And P21-Activated Kinase 1 (Pak1) in Hormone Receptor-Positive Breast Cancer

TARGETING AURORA A KINASE (AURKA) AND P21-ACTIVATED KINASE 1 (PAK1) IN HORMONE RECEPTOR-POSITIVE BREAST CANCER

By Yayi Feng

November 2016

A Dissertation Presented to the Faculty of Drexel University College of Medicine in partial fulfillment of the Requirements for the Degree of Master of Science in Cancer Biology

______Erica Golemis, PhD (Mentor) Mauricio Reginato, PhD (Chair) Adjunct Professor Professor Biochemistry & Molecular Biology Biochemistry & Molecular Biology Drexel University College of Medicine Drexel University College of Medicine

______Jonathan Chernoff, MD, PhD Adjunct Professor Biochemistry & Molecular Biology Drexel University College of Medicine

TABLE OF CONTENTS

LIST OF TABLES ...... iii

LIST OF FIGURES ...... iv

ABSTRACT ...... v

INTRODUCTION ...... 1

MATERIALS AND METHODS ...... 15

Cell lines and cell culture...... 15

Reagents ...... 15

Cell Viability Assay ...... 18

Breast cancer mouse model and tissue collection ...... 18

Preparation of Tumor Lysates ...... 20

Western Blotting ...... 20

Immunoprecipitation and in vitro Kinase Assay ...... 21

Statistical Analysis ...... 23

RESULTS ...... 23

Inhibition of AURKA and PAK1 synergistically hampers cell survival of breast cancer cells in vitro and limits growth of mammary tumor xenografts in vivo ...... 23

Combination of alisertib and FRAX1036 suppresses proliferation and promotes apoptosis in vivo ...... 27

Combined treatment with alisertib and FRAX1036 reduces estrogen receptor α (ERα) phosphorylation and signaling activity in HR-positive breast cancer ...... 31

DISCUSSION ...... 36

LIST OF REFERENCES ...... 44 iii

LISTS OF TABLES

Table 1. List of Antibody Information ...... 17

Table 2. Molecular Phenotypes of BT474 and T47D ...... 23

LISTS OF FIGURES

Figure 1. Structure of estrogen receptor α (ERα) ...... 2

Figure 2. ERα, AURKA, and PAK1 signaling network ...... 14

Figure 3. Dual inhibition of AURKA and PAK1 by alisertib and FRAX1036 impairs survival of breast cancer cells in vitro and limits breast tumor growth in vivo ...... 25

Figure 4. Combination of alisertib and FRAX1036 suppresses proliferation and promotes apoptosis in vivo ...... 29

Figure 5. Concurrent use of alisertib and FRAX1036 decreases phosphorylation of PAK1 and AURKA kinase activity in tamoxifen-resistant breast cancer ...... 34

Figure 6. Combined treatment with alisertib and FRAX1036 reduces ERα phosphorylation and signaling activity in tamoxifen-resistant breast cancer ...... 35

ABSTRACT

Targeting Aurora A Kinase (AURKA) and p21-activated kinase 1 (PAK1) in

hormone receptor-positive breast cancer

Yayi Feng

Advisor: Erica Golemis

Estrogen receptor α (ERα) is a hormone-dependent nuclear and cytoplasmic receptor that regulates many physiological processes, such as reproduction, bone integrity, cardiovascular health, cognition, and behavior. Activation of the ERα pathway plays a crucial role in the development and progression of breast cancer, with 70-80% of breast tumors being positive for hormonal receptors and driven by ERα signaling. Drugs targeting estrogen receptor, such as tamoxifen and fulvestrant, are used in the clinic to treat ERα positive metastatic breast cancer. However, resistance to these therapies ultimately develops and represents a very significant clinical problem.

One of the mechanisms of endocrine resistance is ligand-independent activation of ERα by intracellular protein kinases. Mitotic kinase Aurora A kinase (AURKA) activates ERα through phosphorylation at serine 167 and serine 305. P21-activated kinase 1 (PAK1), a kinase known as a key regulator of MAPK pathway and cytoskeleton remodeling, also modulates ERα activation through phosphorylating serine 118 and serine 305.

Phosphorylation at these serine residues has been correlated with the increased ERα signaling and clinical outcomes. vi

Herein, we show that pharmacological inhibition of AURKA and PAK1 synergistically decreases cell survival of both tamoxifen-resistant and tamoxifen-sensitive breast cancer cell lines in vitro. Further, in vivo study demonstrates that dual inhibition of AURKA and

PAK1 limits growth of breast tumors and leads to increased apoptosis, especially in tamoxifen-resistant xenograft tumors. In vitro experiments show differentiated signaling patterns in tamoxifen-resistant and tamoxifen-sensitive breast cancer upon combination treatment. Taken together, these data suggest that targeting AURKA and PAK1 may be a promising therapeutic strategy for hormone receptor-positive breast cancer, especially in the settings of endocrine resistance. 1

INTRODUCTION

Breast cancer is the most commonly diagnosed malignancy among women in both developing and developed regions (Ferlay, Soerjomataram et al. 2015). In the United

States, it is estimated that about 246,660 women will be diagnosed with breast cancer in

2016 (Howlader, Noone et al. 2016). Although the death rate in the U.S. decreased by 1.9% per year from 2003 to 2012, breast cancer is still the second leading cause of cancer- related death among women after lung cancer, and it is estimated that there will be

40,450 new cases of death from breast cancer in 2016 (Howlader, Noone et al. 2016,

Ryerson, Eheman et al. 2016).

Breast cancer is classified according to the expression of the estrogen receptor (ER), the progesterone receptor (PR), and human epidermal growth factor receptor 2

(HER2/ERBB2/neu) as assessed by immunohistochemistry and in situ hybridization in tumor samples (Hammond, Hayes et al. 2010, Wolff, Hammond et al. 2014). Therefore, breast cancer can be categorized as one of three main subtypes: hormone receptor (HR)- positive (ER or/and PR-positive), HER2/neu-positive, and triple negative (ER, PR and

HER2/neu-negative). The status of these receptors is prognostic and widely used to forecast patients’ clinical outcomes. Since ER and HER2 are well-known therapeutic targets, these biomarkers also determine treatment options.

