equal. Although a little more than

http://erc.endocrinology-journals.org

http://dx.doi.org/10.1530/ERC-14-0232


Review M C Bruce et al. Kinome associated withER-positive status

21 :5 R358

70% of all breast tumors express ER, only about half of

patients with ERC tumors respond to tamoxifen. There-

fore, ERC breast cancers exhibit heterogeneity associated

with prognosis and treatment outcomes. The first step to

resolve this heterogeneity came from the idea that the

measurement of a downstream target of estrogen-

dependent ER signaling such as the progesterone receptor

(PR) would increase confidence that the pathway was

intact (Horwitz et al. 1975). This increased the accuracy of

treatment prediction, but was obviously still imprecise as

some 20–30% of ERC/PRC tumors are de novo resistant to

the endocrine therapies. Furthermore, initial response to

endocrine therapies is often followed by acquired resist-

ance despite the continued expression of ER (Encarnacion

et al. 1993, Bachleitner-Hofmann et al. 2002).

The next significant insight into the heterogeneity of

ERC breast cancer came with the identification of HER2

(ERBB2) amplification. Approximately 20% of all breast

cancers have amplified, overexpressed HER2, and 40–50%

of these will also be ERC. Interestingly, ERC/HER2C

tumors are more likely to be resistant to endocrine

therapy, in particular tamoxifen, thus providing

important insight into the relevance of crosstalk of

signaling pathways initiated at the level of the plasma

membrane with many aspects of the ER signaling

pathway. This may cause ligand-independent activation

of ER signaling and hormone therapy resistance.

The molecular detailing that has become possible

through the Human Genome Project and new high-

throughput/high-content technologies in the last decade

initially established five intrinsic molecular subgroups

(Sorlie et al. 2003) of which three were significantly

populated with ERC tumors. Most recently, at least ten

molecular subgroups of human breast cancer have been

described (Curtis et al. 2012) and eight of these appeared to

be significantly populated with ERC tumors (Curtis et al.

2012). Associated with these studies has been the frequent

identification of kinases, either mutated and/or structurally

altered in large cohorts of breast tumors (Banerji et al. 2012,

Curtis et al. 2012, Shah et al. 2012, Stephens et al. 2012).

Some of these have also been found frequently altered and

associated with AI sensitivity (Ellis et al. 2012).

ER and its many coactivators are regulated by

phosphorylation as well as other post-translational

modifications (PTMs; Rowan et al. 2000, York et al. 2010,

Le Romancer et al. 2011, Zhang et al. 2013). The discovery

that the ER and its coactivators are substrates of several

kinases (enzymes causing phosphorylation of specific

substrates), which are regulated by signaling pathways

frequently mutated or structurally altered in breast cancer


(Yamnik et al. 2009, Yamnik & Holz 2010, Murphy et al.

2011), as well as the demonstration that a clinically

relevant phosphorylation profile of ERs can be identified

in human breast cancer (Skliris et al. 2010a), suggests that

kinases and/or phosphatases associated with ERC breast

cancer could provide a wealth of potential drug targets to

complement existing endocrine therapies or generate new

endocrine therapies. The following is a review of kinases

that have been identified as associated with ER status in

breast tumors or those that have been implicated in the

regulation of estrogen signaling and/or modifying sensi-

tivity to estrogen and its antagonists in breast cancer.

Kinases identified as mutated or structurallyaltered in breast cancer in large breast cancercohorts

Genome-wide analyses of large cohorts of breast cancer

cases are providing detailed, comprehensive analyses of

genomic aberrations in breast cancer at a population level.

Some studies have also provided data concerning their

impact on clinical characteristics. These studies have

shown that many of the frequently altered (amplified,

fused, deleted, or mutated) genes encode kinases. Some of

these frequently altered genes, such as HER2, were

previously known but others, such as MAP3K1 and its

substrate MAP2K4, have not previously been identified to

have functional roles in breast cancer. However, given that

enzymes, kinases in particular, have proven to be clinically

efficacious therapeutic targets, a wealth of data has now

been generated not only to understand the complex

biology of the disease but also to identify new treatments

for specific cohorts.

The recently published METABRIC cohort of breast

cancers, in which 2000 individual breast cancers were

interrogated, identified ten integrative clusters, each with

distinct molecular characteristics associated with clinical

outcome (Curtis et al. 2012). Often, altered genes encoding

kinases and phosphatases dominate individual clusters

(Table 1). Furthermore, the original intrinsic subtypes, in

particular the ERC luminal A and luminal B (Perou et al.

2000, Ignatiadis & Sotiriou 2013) subtypes, have been

further subdivided due to the METABRIC study (Curtis et al.

2012, Dawson et al. 2013). Therefore, a brief description of

this follows as it relates to clusters that have significant

ERC components.

METABRIC integrative cluster 1 (IntClust1), represent-

ing 7% of breast cancer (Curtis et al. 2012, Dawson et al.

