The Kinome Associated with Estrogen Receptor-Positive Status in Human Breast Cancer

M C Bruce et al. Kinome associated with 21:5 R357–R370 Review ER-positive status

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

Correspondence M Christine Bruce, Danielle McAllister and Leigh C Murphy should be addressed to L C Murphy Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, University of Manitoba Email and CancerCare Manitoba, 675 McDermot Avenue, Winnipeg, Manitoba, Canada R3E 0V9 Leigh.Murphy@ med.umanitoba.ca

Abstract

Estrogen receptor alpha (ERa) regulates and is regulated by kinases involved in several Key Words functions associated with the hallmarks of cancer. The following literature review strongly " estrogen receptors suggests that distinct kinomes exist for ERa-positive and -negative human breast cancers. " breast cancer Importantly, consistent with the known heterogeneity of ERa-positive cancers, different " phosphorylation subgroups exist, which can be deﬁned by different kinome signatures, which in turn are " kinases correlated with clinical outcome. Strong evidence supports the interplay of kinase networks, " endocrine therapy sensitivity suggesting that targeting a single node may not be sufﬁcient to inhibit the network. " biomarker 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, Endocrine-Related Cancer for improved overall outcome. Endocrine-Related Cancer (2014) 21, R357–R370

Introduction

The idea of personalized approaches to therapy in breast therapy was used for breast cancer at least in the form of cancer based on the molecular nature of the tumor can be ablation surgery such as ovariectomy dates back well over traced back to the late 1960s and early 1970s, when it was a century, the identification of ERs in breast cancers discovered that some but not all human breast tumors provided a molecular mechanism and rationale for the use express estrogen receptors (ERs; Jensen et al. 1971). This of hormonal therapies (Jensen & Jordan 2003). This led heralded the beginning of an era of targeted therapies for directly to the development of the successful modern breast cancer, identified the first biomarker used clinically endocrine therapies such as tamoxifen and other selective to predict the biological behavior of breast cancer, and estrogen receptor modulators (SERMs), which bind to the established the beginnings of understanding molecular ERs and induce conformational changes that modify and mechanisms by which the ovarian hormone, estrogen, in some cases inactivate the ERs (Jensen & Jordan 2003). drives the growth and survival of the majority of human The newer endocrine therapies, the aromatase inhibitors breast cancers (Jensen & Jordan 2003), at least initially. (AIs), which inhibit the aromatase enzyme, eliminate the Inhibiting the activity of ERs with the antiestrogen production of estrogen and therefore inhibit estrogen’s tamoxifen was the first targeted therapy in breast cancer. proliferative action (Goss et al. 2011). Although the knowledge that female hormones were It was evident from the start that not all ERC breast involved in breast cancer and the hormonal/endocrine tumors were created equal. Although a little more than

http://erc.endocrinology-journals.org q 2014 Society for Endocrinology Published by Bioscientiﬁca Ltd. DOI: 10.1530/ERC-14-0232 Printed in Great Britain Downloaded from Bioscientifica.com at 09/26/2021 01:37:02PM via free access Review M C Bruce et al. Kinome associated with 21:5 R358 ER-positive status