The HR-positive subtype accounts for approximately 80% of all breast cancer cases

(Howlader, Altekruse et al. 2014, Wu, Cheng et al. 2016). Anti-hormonal therapy, also 2 known as endocrine therapy, suppresses HR signaling either by decreasing production of

ER ligands, or by inhibiting ER activity. Tamoxifen, a selective ER modulator, is one of the endocrine agents widely used in clinics for decades. However, some patients do not respond to tamoxifen (de novo resistance), and a large proportion of individuals who initially benefit from tamoxifen treatment, develop resistance over time (acquired resistance) (Ring and Dowsett 2004). Several mechanisms underlying de novo and acquired endocrine resistance have been proposed based on dysfunction of ERα signaling, including mutations of ESR1 (ERα-coding gene), loss of ER expression, and ligand- independent activation of ERα by intracellular protein kinases(Lapidus, Nass et al. 1998,

Robinson, Wu et al. 2013, Nardone, De Angelis et al. 2015).

Figure 1. Structure of estrogen receptor α (ERα). AF – activation function domain, DBD – DNA binding domain, H – hinge domain, LBD – ligand binding domain. (Modified from(Shagisultanova, Korobeynikov et al. 2016)

ERs are found mainly in nucleus and cytoplasm, although a small fraction of ERs may be localized near or at the plasma membrane (Arpino, Wiechmann et al. 2008). There are two types of ER, ERα and ERβ, encoded by two different genes, ESR1 and ESR2, respectively (Menasce, White et al. 1993, Enmark, Pelto-Huikko et al. 1997). Both ER subtypes consist of four functional domains (Fig.1): a ligand-independent activation function-1 (AF-1) domain next to the N-terminus, a DNA binding domain (DBD), a 3 hinge domain (H), and an activation function-2 (AF-2) domain/ligand-binding domain adjacent to the C-terminus (Deroo and Korach 2006). ERα and ERβ are expressed in various tissues including mammary gland, and play important roles in several physiological processes, such as reproduction, cardiovascular health, bone integrity, cognition, and behavior (Enmark, Pelto-Huikko et al. 1997, Deroo and Korach 2006).

Both ERα and ERβs are co-expressed in normal breast epithelial cells, and ERβ expression is predominant in normal human mammary gland (Enmark, Pelto-Huikko et al.

1997, Huang, Omoto et al. 2014). Expression of ERα is significantly increased in ductal carcinoma in situ (DCIS), whereas ERβ expression is less abundant in breast cancer, and is lost in most of invasive ductal carcinoma (IDC) samples (Huang, Omoto et al. 2014).

Therefore, ERα plays an important role in promoting breast malignancies, whereas ERβ might function as tumor suppressor (Osborne, Schiff et al. 2001).

As a pivotal regulator involved in the development and progression of breast cancer, ERα can exert genomic effects as a ligand-dependent transcription factor that modulates transcription of genes involved in cell cycle regulation, apoptosis, and tumor progression.

In genomic signaling, ERα ligands such as 17β-estradiol (E2) diffuse into cells and bind to nuclear ERα at the ligand-binding domain, which induces conformational change and dimerization of ERα (Osborne, Schiff et al. 2001, Arpino, Wiechmann et al. 2008). The

ER dimer translocates to palindromic estrogen response elements (EREs) in promoter region or 3’-flanking region of target genes (Carroll, Meyer et al. 2006, Kwon, Garcia-

Bassets et al. 2007). This leads to recruitment of co-activator or co-repressor proteins that enhance or suppress transcription of the ER-targeted genes, such as VEGF and PS2 4

(Stack, Kumar et al. 1988, Applanat, Buteau-Lozano et al. 2008). The activated ERα dimer also modulates transcription of E2-responsive genes through direct protein-protein interaction with other transcription factors including Sp1, AP1, and NF-κB (Porter, Wang et al. 1996, Paech, Webb et al. 1997, Glidewell-Kenney, Weiss et al. 2005). Transcription of several oncogenic genes such as c-Myc, CCND1, and Bcl-2 are regulated through this

“tethering mechanism” (Dong, Wang et al. 1999, Glidewell-Kenney, Weiss et al. 2005,

Wang, Mayer et al. 2011). Promoters of ER-targeted genes are usually activated synergistically by AF-1 and AF-2 domains of ERα. The AF-1 domain is constitutively active or exerts transactivation effect in a ligand-independent manner, whereas transcriptional activity of AF-2 domain relies on ligand binding (Arpino, Wiechmann et al. 2008).

Beside the genomic effect, ERα also rapidly (in minutes) influences several signaling pathways independent of its function as a transcription factor (Arpino, Wiechmann et al.

2008). These non-genomic effects, at least in part, are induced by extra-nuclear full- length ERα or its variants (Li, Haynes et al. 2003, Wang, Zhang et al. 2006). In the non- genomic pathway, ligand-bound ERα binds to membrane proteins such as caveolin-1 and flotillin-2, tethered on the inner face of plasma membrane (Marquez, Chen et al. 2006,

Pedram, Razandi et al. 2007). Membrane-bound ERα interacts directly and forms large complexes with other membrane receptors including HER2, IGF-1R, and EGFR

(Márquez, Lee et al. 2001, Song, Barnes et al. 2004, Marquez, Chen et al. 2006). Several downstream signaling molecules and functional domains such as c-Src (Razandi, Pedram et al. 2000), Shc (Song, Barnes et al. 2004), distinct G protein subunits (Wyckoff, 5

Chambliss et al. 2001, Razandi, Pedram et al. 2003), and NMAR (Barletta, Wong et al.

2004) interact with ERα. The formation of a large “signalosome” complex results in bi- directional crosstalk between ERα and other receptors, as well as subsequent rapid signal transduction through routes like PI3K/AKT and RAS/RAF/MEK/ERK pathways (Arpino,

Wiechmann et al. 2008).

ERα dimerization, localization and function are regulated through post-translational modification. ERα can be phosphorylated on serine and tyrosine residues by intracellular protein kinases including AKT, PKA, MAPK, P21-activated protein 1 (PAK1), and

Aurora A kinase (AURKA) (Joel, Smith et al. 1998, Chen, Pace et al. 1999, Campbell,

Bhat-Nakshatri et al. 2001, Wang, Mazumdar et al. 2002, Michalides, Griekspoor et al.

2004, Rayala, Talukder et al. 2006, Thomas, Sarwar et al. 2008, Zheng, Guo et al. 2014).

Multiple ERα phosphorylation sites (S104/106, S118, S167, S282, S294, S305, T311, and S559) have been identified, and phosphorylation at some of these sites is correlated with clinical outcomes (Skliris, Nugent et al. 2010, Murphy, Seekallu et al. 2011).