2013), contains predominantly ERC tumors with luminal B

features and is characterized by amplification of the




Table 1 Kinases and phosphatases altered in ERC-dominated

breast cancer clusters from METABRIC (Curtis et al. 2012,

Dawson et al. 2013)

METABRIC

integrative clusters

Kinases or phosphatases

identified Alteration

IntClust1 Ribosomal protein S6kinase 1 (RPS6KB1)

Amplification/overexpression

Protein phosphatase1D (PPM1D)

Amplification/overexpression

IntClust2 PAK1 Amplification/overexpression

PIK3CA MutationIntClust3 PIK3CA Mutation

MAP3K1 MutationIntClust5 HER2 Amplification/

overexpressionPIK3CA Mutation

IntClust6 FGFR1 AmplificationIntClust7 PI3K Mutation

MAP3K1 MutationIntClust8 MAP2K4 Mutation

PI3K MutationIntClust9 Regulatory subunit B

of proteinphosphatase 2alpha (PPP2R2A)

Deletion



21 :5 R359

17q23 region. Two genes in this amplicon associated with

increased expression are ribosomal protein S6 kinase beta 1

(RPS6KB1 and p70S6K1) and protein phosphatase 1D

(PPM1D). Interestingly, expression of both of these genes

has been shown to be regulated by estrogen in ERC breast

cancer cells at the transcriptional level (Han et al. 2009,

Yamnik et al. 2009, Yamnik & Holz 2010). In addition,

the protein product of RPS6KB1 (p70S6K1) has

been shown to phosphorylate ERa (Yamnik & Holz 2010)

and under IGF1 stimulation p70S6K1 can be

co-immunoprecipitated with ERa in ERC breast cancer

cells (Becker et al. 2011). PPM1D/Wip1 is known for its p53

inhibitory effects and, in ERC MCF7 cells, has been shown

to be regulated by estrogen and to increase the transcrip-

tional activity of ERa (ESR1) as well as other steroid hormone

receptors (Proia et al. 2006), possibly by modulating

phosphorylation of coactivators such as SRC1 and/or

p300/CBP (Proia et al. 2006).

Integrative cluster 2 (IntClust2), representing 4% of

breast tumors, is also dominated by ERC tumors with

characteristics of both luminal A and luminal B subtypes

(Dawson et al. 2013). Amplification of chromosome region

11q13/14 characterizes this cluster and high expression of

another kinase, PAK1, known to affect ERa phosphory-

lation and activity (Wang et al. 2002, Mazumdar & Kumar

2003, Holm et al. 2009, Kok et al. 2010) is associated with


its amplification in this region. A high frequency (w50%)

of PI3K catalytic subunit p110a (PIK3CA) mutations is also

observed in this group.

ERC tumors with luminal A characteristics predo-

minate integrative cluster 3 (IntClust3), which represents

15% of all breast tumors (Dawson et al. 2013). Tumors in

this cluster tend to have few structural alterations, low

genomic instability, and usually an excellent prognosis.

Interestingly, they also have the highest frequency of

PIK3CA mutations (w58%). As well, similar to other

clusters dominated by ERC tumors, a high level of

MAP3K1 mutations (w15%) is observed in IntClust3.

IntClust5 represents 10% of all breast tumors and is

characterized by HER2 amplification with w42% of them

also expressing ERa (Dawson et al. 2013). Approximately

30% of tumors in this cohort also have PIK3CA mutations.

ERC tumors with overexpressed HER2 are more likely to

be resistant to endocrine therapies. One reason for this is

thought to be the constitutive activation of several kinases

downstream of HER2, such as ERK1/2, PI3K, and AKT, all

of which can phosphorylate ERa and several ER coacti-

vators to enhance the ligand-independent activity of ERa

as well as to enhance the agonist activity of tamoxifen

(Wu et al. 2005, Le Romancer et al. 2011).

IntClust6, representing 4% of breast tumors, is another

predominantly ERC group with both luminal A and

luminal B characteristics (Dawson et al. 2013). Tumors of this

group show amplification of the chromosome 8p12 locus and

are genomically unstable with an intermediate prognosis.

Less than 20% of tumors in this group have PIK3CA

mutations, which is low compared to other ERC groups.

The gene for FGFR1 is located in the chromosome 8p12

region. FGFR1 amplification and overexpression have been

directly linked to endocrine therapy resistance (Turner et al.

2010, Balko et al. 2012). FGFR1 also regulates several kinases

such as PI3K and AKT, which are known to phosphorylate

ERa and its coactivators (Le Romancer et al. 2011).

IntClust7 (10% of breast tumors) and IntClust8 (15%

of breast tumors) are mainly ERC groups and while similar

in many ways tumors in Intclust8 display higher genomic

instability, with an associated inferior prognosis than

tumors in IntClust7 (Dawson et al. 2013). Interestingly,

both again have a high frequency of PI3K (PIK3CA)

mutations. IntClust7 has a high frequency of MAP3K1

mutations and IntClust8 has the highest frequency of

MAP2K4 mutations, a downstream target of MAP3K1.