70% of all breast tumors express ER, only about half of (Yamnik et al. 2009, Yamnik & Holz 2010, Murphy et al. patients with ERC tumors respond to tamoxifen. There- 2011), as well as the demonstration that a clinically fore, ERC breast cancers exhibit heterogeneity associated relevant phosphorylation profile of ERs can be identified with prognosis and treatment outcomes. The first step to in human breast cancer (Skliris et al. 2010a), suggests that resolve this heterogeneity came from the idea that the kinases and/or phosphatases associated with ERC breast measurement of a downstream target of estrogen- cancer could provide a wealth of potential drug targets to dependent ER signaling such as the progesterone receptor complement existing endocrine therapies or generate new (PR) would increase confidence that the pathway was endocrine therapies. The following is a review of kinases intact (Horwitz et al. 1975). This increased the accuracy of that have been identified as associated with ER status in treatment prediction, but was obviously still imprecise as breast tumors or those that have been implicated in the some 20–30% of ERC/PRC tumors are de novo resistant to regulation of estrogen signaling and/or modifying sensi- the endocrine therapies. Furthermore, initial response to tivity to estrogen and its antagonists in breast cancer. endocrine therapies is often followed by acquired resistance despite the continued expression of ER (Encarnacion Kinases identified as mutated or structurally et al. 1993, Bachleitner-Hofmann et al. 2002). altered in breast cancer in large breast cancer The next significant insight into the heterogeneity of cohorts ERC breast cancer came with the identification of HER2 (ERBB2) amplification. Approximately 20% of all breast Genome-wide analyses of large cohorts of breast cancer cancers have amplified, overexpressed HER2, and 40–50% cases are providing detailed, comprehensive analyses of of these will also be ERC. Interestingly, ERC/HER2C genomic aberrations in breast cancer at a population level. tumors are more likely to be resistant to endocrine Some studies have also provided data concerning their therapy, in particular tamoxifen, thus providing impact on clinical characteristics. These studies have important insight into the relevance of crosstalk of shown that many of the frequently altered (amplified, signaling pathways initiated at the level of the plasma fused, deleted, or mutated) genes encode kinases. Some of membrane with many aspects of the ER signaling these frequently altered genes, such as HER2,were pathway. This may cause ligand-independent activation previously known but others, such as MAP3K1 and its of ER signaling and hormone therapy resistance. substrate MAP2K4, have not previously been identified to The molecular detailing that has become possible have functional roles in breast cancer. However, given that Endocrine-Related Cancer through the Human Genome Project and new high- enzymes, kinases in particular, have proven to be clinically throughput/high-content technologies in the last decade efficacious therapeutic targets, a wealth of data has now initially established five intrinsic molecular subgroups been generated not only to understand the complex (Sorlie et al. 2003) of which three were significantly biology of the disease but also to identify new treatments populated with ERC tumors. Most recently, at least ten for specific cohorts. molecular subgroups of human breast cancer have been The recently published METABRIC cohort of breast described (Curtis et al.2012) and eight of these appeared to cancers, in which 2000 individual breast cancers were be significantly populated with ERC tumors (Curtis et al. interrogated, identified ten integrative clusters, each with 2012). Associated with these studies has been the frequent distinct molecular characteristics associated with clinical identification of kinases, either mutated and/or structurally outcome (Curtis et al.2012). Often, altered genes encoding altered in large cohorts of breast tumors (Banerji et al.2012, kinases and phosphatases dominate individual clusters Curtis et al.2012, Shah et al.2012, Stephens et al.2012). (Table 1). Furthermore, the original intrinsic subtypes, in Some of these have also been found frequently altered and particular the ERC luminal A and luminal B (Perou et al. associated with AI sensitivity (Ellis et al.2012). 2000, Ignatiadis & Sotiriou 2013) subtypes, have been ER and its many coactivators are regulated by further subdivided due to the METABRIC study (Curtis et al. phosphorylation as well as other post-translational 2012, Dawson et al.2013). Therefore, a brief description of modifications (PTMs; Rowan et al. 2000, York et al. 2010, this follows as it relates to clusters that have significant Le Romancer et al. 2011, Zhang et al. 2013). The discovery ERC components. that the ER and its coactivators are substrates of several METABRIC integrative cluster 1 (IntClust1), represent- kinases (enzymes causing phosphorylation of specific ing 7% of breast cancer (Curtis et al.2012, Dawson et al. substrates), which are regulated by signaling pathways 2013), contains predominantly ERC tumors with luminal B frequently mutated or structurally altered in breast cancer features and is characterized by amplification of the

Table 1 Kinases and phosphatases altered in ERC-dominated its amplification in this region. A high frequency (w50%) breast cancer clusters from METABRIC (Curtis et al.2012, of PI3K catalytic subunit p110a (PIK3CA) mutations is also Dawson et al. 2013) observed in this group. ERC tumors with luminal A characteristics predo- METABRIC Kinases or phosphatases minate integrative cluster 3 (IntClust3), which represents integrative clusters identified Alteration 15% of all breast tumors (Dawson et al. 2013). Tumors in IntClust1 Ribosomal protein S6 Amplification/ this cluster tend to have few structural alterations, low kinase 1 (RPS6KB1) overexpression Protein phosphatase Amplification/ genomic instability, and usually an excellent prognosis. 1D (PPM1D) overexpression Interestingly, they also have the highest frequency of IntClust2 PAK1 Amplification/ PIK3CA mutations (w58%). As well, similar to other overexpression C PIK3CA Mutation clusters dominated by ER tumors, a high level of IntClust3 PIK3CA Mutation MAP3K1 mutations (w15%) is observed in IntClust3. MAP3K1 Mutation IntClust5 represents 10% of all breast tumors and is IntClust5 HER2 Amplification/ w overexpression characterized by HER2 amplification with 42% of them PIK3CA Mutation also expressing ERa (Dawson et al. 2013). Approximately IntClust6 FGFR1 Amplification 30% of tumors in this cohort also have PIK3CA mutations. IntClust7 PI3K Mutation C MAP3K1 Mutation ER tumors with overexpressed HER2 are more likely to IntClust8 MAP2K4 Mutation be resistant to endocrine therapies. One reason for this is PI3K Mutation thought to be the constitutive activation of several kinases IntClust9 Regulatory subunit B Deletion of protein downstream of HER2, such as ERK1/2, PI3K, and AKT, all phosphatase 2 of which can phosphorylate ERa and several ER coacti- alpha (PPP2R2A) 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). 17q23 region. Two genes in this amplicon associated with IntClust6, representing 4% of breast tumors, is another increased expression are ribosomal protein S6 kinase beta 1 predominantly ERC group with both luminal A and (RPS6KB1 and p70S6K1) and protein phosphatase 1D luminal B characteristics (Dawson et al. 2013). Tumors of this PPM1D ( ). Interestingly, expression of both of these genes group show amplification of the chromosome 8p12 locus and Endocrine-Related Cancer C has been shown to be regulated by estrogen in ER breast are genomically unstable with an intermediate prognosis. cancer cells at the transcriptional level (Han et al.2009, Less than 20% of tumors in this group have PIK3CA Yamnik et al.2009, Yamnik & Holz 2010). In addition, mutations, which is low compared to other ERC groups. the protein product of RPS6KB1 (p70S6K1) has The gene for FGFR1 is located in the chromosome 8p12 been shown to phosphorylate ERa (Yamnik & Holz 2010) region. FGFR1 amplification and overexpression have been and under IGF1 stimulation p70S6K1 can be directly linked to endocrine therapy resistance (Turner et al. C co-immunoprecipitated with ERa in ER breast cancer 2010, Balko et al. 2012). FGFR1 also regulates several kinases cells (Becker et al.2011). PPM1D/Wip1 is known for its p53 such as PI3K and AKT, which are known to phosphorylate C inhibitory effects and, in ER MCF7 cells, has been shown ERa and its coactivators (Le Romancer et al. 2011). to be regulated by estrogen and to increase the transcrip- IntClust7 (10% of breast tumors) and IntClust8 (15% tional activity of ERa (ESR1) as well as other steroid hormone of breast tumors) are mainly ERC groups and while similar receptors (Proia et al. 2006), possibly by modulating in many ways tumors in Intclust8 display higher genomic phosphorylation of coactivators such as SRC1 and/or instability, with an associated inferior prognosis than p300/CBP (Proia et al.2006). tumors in IntClust7 (Dawson et al. 2013). Interestingly, Integrative cluster 2 (IntClust2), representing 4% of both again have a high frequency of PI3K (PIK3CA) breast tumors, is also dominated by ERC tumors with mutations. IntClust7 has a high frequency of MAP3K1 characteristics of both luminal A and luminal B subtypes mutations and IntClust8 has the highest frequency of (Dawson et al. 2013). Amplification of chromosome region MAP2K4 mutations, a downstream target of MAP3K1. 11q13/14 characterizes this cluster and high expression of The structural alterations usually found in MAP3K1 another kinase, PAK1, known to affect ERa phosphory- and MAP2K4 are deletions and mutations associated lation and activity (Wang et al. 2002, Mazumdar & Kumar with loss of function (Teng et al. 1997, Pham et al. 2013), 2003, Holm et al. 2009, Kok et al. 2010) is associated with suggesting that the pathways they regulate have