Intracellular protein kinases AURKA and PAK1 phosphorylate ERα at S167 and S118, respectively, and both phosphorylate S305 (Wang, Mazumdar et al. 2002, Rayala,

Talukder et al. 2006, Zheng, Guo et al. 2014). Expression of AURKA (Zheng, Guo et al.

2014) and PAK1 (Holm, Rayala et al. 2006, Kok, Zwart et al. 2011) are associated with tamoxifen resistance, leading to the hypothesis that these activities may involve control of ERα activity.

AURKA is a serine/threonine protein kinase that belongs to the Aurora/IPL1-related kinase family. It is a key regulator of mitosis that controls all aspects of cell division including centrosome maturation and separation, assembly and control of a bipolar spindle, timing of mitotic entry, chromosome alignment in the metaphase, and cytokinesis(Nikonova, Astsaturov et al. 2013). Beside its crucial role in maintaining mitosis and genetic integrity, AURKA influences other cellular processes such as microtubule dynamics, cell motility, cell polarity, ciliary disassembly, and calcium regulation (Nikonova, Astsaturov et al. 2013).

AURKA consists of three domains: an N-terminal regulatory domain, a highly conserved catalytic kinase domain, and a short C-terminal domain. Crystallography showed that the catalytic domain contains two lobes (N-lobe and C-lobe), which form a bi-lobal architecture commonly seen in other protein kinases (Nowakowski, Cronin et al. 2002).

An activation loop of AURKA in the C-lobe contains a key threonine residue T288, phosphorylation of which is required for AURKA activation. Two regulatory sequences – a D-box in the C-terminus of the catalytic domain, and an A-box within the N-terminal domain – serve as recognition sites for AURKA degradation at the last stage of mitosis

(Marumoto, Zhang et al. 2005).

Gene expression, activation and degradation of AURKA during cell cycle are tightly regulated both spatially and temporally. During S phase, AURKA begins to gather around the centrosomes, and a small fraction of AURKA becomes activated before chromosome condensation in G2 phase (Marumoto, Zhang et al. 2005). AURKA 7 continues to accumulate, and level of activated AURKA increases dramatically after the cells enter prophase (Carmena and Earnshaw 2003). A small proportion of activated

AURKA moves to nucleus during the chromosome condensation (Marumoto, Zhang et al.

2005). After the breakdown of nuclear envelope, activated AURKA in spindle poles starts to spread along the mitotic spindles to mid-zone during the transition from metaphase to anaphase (Marumoto, Zhang et al. 2005, Nikonova, Astsaturov et al. 2013).

Most of AURKA is inactivated during the transition, and only a small amount of

AURKA are found at the mid-zone and centrosome during telophase (Marumoto, Zhang et al. 2005). AURKA is mostly degraded before the cytokinesis, and protein level of

AURKA is low and undetectable when the cells enter G1 phase (Marumoto, Zhang et al.

2005, Nikonova, Astsaturov et al. 2013).

Proper function of AURKA in cell cycle requires precise cooperation with a variety of other protein partners. AURKA activating partners such as Bora, NEDD9, and TPX2 induce AURKA auto-phosphorylation at T288 within activation loop (Nikonova,

Astsaturov et al. 2013). AURKA works with Bora to promote activation of CDK1/cyclin

B complex through PLK1 phosphorylation, which allows mitotic entry (Seki, Coppinger et al. 2008). Bora and NEDD9 – a scaffold protein involved in focal adhesion – facilitate

AURKA-induced centrosome maturation (Pugacheva and Golemis 2005, Hutterer,

Berdnik et al. 2006). Another AURKA activating protein, TPX2, is released from importin-α/β inhibitory complex by Ran-GTP during the breakdown of nuclear envelope

(Tsai, Wiese et al. 2003). Free TPX2 interacts with AURKA and releases it from inhibition by phosphatase P2A (PP2A). It helps AURKA localization on spindle 8 microtubules, and assists in its interaction with other substrates (Kufer, Sillje et al. 2002,

Carmena and Earnshaw 2003, Eyers, Erikson et al. 2003). AURKA destruction is required for cytokinesis and mitotic exit. Proteasomal degradation of AURKA is mediated by the anaphase-promoting complex/cyclosome (APC/C) and Cdh1 (Taguchi,

Honda et al. 2002). The A-box and D-box of AURKA are required for AURKA ubiquitination during Cdh1-dependent proteolysis (Littlepage and Ruderman 2002). The

E3 ubiquitin ligase Chfr can directly ubiquitinate A-box for AURKA degradation (Yu,

Minter-Dykhouse et al. 2005). PP2A modifies A-box through dephosphorylation of S51, which disrupts AURKA stability and promotes AURKA turnover (Horn, Thelu et al.

2007). Proteasomal degradation of AURKA can be regulated by Aurora A kinase interacting protein 1 (AIP) by promoting interaction of AURKA and Az1, a well-studied mediator of ubiquitination-independent protein degradation pathway (Lim and Gopalan

2007).

High levels of AURKA mRNA and protein expression have been identified in various cancer cell lines and solid tumors including breast carcinoma. AURKA is amplified in approximately 12% of primary breast tumors and breast cancer cell lines and is overexpressed in about 80-90% of breast tumors (Zhou, Kuang et al. 1998). The phenotype of AURKA overexpression includes aneuploidy, supernumerary centrosomes associated with multipolar spindles, increased resistance to apoptosis, and deficient cell cycle checkpoint functions (Nikonova, Astsaturov et al. 2013). AURKA overexpression has a strong correlation with worse survival (Nadler, Camp et al. 2008), especially in patient with ERα positive tumors (Zheng, Guo et al. 2014). Gene expression of AURKA 9 in ER-positive breast cancer is regulated by ERα through activation of transcription factor GATA-3 (Jiang, Katayama et al. 2010). Additionally, AURKA enhances ERα activity through direct ligand-independent phosphorylation of S167 and S305 (Zheng,

Guo et al. 2014). Notably, ERα phosphorylation at S305 is involved in endocrine resistance and enhanced transcription of oncogene CCND1, which is commonly upregulated in breast cancer (Balasenthil, Barnes et al. 2004). Further, AURKA overexpression caused by constitutively active RAF1 induces epithelial- mesenchymal transition (EMT) in ERα-positive breast cancer cells (D'Assoro, Liu et al. 2014) correlating with tumor progression, metastasis, and resistance to standard therapy

(Creighton, Li et al. 2009, Giancotti 2013, Piva, Domenici et al. 2014).