The structural alterations usually found in MAP3K1

and MAP2K4 are deletions and mutations associated

with loss of function (Teng et al. 1997, Pham et al. 2013),

suggesting that the pathways they regulate have






21 :5 R360

tumor-suppressor function. Steroid hormone receptor

signaling, including estrogen and androgen receptors,

shows crosstalk with MAP3K1 in some systems, and it is

the ERC tumors, in particular luminal A type, that have

the highest level of mutation and/or deletion of the

MAP3K1/MAP2K4 pathway (Pham et al. 2013). This

pathway has been implicated in cell death pathways,

which under normal conditions is important for mam-

mary gland involution, hence its loss of function in many

ERC breast cancers may in part be responsible for the

dissociation of estrogen-induced proliferative and survival

pathways from growth-inhibiting differentiation

pathways (Pham et al. 2013). This pathway has been

described as having a molecular switch type function

between survival and cell death, possibly dependent on

cell-type background, subcellular localization, and caspase

3 activity (Pham et al. 2013). Increased ERa expression

combined with a decreased activity of the MAP3K1/

MAP2K4 pathway may, in part, underlie the change in

the balance of estrogen signaling regulation of survival/

proliferation vs cell cycle inhibition that is often

associated with differentiation.

IntClust9 (7% of breast tumors) is characterized by

a high frequency of PPP2R2A deletions (Dawson et al.

2013). PPP2R2A is the regulatory subunit B of protein

phosphatase 2 alpha (PP2A). While IntClust9 contains a

mixture of intrinsic subtypes, its ERC members are mainly

of the luminal B type, and loss of PPP2R2A expression is

associated with a high mitotic index. Interestingly, PP2A

also has been implicated in the regulation of ERa

phosphorylation and activity (Lu et al. 2003a).

The above data underscore that distinctly altered

kinase and phosphatase mutation patterns occur frequently

in ERC breast cancer subgroups with altered clinical

outcome. This would support the idea that combining

endocrine therapies with other targeted therapies informed

by each subgroup’s distinct kinase pattern could provide

better clinical outcome for breast cancer patients.

Kinases associated with the PI3K/AKT/mTORand ER-positive breast cancer

METABRIC and similar studies (Curtis et al. 2012, Ellis et al.

2012, Stephens et al. 2012) as well as previous smaller scale

studies (Miller et al. 2010, 2011a,b) have identified the

frequent structural and mutational alterations in the

PI3K/AKT/mTOR pathway that occur in ERC breast cancer.

IntClust1, IntClust2, IntClust3, and IntClust5 (Dawson

et al. 2013) have w25, 50, 58, and 30% frequencies of

PIK3CA mutations respectively. IntClust7 and IntClust8


also have a high frequency of PIK3CA mutations while by

contrast IntClust6 has less than a 20% frequency of PI3K

pathway mutations (Dawson et al. 2013).

Experimental and clinical data suggest that hyper-

activation of the PI3K/AKT/mTOR pathway is associated

with endocrine resistance. However, gain-of-function

PIK3CA mutations in ERC tumors are often associated

with good prognosis (Miller et al. 2011a). Such apparently

contradictory results may be due to complexities associ-

ated with feedforward and feedback mechanisms involved

in the PI3K/AKT/mTOR pathway and other alterations

co-occurring in regulators of the pathway. This latter idea

is supported by the finding that most often the associ-

ations of PIK3CA mutations with good outcome in ERC

tumors are restricted to those tumors that are also HER2

negative (Fu et al. 2013).

As discussed above, the PIK3CA gene, encoding the

PI3K catalytic subunit p110a, is one of the most frequently

mutated genes in ERC breast cancers (Dawson et al. 2013).

Many of the mutations occur in ‘hot spots’ with the most

frequent mutations being E542K, E545K (both in the

helical domain), and H1047R (in the kinase domain)

(Barbareschi et al. 2007). Generally, the mutations result in

constitutive activation of the kinase (Di Cosimo & Baselga

2009). As mentioned above, the published data largely

suggest that mutations in PIK3CA occur more frequently

in ERC breast cancer with good prognosis and may also be

associated with better clinical outcome in patients treated

with endocrine therapy (Di Cosimo & Baselga 2009, Miller

et al. 2011a). However, this is not a universal finding

(Cuorvo et al. 2014). Recent reviews have discussed this

aspect in detail (Fu et al. 2013).

AKT1 mutations, often associated with PI3K-inde-

pendent constitutive activity, occur in w4% of breast

cancers and are often associated with ERC tumors.

However, the relationship of AKT expression and/or

activation to outcome in breast cancer is inconsistent

(Badve et al. 2010, Aleskandarany et al. 2011). Consi-

dering that the different AKT isoforms have distinct

functions (Dillon & Muller 2010) and that a recent study

using proximity ligation assay (PLA) to distinguish

between pAKT1 and pAKT2 (Spears et al. 2012)

determined the former to be associated with poor

prognosis and the latter with better prognosis, it seems

possible that the relative levels of each isoform may

determine the final read out. Furthermore, the inability

to distinguish the different pAKT isoforms may, at least in

part, underlie the previous lack of consistency (Badve

et al. 2010, Aleskandarany et al. 2011) around their

association with breast cancer outcome.