tumor-suppressor function. Steroid hormone receptor also have a high frequency of PIK3CA mutations while by signaling, including estrogen and androgen receptors, contrast IntClust6 has less than a 20% frequency of PI3K shows crosstalk with MAP3K1 in some systems, and it is pathway mutations (Dawson et al.2013). the ERC tumors, in particular luminal A type, that have Experimental and clinical data suggest that hyper- the highest level of mutation and/or deletion of the activation of the PI3K/AKT/mTOR pathway is associated MAP3K1/MAP2K4 pathway (Pham et al. 2013). This with endocrine resistance. However, gain-of-function pathway has been implicated in cell death pathways, PIK3CA mutations in ERC tumors are often associated which under normal conditions is important for mam- with good prognosis (Miller et al. 2011a). Such apparently mary gland involution, hence its loss of function in many contradictory results may be due to complexities associ- ERC breast cancers may in part be responsible for the ated with feedforward and feedback mechanisms involved dissociation of estrogen-induced proliferative and survival in the PI3K/AKT/mTOR pathway and other alterations pathways from growth-inhibiting differentiation co-occurring in regulators of the pathway. This latter idea pathways (Pham et al. 2013). This pathway has been is supported by the finding that most often the associ- described as having a molecular switch type function ations of PIK3CA mutations with good outcome in ERC between survival and cell death, possibly dependent on tumors are restricted to those tumors that are also HER2 cell-type background, subcellular localization, and caspase negative (Fu et al. 2013). 3 activity (Pham et al. 2013). Increased ERa expression As discussed above, the PIK3CA gene, encoding the combined with a decreased activity of the MAP3K1/ PI3K catalytic subunit p110a, is one of the most frequently MAP2K4 pathway may, in part, underlie the change in mutated genes in ERC breast cancers (Dawson et al. 2013). the balance of estrogen signaling regulation of survival/ Many of the mutations occur in ‘hot spots’ with the most proliferation vs cell cycle inhibition that is often frequent mutations being E542K, E545K (both in the associated with differentiation. helical domain), and H1047R (in the kinase domain) IntClust9 (7% of breast tumors) is characterized by (Barbareschi et al. 2007). Generally, the mutations result in a high frequency of PPP2R2A deletions (Dawson et al. constitutive activation of the kinase (Di Cosimo & Baselga 2013). PPP2R2A is the regulatory subunit B of protein 2009). As mentioned above, the published data largely phosphatase 2 alpha (PP2A). While IntClust9 contains a suggest that mutations in PIK3CA occur more frequently mixture of intrinsic subtypes, its ERC members are mainly in ERC breast cancer with good prognosis and may also be of the luminal B type, and loss of PPP2R2A expression is associated with better clinical outcome in patients treated Endocrine-Related Cancer associated with a high mitotic index. Interestingly, PP2A with endocrine therapy (Di Cosimo & Baselga 2009, Miller also has been implicated in the regulation of ERa et al. 2011a). However, this is not a universal finding phosphorylation and activity (Lu et al. 2003a). (Cuorvo et al. 2014). Recent reviews have discussed this The above data underscore that distinctly altered aspect in detail (Fu et al. 2013). kinase and phosphatase mutation patterns occur frequently AKT1 mutations, often associated with PI3K-inde- in ERC breast cancer subgroups with altered clinical pendent constitutive activity, occur in w4% of breast outcome. This would support the idea that combining cancers and are often associated with ERC tumors. endocrine therapies with other targeted therapies informed However, the relationship of AKT expression and/or by each subgroup’s distinct kinase pattern could provide activation to outcome in breast cancer is inconsistent better clinical outcome for breast cancer patients. (Badve et al. 2010, Aleskandarany et al. 2011). Consi- dering that the different AKT isoforms have distinct Kinases associated with the PI3K/AKT/mTOR functions (Dillon & Muller 2010) and that a recent study and ER-positive breast cancer using proximity ligation assay (PLA) to distinguish between pAKT1 and pAKT2 (Spears et al. 2012) METABRIC and similar studies (Curtis et al.2012, Ellis et al. determined the former to be associated with poor 2012, Stephens et al.2012) as well as previous smaller scale prognosis and the latter with better prognosis, it seems studies (Miller et al.2010, 2011a,b) have identified the possible that the relative levels of each isoform may frequent structural and mutational alterations in the determine the final read out. Furthermore, the inability PI3K/AKT/mTOR pathway that occur in ERC breast cancer. to distinguish the different pAKT isoforms may, at least in IntClust1, IntClust2, IntClust3, and IntClust5 (Dawson part, underlie the previous lack of consistency (Badve et al.2013)havew25, 50, 58, and 30% frequencies of et al. 2010, Aleskandarany et al. 2011)aroundtheir PIK3CA mutations respectively. IntClust7 and IntClust8 association with breast cancer outcome.