Alisertib (MLN8237), a selective AURKA inhibitor, has been tested in preclinical and clinical studies (Cervantes, Elez et al. 2012, Melichar, Adenis et al. 2015). In preclinical experiments, ectopic expression of AURKA induced tamoxifen resistance, while

AURKA depletion and pharmacological inhibition by alisertib sensitized tamoxifen- resistant cells to tamoxifen treatment (Zheng, Guo et al. 2014). Alisertib reverses EMT and suppresses self-renewal ability, and thus inhibits breast cancer metastasis (D'Assoro,

Liu et al. 2014). A phase II clinical study demonstrated that alisertib as a single agent had a promising therapeutic effect in heavily pretreated patients, especially those with HR- positive breast cancer (Melichar, Adenis et al. 2015).

In summary, preclinical and clinical studies demonstrated the signaling interplay between

AURKA and ERα, implicated AURKA activation in tumorigenesis of HR-positive breast 10 cancer, and identified AURKA as a potential therapeutic target in HR-positive breast cancer.

Similar to AURKA, PAK1 is a serine/threonine intracellular protein kinase. It is a member of p21-activated kinase family. PAK1 is a well-known effector for the small

GTPases Cdc42 and Rac. It is involved in upregulation of the MAPK pathway, cytoskeleton rearrangement, cell polarity, motility, and invasion. PAK1 functions as a critical rheostat that integrates and orchestrates several signaling pathways and thus influences cellular processes including proliferation, angiogenesis, stress response, mitogenesis, and apoptosis (Arias-Romero and Chernoff 2008, Wertheimer, Gutierrez-

Uzquiza et al. 2012, Radu, Semenova et al. 2014).

PAK1 has an N-terminal regulatory domain and a conserved C-terminal catalytic domain.

Within the N-terminal domain, a conserved P21-GTPase binding domain (GBD) overlaps with autoinhibitory domain (AID). GBD is the binding region for active form of Cdc42 or Rac GTPases (Radu, Semenova et al. 2014). Two SH3-binding motifs and a non- classical SH3 binding site are also identified within the regulatory domain. Two SH3- binding motifs are binding sites for adaptor proteins NEK and GRB2, respectively, which indicates that PAK1 can be recruited to plasma membrane. The non-classical binding site is responsible for interaction with the guanine-nucleotide-exchange factor PIX and PAK1 translocation to focal adhesion (Arias-Romero and Chernoff 2008). PAK1 has an activation loop within the catalytic domain, and phosphorylation at T423 within the loop is crucial for catalytic function (Gatti, Huang et al. 1999). 11

PAK1 activation is regulated in trans-autoinhibition manner (Lei, Lu et al. 2000). When

PAK1 is in the inactive state, two PAK1 molecules form a homodimer, in which the AID of one PAK1 molecule attaches to the catalytic domain of its partner (Radu, Semenova et al. 2014). Cdc42 and Rac get activated by various stimuli (e.g., stress, integrin, receptor tyrosine kinases, and G-protein coupled receptors), and bind to the GBD of PAK1

(Wertheimer, Gutierrez-Uzquiza et al. 2012). This interaction and coincident binding of phosphoinositide leads to the release of the AID from the catalytic domain of its partner, conformational change of PAK1, and autophosphorylation of PAK1 (Pirruccello,

Sondermann et al. 2006, Arias-Romero and Chernoff 2008). PAK1 further autophosphorylates at multiple sites, including S144, S199, and S204, which enables

PAK1 to maintain a catalytically active state (Gatti, Huang et al. 1999). PAK1 activity can be modulated through phosphorylation by other cellular protein kinases such as AKT

(Zhou, Zhuo et al. 2003), binding with NEK or GRB2 at SH3-binding motifs, and binding with PIX at PIX-binding site (Radu, Semenova et al. 2014).

PAK1 regulates cytoskeleton rearrangement through phosphorylation of downstream targets such as LIM kinase (LIMK), Myosin light chain kinase (MLCK), and Arpc1b

(Edwards, Sanders et al. 1999, Sanders, Matsumura et al. 1999, Vadlamudi, Li et al.

2004). PAK1 phosphorylation and activation of LIMK leads to subsequent phosphorylation of the LIMK substrate Cofilin, which suppresses Cofilin activity to depolymerize actin filaments and thus induces actin-filament stabilization (Yang, Higuchi et al. 1998). MLCK regulates interaction of actin and myosin II by phosphorylation and 12 activation of the regulatory myosin light chain (MLC). PAK1 impairs MLC activity by phosphorylation and inhibition of MLCK and thus induces acto-myosin contraction

(Sanders, Matsumura et al. 1999). PAK1 controls cell motility through Arpc1b, a subunit of Arp2/3 complex. Arpc1b phosphorylation by PAK1 promotes its localization with

Arp2/3 complex, which drives formation of branched actin filament networks and extension of the cell's leading edge (Vadlamudi, Li et al. 2004). Beside its functions in cell morphology, PAK1 prevents apoptosis through RAF and pro-apoptotic protein BAD.

Upon phosphorylation by PAK1, RAF translocates to mitochondria and phosphorylates

Bad (Tran and Frost 2003). PAK1 also can phosphorylate Bad directly (Schurmann,

Mooney et al. 2000). This impairs binding of Bad and Bcl-2, and thus promotes cell survival. Additionally, PAK1 is involved in cell cycle progression. PAK1 is phosphorylated by cyclin B1/Cdc2 complex during mitosis and localizes to chromatin.

Activated PAK1 modifies histone H3 through phosphorylation, which is essential for the initiation of chromosome condensation and cell cycle progression (Li, Adam et al. 2002).

Interestingly, PAK1 activates AURKA through direct phosphorylation of T288 in mitotic cells (Zhao, Lim et al. 2005). PAK1 also facilitates AURKA function by phosphorylating

AURKA activating partners LIMK and Arpc1b, as well as AURKA substrate PLK1

(Edwards, Sanders et al. 1999, Vadlamudi, Li et al. 2004, Maroto, Ye et al. 2008).