21 :5 R361

Owing to the frequency with which components of the

PI3K/AKT/mTOR pathway are altered in ERC tumors, and

the fact that the pathway is a central hub receiving growth

and survival signals from many factors, the detailed

molecular nature of the pathway in different ERC tumors

may be important to guide appropriate therapy options.

Kinases associated with ER status byexpression analyses

Several studies have now been published in which different

kinase expression patterns were found in ERC vs other

clinical subtypes of breast cancer. Using publically available

RNA expression databases, Bianchini et al. (2010) identified

16 kinases that were overexpressed in ERC/HER2K breast

cancer biopsy samples. These were IGF1R, STK32B, ERBB4,

FGFR3, BMPR1B, MAST4, HSPB8, IKBKB, DCLK1, ERBB3,

STK39, MAP3K1, PTK6, PLK2, TEX14, and MST1. Kinases

uniquely overexpressed in ERK and HER2C breast cancer

subtypes were also identified. Two robust kinase clusters

were recognized: a mitosis metagene cluster (12 distinct

kinases) and an immune kinase cluster (15 distinct kinases),

which were present inall clinical subgroups. Overexpression

of kinases composing the mitosis metagene cluster was

mostly found in ERK tumors and was not prognostic in this

subgroup. However, ERC/HER2K tumors that presented a

high mitosis kinase score were associated with a worse

prognosis but showed a higher frequency of pathological

complete response (pCR)toneoadjuvantchemotherapy.On

the other hand, overexpression of kinases from the immune

kinase cluster was associated with better survival in

ERC/HER2K and HER2C subgroups. Interestingly, the

authors also observed that in ERC tumors, many of the

overexpressed kinases were transmembrane growth factor

receptors, while ERK tumors mostly overexpressed intra-

cellular kinases associated with cell proliferation (Bianchini

et al. 2010).

Finetti et al. (2008) undertook a genome-wide

expression analysis of a cohort of primary human breast

tumors focusing mainly on the basal and luminal A

intrinsic breast cancer subtypes. From this analysis, they

extracted kinase genes whose differential expression was

associated with a clinical outcome. Not unexpectedly,

there were substantial differences in the pattern of kinase

gene expression between the basal and the luminal

A intrinsic subtypes. Of more interest, however, was the

discovery that 16 kinases were overexpressed in most of

the basal group and in a few luminal A tumors. These

kinases were AURKA, AURKB, BUB1, BUB1B, CDC2 (CDK1),

CDC7, CHEK1, MASTL, MELK, NEK2, PBK, PLK1, PLK4,


SRPK1, TTK, and VRK1. Many of these kinases have been

previously reported to be involved in the G2 and M phases

of the cell cycle (Malumbres & Barbacid 2009, Knight et al.

2010). Notably, these kinases include the 12 kinases

making up the mitosis metagene cluster identified by

Bianchini et al. (2010). Similarly, Finetti et al. (2008) found

that overexpression of kinases from this 16-kinase

signature in luminal A (ERC) tumors was associated with

a poor clinical outcome. The association of mitosis and

proliferation signatures with poorer prognosis in the

luminal breast cancer subgroups is a consistent theme

(Ribelles et al. 2013).

Unique kinases associated with ER status in breast

cancer cell lines have also been reported (Michalides et al.

2002, Finetti et al. 2008, Midland et al. 2012), some of

which show overlap with studies in tumors (Finetti et al.

2008, Midland et al. 2012), therefore potentially providing

models of ERC breast cancer kinomes in vitro. These data

support the existence of distinct kinomes, not only

associated with ER status per se, but also further define

heterogeneity within ERC breast tumors.

Kinase overexpression associated withsensitivity to endocrine therapies

There are many different kinases that when either over-

expressed or underexpressed in ERC breast cancer cells

lead to altered cell growth and survival responses to both

SERMs such as estrogen and tamoxifen, and AIs. Many of

these are listed in Table 2.

Such data underscore the importance of kinase net-

works, as many of the kinases listed in Table 2 converge on

common hubs associated with cell growth, survival and

cell death. It is not surprising that the interaction of

multiple kinases with ER signaling is observed in breast

cancer, as many of these kinases are key components of

proliferation, survival and cell death pathways and would

be expected to be regulated by estrogen, through the ER,

which is the primary mitogenic/survival signal in the

majority of breast cancers, at least initially (Musgrove &

Sutherland 2009, Osborne & Schiff 2011).

In ERC breast cancer, gene signatures that are based on

proliferation often strongly correlate with Ki67 expression

(Gao et al. 2014), a well-known marker of proliferation.

Furthermore, in ERC breast tumors in particular, it is

Ki67 and not pCR that is the early correlative endpoint

for predicting efficacy of both hormonal therapy or

chemotherapy in ERC breast cancer (Yerushalmi et al.