Owing to the frequency with which components of the SRPK1, TTK, and VRK1. Many of these kinases have been PI3K/AKT/mTOR pathway are altered in ERC tumors, and previously reported to be involved in the G2 and M phases the fact that the pathway is a central hub receiving growth of the cell cycle (Malumbres & Barbacid 2009, Knight et al. and survival signals from many factors, the detailed 2010). Notably, these kinases include the 12 kinases molecular nature of the pathway in different ERC tumors making up the mitosis metagene cluster identified by may be important to guide appropriate therapy options. 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 Kinases associated with ER status by a poor clinical outcome. The association of mitosis and expression analyses proliferation signatures with poorer prognosis in the Several studies have now been published in which different luminal breast cancer subgroups is a consistent theme kinase expression patterns were found in ERC vs other (Ribelles et al. 2013). clinical subtypes of breast cancer. Using publically available Unique kinases associated with ER status in breast RNA expression databases, Bianchini et al.(2010)identified cancer cell lines have also been reported (Michalides et al. 16 kinases that were overexpressed in ERC/HER2K breast 2002, Finetti et al. 2008, Midland et al. 2012), some of cancer biopsy samples. These were IGF1R, STK32B, ERBB4, which show overlap with studies in tumors (Finetti et al. FGFR3, BMPR1B, MAST4, HSPB8, IKBKB, DCLK1, ERBB3, 2008, Midland et al. 2012), therefore potentially providing STK39, MAP3K1, PTK6, PLK2, TEX14,andMST1. Kinases models of ERC breast cancer kinomes in vitro. These data uniquely overexpressed in ERK and HER2C breast cancer support the existence of distinct kinomes, not only subtypes were also identified. Two robust kinase clusters associated with ER status per se, but also further define were recognized: a mitosis metagene cluster (12 distinct heterogeneity within ERC breast tumors. kinases) and an immune kinase cluster (15 distinct kinases), which were present in all clinical subgroups. Overexpression Kinase overexpression associated with of kinases composing the mitosis metagene cluster was sensitivity to endocrine therapies mostly found in ERK tumors and was not prognostic in this subgroup. However, ERC/HER2K tumors that presented a There are many different kinases that when either over- high mitosis kinase score were associated with a worse expressed or underexpressed in ERC breast cancer cells prognosis but showed a higher frequency of pathological lead to altered cell growth and survival responses to both Endocrine-Related Cancer complete response (pCR) to neoadjuvant chemotherapy. On SERMs such as estrogen and tamoxifen, and AIs. Many of the other hand, overexpression of kinases from the immune these are listed in Table 2. kinase cluster was associated with better survival in Such data underscore the importance of kinase net- ERC/HER2K and HER2C subgroups. Interestingly, the works, as many of the kinases listed in Table 2 converge on authors also observed that in ERC tumors, many of the common hubs associated with cell growth, survival and overexpressed kinases were transmembrane growth factor cell death. It is not surprising that the interaction of receptors, while ERK tumors mostly overexpressed intra- multiple kinases with ER signaling is observed in breast cellular kinases associated with cell proliferation (Bianchini cancer, as many of these kinases are key components of et al.2010). proliferation, survival and cell death pathways and would Finetti et al. (2008) undertook a genome-wide be expected to be regulated by estrogen, through the ER, expression analysis of a cohort of primary human breast which is the primary mitogenic/survival signal in the tumors focusing mainly on the basal and luminal A majority of breast cancers, at least initially (Musgrove & intrinsic breast cancer subtypes. From this analysis, they Sutherland 2009, Osborne & Schiff 2011). extracted kinase genes whose differential expression was In ERC breast cancer, gene signatures that are based on associated with a clinical outcome. Not unexpectedly, proliferation often strongly correlate with Ki67 expression there were substantial differences in the pattern of kinase (Gao et al.2014), a well-known marker of proliferation. gene expression between the basal and the luminal Furthermore, in ERC breast tumors in particular, it is A intrinsic subtypes. Of more interest, however, was the Ki67 and not pCR that is the early correlative endpoint discovery that 16 kinases were overexpressed in most of for predicting efficacy of both hormonal therapy or the basal group and in a few luminal A tumors. These chemotherapy in ERC breast cancer (Yerushalmi et al. kinases were AURKA, AURKB, BUB1, BUB1B, CDC2 (CDK1), 2010, Dowsett et al. 2011, Ellis et al.2011, CDC7, CHEK1, MASTL, MELK, NEK2, PBK, PLK1, PLK4, von Minckwitz et al. 2012, Gao et al. 2014). Recently, using