Moreover, PAK1 also promotes cell proliferation through activation of RAF/MEK/ERK signaling and β-catenin translocation to nucleus, which stimulates expression of Cyclin

D1 (encoded by CCND1) driving cell cycle progression (Balasenthil, Sahin et al. 2004,

Radu, Semenova et al. 2014). In addition to migration, cytoskeleton reorganization, proliferation, and apoptosis, PAK1 regulates angiogenesis (Radu, Semenova et al. 2014). 13

Hyperactivation and high expression of PAK1 is found in a variety of cancers, including brain, breast, liver, kidney, colon, lung, and ovarian tumors (Ye and Field 2012).

Transgenic mice with PAK1 overexpression develop invasive breast cancer (Wang,

Zhang et al. 2006). One of the mechanisms underlying dysfunction of PAK1 is gene amplification. PAK1 gene is located in the chromosomal region 11q13, in which other oncogenes such as CCND1 are clustered (Schraml, Schwerdtfeger et al. 2003).

Amplification of this region is found in various types of cancer and correlates with poor prognosis in breast cancer. It should be noted, however, that overexpression of PAK1 does not always coexist with PAK1 gene amplification (Radu, Semenova et al. 2014).

Studies have shown that high PAK1 expression is associated with decreased tamoxifen sensitivity in pre-menopausal breast cancer patients (Holm, Rayala et al. 2006, Rayala,

Talukder et al. 2006, Bostner, Ahnstrom Waltersson et al. 2007). Similar to AURKA,

PAK1 can phosphorylate ERα at serine 305, followed by phosphorylation at serine 118, which suggests that ligand-independent activation of ERα by PAK1 may lead to endocrine resistance. A preclinical study supported this idea by inhibiting PAK1 activity using a histone deacetylase (HDAC) inhibitor FK228, showing that FK228 suppresses estrogen-dependent growth of both tamoxifen-resistant and tamoxifen-sensitive human breast cancer (Hirokawa, Arnold et al. 2005). 14

Figure 2. ERα, AURKA, and PAK1 signaling network. E2 - 17β-estradiol (Modified from(Shagisultanova, Korobeynikov et al. 2016)

Taken together, these studies suggest a signaling network between ERα, AURKA, and

PAK1 (Fig. 2). In this model, PAK1 activates AURKA and its activating partners LIMK and Arpc1b through direct phosphorylation (Edwards, Sanders et al. 1999, Vadlamudi, Li et al. 2004, Zhao, Lim et al. 2005). PAK1 and AURKA activate ERα by phosphorylating

S118 and S167, respectively, and both phosphorylate S305 (Wang, Mazumdar et al. 2002,

Rayala, Talukder et al. 2006, Zheng, Guo et al. 2014). Phosphorylation at these residues is correlated with endocrine resistance in hormone receptor-positive patients. ERα, in turn, upregulates expression of AURKA upon ligand binding (Jiang, Katayama et al. 2010).

Here, we introduce an AURKA selective inhibitor alisertib, and a novel ATP-competitive small molecule inhibitor of group I PAKs, FRAX1036, and demonstrate the synergistic suppression of proliferation in human breast cancer cell lines (tamoxifen-sensitive and tamoxifen-resistant). The antitumor effect of combined inhibition was confirmed in xenograft models. Therefore, these results suggest that dual inhibition of AURKA and

PAK1 is a promising therapeutic strategy in hormone receptor-positive breast cancer, especially in the settings of endocrine resistance. 15

MATERIAL AND METHODS

Cell lines and cell culture

The human breast cancer cell lines BT-474 (ATCC® HTB-20™) and T-47D (ATCC®

HTB-133™) were obtained from ATCC (Manassas, VA). Both cell lines were cultured in

RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% GlutaMAX-I®

(100×), and 100 μg/ml penicillin/ streptomycin antibiotic mix as previously described

(Rusnak, Lackey et al. 2001, Yamashita, Iwase et al. 2003, Neve, Chin et al. 2006).

Unique STR profile was confirmed for both cell lines.

Reagents

Aurora A kinase inhibitor alisertib (MLN8237) was purchased from MedChem Express

(Monmount Junction, NJ). PAK group I inhibitor FRAX1036 was synthesized at the laboratory of Dr. William Wuest (Department of Chemistry, Temple University,

Philadelphia, PA). RPMI 1640 medium, fetal bovine serum, penicillin/ streptomycin antibiotic mix were obtained from Cell Culture Facility, Fox Chase Cancer Center.

GlutaMAX-I® (100×) was purchased from Life Technologies (Grand Island, NY).

Tumor lysis buffer was prepared with T-PER™ Tissue Protein Extraction Reagent

(Thermo Scientific, Rockford, IL) supplemented with PhosSTOP™ phosphatase inhibitor and cOmplete™ ULTRA protease inhibitor cocktails (Roche, Mannheim, Germany).

Luminata™ Western HRP substrates (Classico and Crescendo) were purchased from

EMD Millipore (Billerica, MA) and SuperSignal™ West HRP substrate kits (Pico and 16

Femto) were obtained from Thermo Scientific (Rockford, IL). Immun-Star™ AP substrate was purchased from Bio-Rad (Hercules, CA).

The antibodies against phospho-AKT (Thr 308) and AKT (pan), phospho-ERα (Ser118), phospho-ERα (Ser167), PAK1, and histone H3 were purchased from Cell Signaling

(Danvers, MA). Aurora A kinase antibody was from BD Transduction Laboratories (San

Jose, CA). Antibody against Phospho-PAK 1, 2, 3(Ser141) was purchased from Life

Technologies (Frederick, MD). Phospho-ERα (Ser305) antibody was obtained from EMD

Millipore (Billerica, MA). All the primary antibodies were diluted to 1:1000 in

Superblock T20 Blocking Buffer (Thermo Scientific, Rockford, IL) before incubation, unless stated otherwise in Table 1. HRP-conjugated secondary antibodies were obtained from GE Healthcare (Little Chalfont, UK), and diluted to 1:10000 in blocking Buffer before use. AP-conjugated secondary antibodies were purchased from Jackson

ImmunoResearch (West Grove, PA), and diluted to 1:5000 in blocking buffer prior to use.