2010, Dowsett et al. 2011, Ellis et al. 2011,

von Minckwitz et al. 2012, Gao et al. 2014). Recently, using




Table

2Kinasesexp

erimentallymanipulatedandshownto

influence

tamoxifen(Tam)oraromatase

inhibitor(A

I)sensitivity

Kinase

sGain

offu

nction

Loss

offu

nction

Refe

rence

sEffects

Clinicalco

rrelation

EGFR

Ectopic

ove

rexp

ression

Agthove

netal.(1992)

Tam-resistantgrowth

Yes(G

iltnaneetal.2007

HER2

Ectopic

ove

rexp

ression

Benzetal.(1992)

Tam-resistantgrowth

Yes(O

sborneetal.2003)

AKT

Ectopic

ove

rexp

ression

Campbelletal.(2001),

deGraffenriedetal.(2004)

andSilvaetal.(2007)

Tam-resistantgrowth

Yes(Perez-Te

norioetal.2002)

Akt3

Ectopic

ove

rexp

ression

Faridietal.(2003)

E2-independent,

Tam-stimulated

growth

asxe

nograft

Yes(N

akatanietal.1999)

ERK1/2

MAPK

Yes(G

eeetal.2001)

PKCa

Ectopic

ove

rexp

ression

Chisamore

etal.(2001)and

Frankeletal.(2007)

Tam-resistantgrowth

Yes(Kalstadetal.2010)

PKCd

Ectopic

ove

rexp

ression

Nabhaetal.(2005)

Tam-resistantgrowth

PKA

Ove

rexp

ressionbyreducing

exp

ressionofnegative

regulatory

subunit

PKA-RIalpha

Michalidesetal.(2004)

Tam-resistantgrowth

Yes(M

ichalidesetal.2004)

MKP3

Ectopic

ove

rexp

ression

Cuietal.(2006)

Tam-resistantgrowth

Yes(Cuietal.2006)

CDK10

siRNA

Iornsetal.(2008)

Tam-resistantgrowth

inhibition

Yes(Iornsetal.2008)

CRK7

siRNA

Iornsetal.(2009)

Tam-resistantgrowth

inhibition

No

IKK3

Ectopic

ove

rexp

ression

Guoetal.(2010)

Protectionagainst

Tam-inducedcell

death

No

SphK1

Ectopic

ove

rexp

ression

Sukoch

eva

etal.(2009)

Tam-resistantgrowth

inhibition

Yes(Ruckhaberleetal.2008)

c-ABL

siRNA

Zhaoetal.(2010)

Sensitize

scellsto

Tam

inhibition

Yes(Zhaoetal.2010)

Ronreceptortyrosine

kinase

(MST

1R)

Ectopic

ove

rexp

ression

McC

laineetal.(2010)

Tamoxifenresistance

No

EphA2receptortyrosine

kinase

(EPHA2)

Ectopic

ove

rexp

ression

Luetal.(2003b)

Tamoxifenresistance

Yes(Brantley-Sieders

etal.2011)

cRET

siRNA

Plaza

-Menach

oetal.(2010)

Increasedsensitivity

toTa

mYes(M

orandietal.2013)

HSP

B8

Ectopic

ove

rexp

ression

Gonza

lez-Malervaetal.(2011)

Tam

resistance

Yes(G

onza

lez-Malervaetal.

2011)

LMTK3

siRNA

Giamasetal.(2011)

Increasedsensitivity

toTa

mYes(G

iamasetal.2011)

IGF-R1

Ectopic

ove

rexp

ression

Zhangetal.(2011)

Tam

andfulvestrant

resistance

Yes(Foxetal.2011,W

inder

etal.2014)

FGFR

3Ectopic

ove

rexp

ression

Tomlinsonetal.(2012)

Tam

andfulvestrant

resistance

Yes(Tomlinsonetal.2012)

TBK1

Ectopic

ove

rexp

ression

Weietal.(2014)

Tam

resistance

Yes(W

eietal.2014)



21 :5 R362





A/B

1

S46/47Y52

Y537S102 S118 S167 S236 T311S104 S212 S305

S559

S554

S282S154S106 Y219 S294

180 263 302 595

C D FE

Figure 1

Known phosphorylation sites on estrogen receptor a. Sites identified by

stars are those that have been determined in ERaC breast cancer cases in

the Manitoba Breast Tumor Bank (MBTB) and are the basis of the P7-ERa

score. The black stars are those residues which when phosphorylated are

associatedwith a good clinical outcome in patients treated with tamoxifen.

The open/white stars are those residues which when phosphorylated are



21 :5 R363

neoadjuvant antiestrogen therapy (particularly AIs), it

was observed that decreased detection of survival pathway

activation markers (e.g. phosphorylated AKT and phos-

phorylated mTOR) in tumors after treatment, compared

with pretreatment levels, was predictive of a better clinical

outcome (Generali et al. 2008, 2011), suggesting that

estrogen via the ER was regulating these pathways, directly

or indirectly in vivo. Many preclinical models have shown

that estrogen, via ER signaling, at least in part, regulates the

activities of PI3K/AKT/mTOR, p38MAPK, and MAPK/

ERK1/2 (Zhang et al. 2002, Lee et al. 2005, Yu & Henske

2006, Kazi et al. 2009, Maruani et al. 2012). This suggests that

the use of hormonal therapies in combination with the

most appropriately targeted kinase inhibitors may be more

beneficial at the onset of therapy than application in a

sequential fashion. Indeed, there are ongoing clinical trials

evaluating the use of combination therapies with endo-

crine therapies as first-line approaches (Ciruelos 2014).