http://erc.endocrinology-journals.org q 2014 Society for Endocrinology Published by Bioscientiﬁca Ltd. DOI: 10.1530/ERC-14-0232 Printed in Great Britain Downloaded from Bioscientifica.com at 09/26/2021 01:37:02PM via free access Endocrine-Related Cancer O:1.50EC1-22Pitdi ra Britain Great in Printed 10.1530/ERC-14-0232 DOI: http://erc.endocrinology-journals.org

Table 2 Kinases experimentally manipulated and shown to inﬂuence tamoxifen (Tam) or aromatase inhibitor (AI) sensitivity Review

Kinases Gain of function Loss of function References Effects Clinical correlation

EGFR Ectopic overexpression Agthoven et al. (1992) Tam-resistant growth Yes (Giltnane et al. 2007 HER2 Ectopic overexpression Benz et al. (1992) Tam-resistant growth Yes (Osborne et al. 2003) AKT Ectopic overexpression Campbell et al. (2001), Tam-resistant growth Yes (Perez-Tenorio et al. 2002) deGraffenried et al. (2004) and Silva et al. (2007) Akt3 Ectopic overexpression Faridi et al. (2003) E2-independent, Yes (Nakatani et al. 1999) Tam-stimulated growth as xenograft ERK1/2 MAPK Yes (Gee et al. 2001) PKCa Ectopic overexpression Chisamore et al. (2001) and Tam-resistant growth Yes (Kalstad et al. 2010)

Frankel et al. (2007) with associated Kinome al. et Bruce C M q PKCd Ectopic overexpression Nabha et al. (2005) Tam-resistant growth 04SceyfrEndocrinology for Society 2014 PKA Overexpression by reducing Michalides et al. (2004) Tam-resistant growth Yes (Michalides et al. 2004) expression of negative regulatory subunit PKA-RIalpha MKP3 Ectopic overexpression Cui et al. (2006) Tam-resistant growth Yes (Cui et al. 2006) CDK10 siRNA Iorns et al. (2008) Tam-resistant growth Yes (Iorns et al. 2008) inhibition CRK7 siRNA Iorns et al. (2009) Tam-resistant growth No inhibition IKK3 Ectopic overexpression Guo et al. (2010) Protection against No

Tam-induced cell status ER-positive ulse yBocetﬁaLtd. Bioscientiﬁca by Published death SphK1 Ectopic overexpression Sukocheva et al. (2009) Tam-resistant growth Yes (Ruckhaberle et al. 2008) inhibition c-ABL siRNA Zhao et al. (2010) Sensitizes cells to Yes (Zhao et al. 2010) Tam inhibition Ron receptor tyrosine Ectopic overexpression McClaine et al. (2010) Tamoxifen resistance No kinase (MST1R) EphA2 receptor tyrosine Ectopic overexpression Lu et al. (2003b) Tamoxifen resistance Yes (Brantley-Sieders et al. 2011) kinase (EPHA2) cRET siRNA Plaza-Menacho et al. (2010) Increased sensitivity Yes (Morandi et al. 2013) Downloaded fromBioscientifica.com at09/26/202101:37:02PM to Tam HSPB8 Ectopic overexpression Gonzalez-Malerva et al. (2011) Tam resistance Yes (Gonzalez-Malerva et al. 2011) LMTK3 siRNA Giamas et al. (2011) Increased sensitivity Yes (Giamas et al. 2011) to Tam IGF-R1 Ectopic overexpression Zhang et al. (2011) Tam and fulvestrant Yes (Fox et al. 2011, Winder resistance et al. 2014) 21 FGFR3 Ectopic overexpression Tomlinson et al. (2012) Tam and fulvestrant Yes (Tomlinson et al. 2012) :5 resistance TBK1 Ectopic overexpression Wei et al. (2014) Tam resistance Yes (Wei et al. 2014) R362 via freeaccess Review M C Bruce et al. Kinome associated with 21:5 R363 ER-positive status