Table 1. List of Antibody Information Biological Antibody Company Catalog Number Dilution Ratio Source

Anti-mouse HRP GE Healthcare UK NA931-1ML Sheep 1:10000 conjugated antibody Limited

Anti-rabbit HRP GE Healthcare UK NA9340-1ML Donkey 1:10000 conjugated antibody Limited

Anti-mouse AP Jackson 115-055-003 Goat 1:5000 conjugated antibody ImmunoResearch

Anti-rabbit AP Jackson 115-055-003 Goat 1:5000 conjugated antibody ImmunoResearch

Vinculin Sigma-Aldrich V9131-.5ML Mouse 1:1000

BD Transduction Aurora A (IAK1) #610939 Mouse 1:1000 Laboratories phospho-PAK Life Technologies #44-940G Rabbit 1:5000 1,2,3(Ser141)

PAK 1 Cell Signaling #2602 Rabbit 1:5000

phospho-AKT Cell Signaling #4056 Rabbit 1:1000 (Thr308)

AKT(pan) Cell Signaling #2920 Mouse 1:1000

phospho-ERα Cell Signaling #5587 Rabbit 1:1000 (Ser167) phospho-ERα EMD Millipore #07-962 Rabbit 1:1000 (Ser305)

ERα (D6R2W) Cell Signaling #13258 Rabbit 1:1000

histone H3 (96C10) Cell Signaling #3638 Mouse 1:1000

Cell Viability Assay

Viability of BT474 and T47D cells was evaluated by CellTiter-Glo assay (Promega,

Madison, WI) after 4 different treatments: (1) FRAX1036, (2) alisertib, (3) combination of FRAX1036 and alisertib in 1:1.5 molar ratio, (4) vehicle (DMSO). Each drug was prepared in 8 serial dilutions with the dilution factor of 1.6. Cells were grown on opaque- walled 96-well plates in antibiotic-free media for 24 hours before treatment was applied.

After 96 hours of treatment, 100μl of CellTiter-Glo® Reagent was added each well, the plates were placed on a shaker for 2 minutes to achieve complete lysis of the cells and mixture of the reagents, and then incubated for an additional 8 minutes. The luminosity of each well was measured on GloMax™ 96 Microplate Luminometer. Luminosity of each well reflects the amount of ATP which is directly proportional to the number of live cells present in culture. Each drug concentration was tested in triplicates, and each experiment was performed in three or more biologic repetitions. IC50 for single agents and combination treatment was established for each cell line. Synergy was determined by

Chou-Talaly method as previously described (Chou and Talalay 1984).

Breast cancer mouse model and tissue collection

Six-week-old non-obese diabetic /severe combined immunodeficient (NOD/SCID) female mice were obtained from the Animal Facility of Fox Chase Cancer Center. Prior to cell injection, estrogen pellets (~1mg 17β-estradiol/ pellet) were made and implanted to the mice as previously described (DeRose, Gligorich et al. 2013). Ten million T47D cells were suspended in Matrigel (BD Biosciences, San Jose, CA) (1:1, v/v), and then injected subcutaneously into mammary fat pads of the mice to establish orthotopic 19 xenografts. Body weight was monitored, and tumor length (L, the largest tumor diameter) and width (W, the perpendicular diameter) were measured with calipers three times per week. Tumor volume was calculated as 0.5 × (L × W2) mm3.

When tumor volumes reached 150-160mm3, the mice were randomly assigned to four cohorts: 7.5 mg/kg (T47D) or 15 mg/kg (BT474) alisertib, 20 mg/kg FRAX1036, a combination of both, and Vehicle. Alisertib was dissolved in 10% 2-hydroxypropyl-β- cyclodextrin with 1% sodium bicarbonate, FRAX1036 was formulated in 10% 2- hydroxypropyl-β-cyclodextrin with 50mM citrate buffer (pH=3). Mice treated with vehicle were orally dosed with alisertib solvent in the morning, and FRAX1036 solvent in the afternoon. All the mice were treated for a total of 21 days of treatment, using 5 days on/ 2 days off schedule.

24 hours after the last treatment, mice were euthanized by CO2 asphyxiation, and tumors and organs were harvested. Three quarters of the tumors were snap-frozen in liquid nitrogen, and stored at -80 °C to be used for western blot analysis and in vitro kinase assay. The organs, as well as the remaining quarter of tumor, were fixed in 10% phosphate-buffered formaldehyde (formalin) and processed as paraffin embedded tissues.

These tissues were prepared for pathological assessment as slides and stained with hematoxylin and eosin (H&E) and Ki-67 antibody by Pathology Facility in Fox Chase

Cancer Center and stained with antibody against cleaved caspase-3 by University of

Colorado School of Medicine. The slides were imaged and scored using Vectra 3.0 20

(PerkinElmer, Waltham, MA) and APERIO Image Analysis (Leica Biosystems Imaging,

Buffalo Grove, IL)

All experiments involving mice were approved by The Institutional Animal Care and Use

Committee (IACUC) in Fox Chase Cancer Center.

Preparation of Tumor Lysates

Fresh frozen tumors were weighed and lysed in the tumor lysis buffer (1:5, w/v) at 4 °C .

Then tumors were homogenized by PowerGen 125 Homogenizer (Fisher Scientific,

Pittsburgh, PA), followed by ultrasonication using Model 550 Sonic Dismembrator

(Fisher Scientific, Pittsburgh, PA). The lysates were centrifuged at 13000 rpm, 4 °C for

30 minutes, and the supernatants were collected. Protein concentrations of whole lysates were measured using Pierce BCA assay kit as the manufacturer’s instructions (Thermo

Scientific, Rockford, IL), and adjusted to 5 µg/µl.

Western Blotting

The lysates were mixed with 5× lane marker reducing sample buffer (Thermo Scientific,

Rockford, IL), boiled for 5 minutes at 100 °C , and centrifuged at 13000 rpm, 1 minute.

Then these samples were frozen in a mixture of solid CO2 and ethanol, and stored at -

80 °C until use. 50 µg of proteins were separated by size using BLOT 8% or 10% Bis-

Tris 15 well Mini Protein Gels (Life Technologies, Carlsbad, CA). Then the proteins were transferred to 0.45 µm Immobilon-P PVDF Transfer Membrane (EMD Millipore,

Billerica MA) as the manufacturer’s instructions. Membranes were blocked in at room 21 temperature for 1 hour, and incubated with primary antibodies overnight at 4 °C on a rocker. Membranes were washed 3 times for 10 minutes each in PEST, followed by 1- hour incubation with secondary HRP (1:10000) or AP (1:5000) antibodies at room temperature. The membranes were washed 3 times again. Then the membranes were incubated with HRP substrates or AP substrate for 5 minutes. Chemiluminescence was captured by X-ray films. The films were scanned by Epson Perfection V800 Photo

Flatbed Scanner (Epson America Inc., Long Beach, CA). ImageJ Imaging and Processing

Analysis Software (NIH, Bethesda, MD) was used for densitometric analysis, normalizing signal intensity of detected proteins to that of loading control (Vinculin).