A major challenge of using kinase inhibitors is that

kinase networks especially those associated with growth and

survival are often interconnected and inhibition of one may

have effects beyond the immediate targets. Adaptive

reprogramming of cancer kinomes due to single kinase

inhibitors has been demonstrated (Duncan et al. 2012) and

an argument for targeted combination therapy approaches

initially has been put forward (Stuhlmiller et al. 2014). The

development of resistance to hormonal therapies most

often occurs despite the continued expression of ERa

(Encarnacion et al. 1993, Bachleitner-Hofmann et al.

2002). In addition, many laboratory studies suggest that

adaptive responses of breast cancer cells, rather than

selection of existing ER-negative cells in the original

heterogeneous population, frequently occur (Santen et al.

2003, Browne et al. 2013). In such cases, increased

expression/activity of kinases also frequently occurs (Coutts

& Murphy 1998, Santen et al. 2003, 2004), supportive of the

concept that adaptive reprogramming of the breast cancer

kinome, in part, underlies some mechanisms of estrogen

independence and anti-estrogen resistance (Stuhlmiller

et al. 2014). The challenge will be to understand this and

its potential plasticity within the underlying heterogeneity

of ERC breast cancers and at the individual patient level.

associated with a poor clinical outcome in patients treated with tamoxifen

(Skliris et al. 2010a). It should be noted that others have determined that

S305 when phosphorylated is associated with a poor clinical outcome in

tamoxifen-treated patients (Le Romancer et al. 2011) and we have found

that tyrosine (Y) 537 when phosphorylated is also associated with a poor

clinical outcome in tamoxifen-treated patients (Skliris et al. 2010b). These

latter two results (textured stars) are consistent with our observation that

phosphorylation in N-terminal residues is associated with a good clinical

outcome, but phosphorylation in C-terminal residues is associated with a

poor clinical outcome.

Kinases known to phosphorylate ERa

In addition to ER signaling regulating kinase pathway

expression and/or activity, ER activity can also be

regulated by kinases.

ERa can be phosphorylated on many residues (Fig. 1)

and ERa has been identified as a substrate for several


kinases with known functions in breast cancer develop-

ment and progression. Dysregulation including mutation

and structural alterations of kinases themselves or the

signaling pathways in which they function is thought, in

part, to contribute to the dysregulation of ER signaling

that is associated with the development of breast cancer as

well as its progression to endocrine resistance in vivo.

Multiple aspects of ER activity can be affected by

phosphorylation; however, the focus of the majority of

studies addressing this issue has been on the transcrip-

tional activity (Le Romancer et al. 2011) and, not

surprisingly, it has been observed that differential phos-

phorylation of ERa (e.g. PKA activation) can alter the

chromatin-binding pattern (cistrome) of the receptor, in

turn altering the ERa transcriptome (de Leeuw et al. 2013).

The function of phosphorylation on ER activity and that

of other steroid hormone receptors have been recently

extensively reviewed (Le Romancer et al. 2011, Trevino &

Weigel 2013) and will not be reviewed herein.

Multiple different kinases can phosphorylate the same

ERa residue (e.g. serine amino acid residue 167 (S167) can

be phosphorylated by AKT, p70S6K, Aurora A, and

p90RSK) (Le Romancer et al. 2011). Similarly, one kinase

can phosphorylate several different residues on ERa

(e.g. MAPK can phosphorylate S104/106, S118, and S167)

(Le Romancer et al. 2011). Many kinases known to

phosphorylate ERa can also phosphorylate various






21 :5 R364

coactivators and coregulators (Wu et al. 2005), impacting

ER (ESR1) transcriptional activity. This provides a

potential mechanism allowing the integration of multiple

pathways to regulate the biology and/or patho-biology of

ERa in the mammary gland. Support for this idea, at least

in breast cancer, is provided by the existence of a

phosphorylation code for ERa (Skliris et al. 2010a) and at

least one of its coactivators, SRC3/AIB1 (Wu et al. 2004,

York et al. 2010). As ERa can undergo several other types of

PTMs, e.g. acetylation, methylation, ubiquitylation, etc.

(Le Romancer et al. 2011), one can envisage a broader ERa

PTM or epigenetic-like code.