neoadjuvant antiestrogen therapy (particularly AIs), it kinases with known functions in breast cancer develop- was observed that decreased detection of survival pathway ment and progression. Dysregulation including mutation activation markers (e.g. phosphorylated AKT and phos- and structural alterations of kinases themselves or the phorylated mTOR) in tumors after treatment, compared signaling pathways in which they function is thought, in with pretreatment levels, was predictive of a better clinical part, to contribute to the dysregulation of ER signaling outcome (Generali et al. 2008, 2011), suggesting that that is associated with the development of breast cancer as estrogen via the ER was regulating these pathways, directly well as its progression to endocrine resistance in vivo. or indirectly in vivo. Many preclinical models have shown Multiple aspects of ER activity can be affected by that estrogen, via ER signaling, at least in part, regulates the phosphorylation; however, the focus of the majority of activities of PI3K/AKT/mTOR, p38MAPK, and MAPK/ studies addressing this issue has been on the transcrip- ERK1/2 (Zhang et al. 2002, Lee et al. 2005, Yu & Henske tional activity (Le Romancer et al.2011)and,not 2006, Kazi et al.2009, Maruani et al.2012). This suggests that surprisingly, it has been observed that differential phos- the use of hormonal therapies in combination with the phorylation of ERa (e.g. PKA activation) can alter the most appropriately targeted kinase inhibitors may be more chromatin-binding pattern (cistrome) of the receptor, in beneficial at the onset of therapy than application in a turn altering the ERa transcriptome (de Leeuw et al. 2013). sequential fashion. Indeed, there are ongoing clinical trials The function of phosphorylation on ER activity and that evaluating the use of combination therapies with endo- of other steroid hormone receptors have been recently crine therapies as first-line approaches (Ciruelos 2014). extensively reviewed (Le Romancer et al. 2011, Trevino & A major challenge of using kinase inhibitors is that Weigel 2013) and will not be reviewed herein. kinase networks especially those associated with growth and Multiple different kinases can phosphorylate the same survival are often interconnected and inhibition of one may ERa residue (e.g. serine amino acid residue 167 (S167) can have effects beyond the immediate targets. Adaptive be phosphorylated by AKT, p70S6K, Aurora A, and reprogramming of cancer kinomes due to single kinase p90RSK) (Le Romancer et al. 2011). Similarly, one kinase inhibitors has been demonstrated (Duncan et al.2012)and can phosphorylate several different residues on ERa an argument for targeted combination therapy approaches (e.g. MAPK can phosphorylate S104/106, S118, and S167) initially has been put forward (Stuhlmiller et al.2014). The (Le Romancer et al.2011). Many kinases known to development of resistance to hormonal therapies most phosphorylate ERa can also phosphorylate various often occurs despite the continued expression of ERa Endocrine-Related Cancer (Encarnacion et al. 1993, Bachleitner-Hofmann et al. S554 2002). In addition, many laboratory studies suggest that S106 Y219 S294 S104 S154 S212 S282 S305 adaptive responses of breast cancer cells, rather than S102 S118 S167 S236 T311 Y537 S559 Y52 selection of existing ER-negative cells in the original S46/47 heterogeneous population, frequently occur (Santen et al. 1 180 263 302 595 2003, Browne et al.2013). In such cases, increased expression/activity of kinases also frequently occurs (Coutts A/B CDE F &Murphy1998, Santen et al.2003, 2004), supportive of the concept that adaptive reprogramming of the breast cancer kinome, in part, underlies some mechanisms of estrogen Figure 1 independence and anti-estrogen resistance (Stuhlmiller Known phosphorylation sites on estrogen receptor a. Sites identified by stars are those that have been determined in ERaC breast cancer cases in et al.2014). The challenge will be to understand this and the Manitoba Breast Tumor Bank (MBTB) and are the basis of the P7-ERa its potential plasticity within the underlying heterogeneity score. The black stars are those residues which when phosphorylated are of ERC breast cancers and at the individual patient level. associated with a good clinical outcome in patients treated with tamoxifen. The open/white stars are those residues which when phosphorylated are associated with a poor clinical outcome in patients treated with tamoxifen (Skliris et al. 2010a). It should be noted that others have determined that Kinases known to phosphorylate ERa S305 when phosphorylated is associated with a poor clinical outcome in tamoxifen-treated patients (Le Romancer et al. 2011) and we have found In addition to ER signaling regulating kinase pathway that tyrosine (Y) 537 when phosphorylated is also associated with a poor expression and/or activity, ER activity can also be clinical outcome in tamoxifen-treated patients (Skliris et al. 2010b). These regulated by kinases. latter two results (textured stars) are consistent with our observation that phosphorylation in N-terminal residues is associated with a good clinical ERa can be phosphorylated on many residues (Fig. 1) outcome, but phosphorylation in C-terminal residues is associated with a and ERa has been identified as a substrate for several poor clinical outcome.