Immunoprecipitation and in vitro Kinase Assay

Protein A/G Agarose beads (Pierce by Thermo Scientific, Rockford, IL) were mixed gently with cold PTY buffer (50 mM HEPES, 50 mM NaCl, 5 mM EDTA, 1% Triton X-

100, and 50 mM NaF). Then antibody against AURKA (BD Transduction Laboratories,

San Jose, CA) were added to the mixture and incubated on the rotator at 4 °C for 4 hours.

Mouse Immunoglobulin G (IgG) (Genetex, Irvine, CA) was used as negative control for immunoprecipitation.

After antibody conjugation to the beads, beads were centrifuged at 7000 rpm, 4 °C for 3 minutes, and supernatant was aspirated. The beads were then washed gently in cold PTY buffer, and centrifuged again. After 3 washes, immobilized bead conjugates were mixed with 500 µg tumor lysates in cold PTY buffer. For positive control, purified recombinant 22

AURKA was mixed with conjugates in PTY buffer. The mixtures were then incubated overnight on the rotator at 4 °C .

After incubation mixtures were centrifuged, and supernatants were aspirated. The bead conjugates were mixed with 5 × kinase buffer containing purified AURKA substrate, histone H3 (Upstate, Charlottesville, VA), and [γ-32P] ATP mix that contains [γ-32P]

ATP (BLU502A001MC, Perkin Elmer), Mg2+/ATP cocktail (20-113, EMD Millipore), and Milli-Q H2O (Cell Culture Facility, Fox Chase Cancer Center). Mixtures were then incubated at 32 °C for 30 minutes. Incubation was followed by addition of 2× lane marker reducing sample buffer (Thermo Scientific, Rockford, IL), and denaturation at

100 °C for 5 minutes.

After centrifuging at 13000 rpm for 1 minute supernatants were resolved by BOLT 12%

Bis-Tris 17 well Mini Protein Gels (Novex by Life Technologies, Carlsbad, CA), and transferred to 0.45 µm Immobilon-P PVDF Transfer Membranes. The mini protein gels were stained with Simply Blue Safe Stain (LC6060, Thermo Scientific, Rockford, IL) after transfer. Membranes were then placed within a transparent plastic folder in an X-

Ray film cassette, and kept at -80°C in order to capture exposure on X-Ray films. After obtaining multiple exposures, membranes were incubated with primary antibody against histone H3 overnight and then processed as the aforementioned Western Blotting instructions. Signal intensity of phospho-histone H3 on 32P films was normalized to that of histone H3 on immunoblot films.

Statistical Analysis

Statistical analysis was performed by Biostatistician Brian Egleston, or through Prism 5

(GraphPad Software, San Diego, CA) by Yayi Feng, Elena Shagisultanova, and Vladislav

Korobeynikov.

RESULTS

Inhibition of AURKA and PAK1 synergistically hampers cell survival of breast cancer cells in vitro and limits growth of mammary tumor xenografts in vivo.

To investigate the effect of dual inhibition of AURKA and PAK1 on cell proliferation,

CellTiter-Glo cell viability assays in tamoxifen-resistant and tamoxifen-sensitive human breast cancer cell lines BT474 and T47D (Table 2) were conducted by my collaborator

Dr. Elena Shagisultanova (Shagisultanova, Korobeynikov et al. 2016). Herein, we pharmacologically targeted AURKA and PAK1 by utilizing alisertib and FRAX1036 in both cell lines for 96 hours. Drug synergy was determined by Chou-Talaly method.

Results demonstrated a dramatic synergistic effect of the alisertib and FRAX1036 combination in reducing cell survival in both cell lines (Fig. 3A).

Table 2. Molecular Phenotypes of BT474 and T47D 11q13 Cell lines PR ER HER2 p53 PI3KCA Sensitivity to tamoxifen amplification BT474 + + + amp mut mut tamoxifen-resistant

T47D + + - non-amp mut mut tamoxifen-sensitive

*amp - amplified, mut - mutated 24

To confirm the inhibitory effect on proliferation of these breast cancer cells in vivo.

BT474 and T47D cells were injected into the mammary fat pads of 6-week-old

NOD/SCID mice to establish orthotopic xenograft tumor models. Once implanted tumor volume reached 150 mm3, the mice were assigned into four cohorts and administered following treatments for 21 days: Vehicle, FRAX1036 20 mg/kg, alisertib 15 mg/kg

(BT474) or 7.5 mg/kg BID (T47D), and a combination of FRAX1036 and alisertib. We observed more rapid growth of BT474 tumors in vehicle-treated group comparing to treatment cohorts: in the Vehicle group, the average tumor volume reached 930 mm3 by the end of treatment (Fig. 3B) (Shagisultanova, Korobeynikov et al. 2016). Mice dosed with either FRAX1036 or alisertib had smaller average tumor size (FRAX1036: 826 mm3, alisertib: 188 mm3), whereas combined FRAX-alisertib treatment significantly suppressed growth of BT474 tumors (55 mm3). T47D xenograft mice dosed with vehicle harbored smaller tumors (276 mm3) in the end of treatment compared to BT474 xenograft.

Alisertib at 7.5 mg/kg and combination treatment completely eliminated tumor growth.

FRAX1036 treatment alone showed a trend to retard the tumor progression at the beginning of the dosing. Therefore, the in vivo experiment demonstrates that FRAX1036 or alisertib decreased tumor burden, and combination therapy further suppressed tumor growth in BT474 xenograft mouse model, which is statistically significantly better than either of the single agents. In T47D xenograft model, combination treatment overall resulted in a better inhibition of tumor growth compared to single agents. However, comparison of the effect of combination treatment to that of alisertib alone did not reach statistical significance.