The identification of kinases phosphorylating ERa has

been achieved using multiple methods, alone or in

combination: in vitro studies using recombinant proteins,

mass spectroscopy, the use of selective small-molecular-

weight kinase activators or inhibitors, and overexpression

or knockdown of expression using cells in culture or as

xenografts. Furthermore, it is reasonable to assume, while

not proving, that if ER can be directly phosphorylated by a

kinase, the two proteins would directly bind under

appropriate circumstances. Table 3 lists a number of kinases

that have been found to undergo protein:protein

interactions with ERa using different methodologies,

e.g. co-immunoprecipitation (Wierer et al. 2013) and

other pull-down-type assays (Kanaujiya et al. 2013), PLA

(Poulard et al. 2012), yeast-2-hybrid assay (Paul et al. 2014),

Table 3 Kinases shown to interact directly with estrogen receptor

Kinases Assays

ERK1/2 CoIP; ChIP/reChIPp70S6K1 Co-IPGSK3 Co-IPCDK2 GST pull-down, in vitro kinase assIKKa Co-IP ChIP-reChIPCDK7 In vitro Co-IPAKT In vitro kinase assayPI3K Proximity ligation assay Co-IPpp90RSK1 In vitro kinase assays Co-IPPKA FRET in vitro kinase assayILK GST-down experiments Co-IPEGFR Co-IP in vitro kinase assayIGFR Co-IPDNA-PK Co-IPcAbl Co-IP in vitro kinase assayCK2 In vitro kinase assayIKK3 Co-IP in vitro kinase assayPAK1 In vitro kinase assay GST-pull-dow

experiments Co-IPc-SRC Proximity ligation assay Co-IP in v

kinase assayp38 MAPK Immunocomplex kinase assay in v

kinase assay shows no phosphoPLK1 Co-IP ChIP/reChIP


and fluorescence resonance energy transfer (FRET) (Zwart

et al. 2007a,b). Direct interaction of ERa with kinases can

occur in the cytoplasm (Poulard et al. 2012), at the plasma

membrane (Poulard et al. 2012), as well as in the nucleus on

chromatin (Madak-Erdogan et al. 2011, 2014, Wierer et al.

2013), with some methodologies, such as PLA, allowing the

determination of the subcellular localization of any

interactions that occur (Poulard et al. 2012).

There is no doubt that phosphorylation and other

PTMs (Wu et al. 2007, Le Romancer et al. 2011) are

important for the regulation of ER highlighted by the

existence of a phosphorylation code for ERa in human

breast tumors (Skliris et al. 2010a). However, it is still

unclear as to which kinases and phosphatases are involved

in regulating ERa in vivo. Correlations of expression and/or

activity of some kinases with ER or its phosphorylated

forms in human breast tumors have been reported,

supporting a possible role in vivo (Murphy et al. 2004,

Gee et al. 2005, Jiang et al. 2007, Shrivastav et al. 2014).

Many of the kinases that can directly phosphorylate

ERa and its coregulators can also be regulated by estrogen

signaling under some circumstances. Many such kinases are

components of key signaling pathways involved in

development/differentiation, proliferation, cell cycle,

motility/invasion, and cell death. Whether or not they are

regulated by estrogen or in turn regulate ERa may depend on

levels of expression of both substrate and kinase, and on

a

References

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ay Rogatsky et al. (1999)Park et al. (2005)Chen et al. (2000)Campbell et al. (2001) and Sun et al. (2001)Sun et al. (2001) and Poulard et al. (2012)Joel et al. (1998)Chen et al. (1999), Zwart et al. (2007a,b)Acconcia et al. (2006)Marquez et al. (2001)Mendez et al. (2003)Medunjanin et al. (2010)He et al. (2010)Williams et al. (2009)Guo et al. (2010)

n Wang et al. (2002)

itro Arnold et al. (1995b, Poulard et al. (2012) andSun et al. (2012))

itrorylation

Lee & Bai (2002)

Wierer et al. (2013)






21 :5 R365

mutations or structural alterations that may eliminate or

introduce new regulatory factors. The ability of the

estrogen/ERa complex to activate kinases that in turn

can phosphorylate ER and modify function provides a

powerful feed-forward amplification system that may

drive many human breast cancers. Understanding how

this occurs and how it can be perturbed to drive

progression to therapy resistance would provide funda-

mental information to inform early combination

approaches, for preventing and/or treating resistant tumors.

Interestingly, although in primary breast tumors, few

ERa mutations have been found, one known mutation

K303R changes the amino acid lysine that can be

acetylated (another type of PTM) to an arginine residue

that cannot be acetylated. This in turn alters regulation of

S305 phosphorylation on ERa, a site of ERa phosphory-

lation with established relevance in vivo and in AI

resistance (Herynk & Fuqua 2004, Barone et al. 2010).

There are also other examples of the co-regulation of

phosphorylation with other PTMs to regulate ERa

expression and/or activity. For example, c-Src can phos-

phorylate ERa at Y537 that in turn facilitates its

interaction with E3 ubiquitin ligases such as E6AP,

resulting in ubiquitin-dependent degradation of ERa

(Zhou & Slingerland 2014). Most recently, however,

frequent somatic mutations in ERa have been identified

in metastases from ERC breast cancer. This is more

frequently associated with metastases that occur during

or following endocrine therapy (Zhang et al. 1997,

Robinson et al. 2013, Jeselsohn et al. 2014, Segal & Dowsett

2014). The most frequent mutation identified was at Y537

or the residue beside it, D538; therefore, affecting directly

or indirectly a well-known phosphorylation site on ERa

(Arnold et al. 1995a, Nettles et al. 2008, Skliris et al. 2010b)

with known clinical relevance (Skliris et al. 2010b). Such

data underscore the importance of ERa phosphorylation

and other PTMs to breast cancer progression.