coactivators and coregulators (Wu et al. 2005), impacting and ﬂuorescence resonance energy transfer (FRET) (Zwart ER (ESR1) transcriptional activity. This provides a et al.2007a,b). Direct interaction of ERa with kinases can potential mechanism allowing the integration of multiple occur in the cytoplasm (Poulard et al.2012), at the plasma pathways to regulate the biology and/or patho-biology of membrane (Poulard et al.2012), as well as in the nucleus on ERa in the mammary gland. Support for this idea, at least chromatin (Madak-Erdogan et al.2011, 2014, Wierer et al. in breast cancer, is provided by the existence of a 2013), with some methodologies, such as PLA, allowing the phosphorylation code for ERa (Skliris et al. 2010a) and at determination of the subcellular localization of any least one of its coactivators, SRC3/AIB1 (Wu et al. 2004, interactions that occur (Poulard et al.2012). York et al. 2010). As ERa can undergo several other types of There is no doubt that phosphorylation and other PTMs, e.g. acetylation, methylation, ubiquitylation, etc. PTMs (Wu et al. 2007, Le Romancer et al. 2011) are (Le Romancer et al. 2011), one can envisage a broader ERa important for the regulation of ER highlighted by the PTM or epigenetic-like code. existence of a phosphorylation code for ERa in human The identiﬁcation of kinases phosphorylating ERa has breast tumors (Skliris et al. 2010a). However, it is still been achieved using multiple methods, alone or in unclear as to which kinases and phosphatases are involved combination: in vitro studies using recombinant proteins, in regulating ERa in vivo. Correlations of expression and/or mass spectroscopy, the use of selective small-molecular- activity of some kinases with ER or its phosphorylated weight kinase activators or inhibitors, and overexpression forms in human breast tumors have been reported, or knockdown of expression using cells in culture or as supporting a possible role in vivo (Murphy et al. 2004, xenografts. Furthermore, it is reasonable to assume, while Gee et al. 2005, Jiang et al. 2007, Shrivastav et al. 2014). not proving, that if ER can be directly phosphorylated by a Many of the kinases that can directly phosphorylate kinase, the two proteins would directly bind under ERa and its coregulators can also be regulated by estrogen appropriate circumstances. Table 3 lists a number of kinases signaling under some circumstances. Many such kinases are that have been found to undergo protein:protein components of key signaling pathways involved in interactions with ERa using different methodologies, development/differentiation, proliferation, cell cycle, e.g. co-immunoprecipitation (Wierer et al. 2013) and motility/invasion, and cell death. Whether or not they are other pull-down-type assays (Kanaujiya et al.2013), PLA regulated by estrogen or in turn regulate ERa may depend on (Poulard et al.2012), yeast-2-hybrid assay (Paul et al.2014), levels of expression of both substrate and kinase, and on Endocrine-Related Cancer Table 3 Kinases shown to interact directly with estrogen receptor a

Kinases Assays References

ERK1/2 CoIP; ChIP/reChIP Madak-Erdogan et al. (2011) p70S6K1 Co-IP Becker et al. (2011) GSK3 Co-IP Medunjanin et al. (2005) CDK2 GST pull-down, in vitro kinase assay Rogatsky et al. (1999) IKKa Co-IP ChIP-reChIP Park et al. (2005) CDK7 In vitro Co-IP Chen et al. (2000) AKT In vitro kinase assay Campbell et al. (2001) and Sun et al. (2001) PI3K Proximity ligation assay Co-IP Sun et al. (2001) and Poulard et al. (2012) pp90RSK1 In vitro kinase assays Co-IP Joel et al. (1998) PKA FRET in vitro kinase assay Chen et al. (1999), Zwart et al. (2007a,b) ILK GST-down experiments Co-IP Acconcia et al. (2006) EGFR Co-IP in vitro kinase assay Marquez et al. (2001) IGFR Co-IP Mendez et al. (2003) DNA-PK Co-IP Medunjanin et al. (2010) cAbl Co-IP in vitro kinase assay He et al. (2010) CK2 In vitro kinase assay Williams et al. (2009) IKK3 Co-IP in vitro kinase assay Guo et al. (2010) PAK1 In vitro kinase assay GST-pull-down Wang et al. (2002) experiments Co-IP c-SRC Proximity ligation assay Co-IP in vitro Arnold et al. (1995b, Poulard et al. (2012) and kinase assay Sun et al. (2012)) p38 MAPK Immunocomplex kinase assay in vitro Lee & Bai (2002) kinase assay shows no phosphorylation PLK1 Co-IP ChIP/reChIP Wierer et al. (2013)