Our understanding of estrogen-regulated ERa signaling

in human breast cells comes from model systems that are all

cancer cells. Moreover, all ERC and ERK cell lines were

originally obtained from breast cancer metastases usually

pleural and ascitic effusions (Soule et al. 1973, Lippman

et al. 1976). We have little if any understanding of ERa

signaling in normal human breast epithelial cells, mainly

due to the lack of appropriate ERC models. This remains a

large gap in our understanding, potentially limiting the

development of more efficacious prevention strategies. This

is especially important as increased ERa expression in

normal human breast is associated with an increased cancer

risk (Khan et al. 2005) and elevated ERa expression is one of


the earliest alterations occurring in ‘precursors’ of breast

cancer (Shoker et al. 1999, Lee et al. 2006). ERa is highly

expressed in most atypical ductal hyperplasia (ADH), ductal

carcinoma in situ (DCIS), and invasive breast cancers

(Shoker et al. 1999, Allred et al. 2001). However, the

mechanisms leading to increased ERa expression during

tumorigenesis are poorly understood. Interestingly, beside

alterations in ER gene transcription (Muscat et al. 2013),

altered kinase expression affecting ERa protein stability has

been suggested (Henrich et al. 2003, Antoon et al. 2013)

and, therefore, highlights the potential link between

phosphorylation and estrogen signaling.

The challenge now is to identify those kinases and

their associated networks that phosphorylate and regulate

ERa function in ERC tumors and potentially normal

breast in vivo, as such information will identify the most

efficacious targeted treatment approaches upfront and

may inform better prevention approaches.

Conclusions

ERa regulates and is regulated by kinases involved in several

functions associated with the hallmarks of cancer (Hanahan

& Weinberg 2011). The presence of a phosphorylation code

for ERa in breast cancers associated with clinical outcome

(Skliris et al. 2010a) and the knowledge that there are also

other PTMs associated with ERa (Le Romancer et al. 2011)

in vivo suggest that there is an epigenetic-like code for ERa,

which can regulate and is in part regulated by distinct

kinomes. The literature reviewed above strongly suggests that

distinct kinomes exist for ER-positive and -negative breast

cancers. Even within ERC cancers, different subgroups exist,

defined by different kinome signatures, which can be

correlated with clinical outcome. Strong evidence supports

the interplay of kinase networks, suggesting that targeting a

single node may not be sufficient to inhibit the network.

Therefore, identifying the important hubs/nodes associated

with each clinically relevant kinome in ERC tumors could

offer the ability to implement the best therapy options at

diagnosis, either endocrine therapy alone or together with

other targeted therapies for an improved overall outcome.

Declaration of interest

The authors declare that there is no conflict of interest that could be

perceived as prejudicing the impartiality of the review.

Funding

This work was made possible through grant support to L C Murphy from the

Canadian Institutes of Health Research (CIHR), the Canadian Breast Cancer

Foundation (CBCF), and the Canadian Cancer Society Research Institute






21 :5 R366

(CCSRI). M C Bruce is funded by a CBCF-Prairies/NWT region Postdoctoral

Fellowship. Morerover, some of published works referred to in this review

used breast tumor samples from theManitoba Breast Tumor Bank, a member

of the Canadian Tumor Repository Network, which are funded in part by the

CancerCare Manitoba Foundation (CCMF) and CIHR, respectively.

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Received in final form 9 July 2014Accepted 23 July 2014Made available online as an Accepted Preprint23 July 2014


http://dx.doi.org/10.1002/path.4022


http://dx.doi.org/10.1038/clpt.2014.8

http://dx.doi.org/10.1210/en.2009-0391

http://dx.doi.org/10.1210/me.2012-1140

http://dx.doi.org/10.1002/ijc.26304

http://dx.doi.org/10.1016/j.tem.2013.05.008



http://dx.doi.org/10.1093/emboj/cdf543



http://dx.doi.org/10.1016/j.celrep.2013.05.024

http://dx.doi.org/10.1186/1471-2091-10-36

http://dx.doi.org/10.1038/tpj.2013.8

http://dx.doi.org/10.1016/j.molcel.2004.08.019

http://dx.doi.org/10.1210/er.2004-0018


http://dx.doi.org/10.1016/j.febslet.2009.11.041


http://dx.doi.org/10.1016/S1470-2045(09)70262-1



http://dx.doi.org/10.1016/S0006-291X(02)00348-0




http://dx.doi.org/10.1038/sj.emboj.7601791

http://dx.doi.org/10.1158/1535-7163.MCT-06-0750



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