mutations or structural alterations that may eliminate or the earliest alterations occurring in ‘precursors’ of breast introduce new regulatory factors. The ability of the cancer (Shoker et al.1999, Lee et al.2006). ERa is highly estrogen/ERa complex to activate kinases that in turn expressed in most atypical ductal hyperplasia (ADH), ductal can phosphorylate ER and modify function provides a carcinoma in situ (DCIS), and invasive breast cancers powerful feed-forward amplification system that may (Shoker et al.1999, Allred et al.2001). However, the drive many human breast cancers. Understanding how mechanisms leading to increased ERa expression during this occurs and how it can be perturbed to drive tumorigenesis are poorly understood. Interestingly, beside progression to therapy resistance would provide funda- alterations in ER gene transcription (Muscat et al.2013), mental information to inform early combination altered kinase expression affecting ERa protein stability has approaches, for preventing and/or treating resistant tumors. been suggested (Henrich et al.2003, Antoon et al.2013) Interestingly, although in primary breast tumors, few and, therefore, highlights the potential link between ERa mutations have been found, one known mutation phosphorylation and estrogen signaling. K303R changes the amino acid lysine that can be The challenge now is to identify those kinases and acetylated (another type of PTM) to an arginine residue their associated networks that phosphorylate and regulate that cannot be acetylated. This in turn alters regulation of ERa function in ERC tumors and potentially normal S305 phosphorylation on ERa, a site of ERa phosphory- breast in vivo, as such information will identify the most lation with established relevance in vivo and in AI efficacious targeted treatment approaches upfront and resistance (Herynk & Fuqua 2004, Barone et al. 2010). may inform better prevention approaches. There are also other examples of the co-regulation of phosphorylation with other PTMs to regulate ERa Conclusions expression and/or activity. For example, c-Src can phos- ERa regulates and is regulated by kinases involved in several a phorylate ER at Y537 that in turn facilitates its functions associated with the hallmarks of cancer (Hanahan interaction with E3 ubiquitin ligases such as E6AP, & Weinberg 2011).Thepresenceofaphosphorylationcode a resulting in ubiquitin-dependent degradation of ER for ERa in breast cancers associated with clinical outcome (Zhou & Slingerland 2014). Most recently, however, (Skliris et al.2010a) and the knowledge that there are also a frequent somatic mutations in ER have been identified other PTMs associated with ERa (Le Romancer et al.2011) C in metastases from ER breast cancer. This is more in vivo suggest that there is an epigenetic-like code for ERa, frequently associated with metastases that occur during Endocrine-Related Cancer which can regulate and is in part regulated by distinct or following endocrine therapy (Zhang et al. 1997, kinomes. The literature reviewed above strongly suggests that Robinson et al. 2013, Jeselsohn et al. 2014, Segal & Dowsett distinct kinomes exist for ER-positive and -negative breast 2014). The most frequent mutation identified was at Y537 cancers. Even within ERC cancers, different subgroups exist, or the residue beside it, D538; therefore, affecting directly defined by different kinome signatures, which can be a or indirectly a well-known phosphorylation site on ER correlated with clinical outcome. Strong evidence supports (Arnold et al. 1995a, Nettles et al. 2008, Skliris et al. 2010b) the interplay of kinase networks, suggesting that targeting a with known clinical relevance (Skliris et al. 2010b). Such single node may not be sufficient to inhibit the network. a data underscore the importance of ER phosphorylation Therefore, identifying the important hubs/nodes associated and other PTMs to breast cancer progression. with each clinically relevant kinome in ERC tumors could a Our understanding of estrogen-regulated ER signaling offer the ability to implement the best therapy options at in human breast cells comes from model systems that are all diagnosis, either endocrine therapy alone or together with C K cancer cells. Moreover, all ER and ER cell lines were other targeted therapies for an improved overall outcome. originally obtained from breast cancer metastases usually pleural and ascitic effusions (Soule et al.1973, Lippman et al a .1976). We have little if any understanding of ER Declaration of interest signaling in normal human breast epithelial cells, mainly The authors declare that there is no conflict of interest that could be due to the lack of appropriate ERC models. This remains a perceived as prejudicing the impartiality of the review. large gap in our understanding, potentially limiting the development of more efficacious prevention strategies. This is especially important as increased ERa expression in Funding This work was made possible through grant support to L C Murphy from the normal human breast is associated with an increased cancer Canadian Institutes of Health Research (CIHR), the Canadian Breast Cancer risk (Khan et al.2005) and elevated ERa expression is one of Foundation (CBCF), and the Canadian Cancer Society Research Institute

(CCSRI). M C Bruce is funded by a CBCF-Prairies/NWT region Postdoctoral Benz CC, Scott GK, Sarup JC, Johnson RM, Tripathy D, Coronado E, Fellowship. Morerover, some of published works referred to in this review Shepard HM & Osborne CK 1992 Estrogen-dependent, tamoxifen- used breast tumor samples from the Manitoba Breast Tumor Bank, a member resistant tumorigenic growth of MCF-7 cells transfected with HER2/neu. of the Canadian Tumor Repository Network, which are funded in part by the Breast Cancer Research and Treatment 24 85–95. (doi:10.1007/BF01961241) CancerCare Manitoba Foundation (CCMF) and CIHR, respectively. Bianchini G, Iwamoto T, Qi Y, Coutant C, Shiang CY, Wang B, Santarpia L, Valero V, Hortobagyi GN, Symmans WF et al. 2010 Prognostic and therapeutic implications of distinct kinase expression patterns in different subtypes of breast cancer. Cancer Research 70 8852–8862. 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Received in ﬁnal form 9 July 2014 Accepted 23 July 2014 Made available online as an Accepted Preprint 23 July 2014