Figure 3. Dual inhibition of AURKA and PAK1 by alisertib and FRAX1036 impairs survival of breast cancer cells in vitro and limits breast tumor growth in vivo. Cell viability assays were performed in BT474 andT47D cells after four different treatments: FRAX1036, alisertib, combination of FRAX1036 and alisertib in 3:2 molar ratio, and vehicle (DMSO). Synergy was analyzed by the Chou-Talalay method. In in vivo study, BT474 and T47D cells were injected to mammary fat pads of NOD/SCID mice. When the tumor volume reached 150 mm3, mice were administered with vehicle, FRAX1036 or/and alisertib as described in “Materials and Methods”. The tumor growth was monitored three times per week. A. Combined use of alisertib and FRAX1036 exerted synergistic effect on cell death in T47D and BT474 breast cancer cell lines. IC50 of each treatment is indicated in the graphs. Chou Talalay analysis of synergy indicated that alisertib and FRAX1036 were synergistic (CI<1) or had at least additive effect (CI=1) in the tested concentration range (CI – combination index; synergy: CI<1; additive effect: CI=1; antagonistic effect: CI>1). B. Alisertib impeded tumor growth, and combination of alisertib and FRAX1036 led to a decline of tumor burden in BT474 and T47D breast cancer xenograft models. **P<0.01.

Combination of alisertib and FRAX1036 suppresses proliferation and promotes apoptosis in vivo

In order to determine the changes in cell proliferation and apoptosis caused by alisertib and FRAX1036, tumors were harvested at the end of treatment, and histopathological assessment performed. Immunohistochemical (IHC) staining was performed using antibodies against Ki-67 to indicate proliferation and cleaved caspase-3 to indicate apoptosis. As expected, FRAX and alisertib cooperatively led to a significant decline in proliferative cell population, and induced caspase-3 cleavage in BT474 orthotopic tumors

(Fig. 4B-E) (Shagisultanova, Korobeynikov et al. 2016). Assessment of hematoxylin and eosin (H&E) stained slides demonstrated many few cancer cells and larger fibrotic and necrotic areas in tumors of the combination-treated cohort as compared with those in other treatment groups (Fig. 4A). Alisertib alone at the dose of 15mg/kg also decreased proliferation and promoted apoptosis in tumors collected from BT474 xenograft. H&E staining revealed that alisertib as well as FRAX1036 led to an increase in fibrotic area in

BT474 tumor samples, albeit to a lesser degree compared to combination treatment. IHC analysis in tumors after 21 days of FRAX1036 treatment did not show significant changes in Ki-67 and cleaved caspase-3. This is in accordance with the fact that FRAX initially suppressed growth of BT474 xenografts, however, at the end of the experiment this effect was lost. These data suggest that alisertib alone was used at a concentration sufficient to inhibit proliferation and induce cell death in this model, while concurrent use of alisertib and FRAX1036 diminished proliferation and enhanced apoptosis in BT474 xenograft.

Previously we observed T47D tumors shrank during the combination therapy (Fig. 3B).

However, there was no discernible difference in Ki-67 positive cell populations between all the treatment groups of T47D xenograft (Fig. 4B, D). Interestingly, tumors with alisertib treatment alone had a decrease in cleaved caspace-3 staining compared to those with FRAX1036 treatment (Fig. 4C, E).

Figure 4. Combination of alisertib and FRAX1036 suppresses proliferation and promotes apoptosis in vivo. Orthotopic tumors were collected after the 21-day treatment, and processed as paraffin embedded tissues, which was followed by H&E staining (A), IHC staining using anti-Ki-67 (B) and anti-cleaved caspase 3 antibodies (C). Quantification of Ki-67 and cleaved caspase 3 stained cells were performed using APERIO e-pathology (D, E). A. In BT474, FRAX1036 or alisertib treatment led to an increase in fibrotic area, and combination treatment effectively decreased tumor cell population resulting in large areas of fibrosis. In T47D, FRAX1036 alone did not result in the increase of fibrotic area, while both Alisertib and combination treatment resulted in the increased fibrosis. B, D. Alisertib and combination treatment suppressed proliferation in BT474 orthotopic tumors as shown by decrease in Ki-67, which was statistically significantly different between vehicle and combination treatment, FRAX1036 and alisertib, and FRAX1036 and combination treatment. This effect was lost after 21-day treatment in T47D xenograft mice. C, E. Apoptosis was induced in BT474 tumors upon combination treatment, which was statistically significantly different from those with vehicle treatment. No difference between vehicle and treatment groups were found in T47D tumors, although and FRAX1036 exerted stronger pro-apoptotic effect in T47D tumors compared to alisertib. **P<0.01.

Combined treatment with alisertib and FRAX1036 reduces estrogen receptor α

(ERα) phosphorylation and signaling activity in HR-positive breast cancer.

To further explore the short-term effect of alisertib and FRAX1036 in HR-positive breast cancer, we established BT474 and T47D xenograft models for a three-day treatment in order to confirm results of in vitro cell viability experiments based on 96 hours of drug exposure (Fig. 3A). Using Western blotting to detect protein levels in tumor lysates, we found that FRAX1036 as a single agent effectively reduced levels of phosphorylated

PAK1 in both BT474 and T47D orthotopic tumors (Fig. 5A). Alisertib enhanced the

FRAX1036 inhibitory effect, resulting in a depletion of phosphorylated PAK1 in BT474 tumors. T47D xenograft tumors treated with FRAX1036 alone or in combination with alisertib also had suppressed phosphorylation of PAK1. However, in contrast to BT474,

T47D tumors had increased expression of total PAK1 after treatment with our targeted agents, which could have been a compensatory mechanism diminishing antitumor activity of FRAX1036 alone or in combination with alisertib in T47D tumor xenograft model.

We validated inhibition of AURKA by alisertib using an in vitro kinase assay. BT474 and T47D tumor lysates were subjected to immunoprecipitation by protein A/G agarose beads conjugated with anti-AURKA antibody. Histone H3 was used as AURKA substrate to incubate with the immunoprecipitates and 32P-labeled ATP, as it is an established substrate phosphorylated by AURKA in vitro (Crosio, Fimia et al. 2002). The in vitro kinase assay and coupled Western Blot analysis demonstrated that alisertib treatment suppressed AURKA activity in both xenograft models, and reduced up to 70% 32 of phospho-histone H3 in the presence of FRAX1036 in BT474, but not T47D orthotopic tumors (Fig. 5B).

Previous published studies revealed that both PAK1 and AURKA interact with ERα and modulate ERα transactivation activity through phosphorylation at serine residues, S118 and S167 in the AF-1 domain, respectively, and S305 at the hinge domain (Rayala,

Talukder et al. 2006, Zheng, Guo et al. 2014). The phosphorylation of these residues is involved in ligand-independent activation of ERα signaling. Therefore, we wanted to determine if inhibition of PAK1 and AURKA by FRAX1036 and alisertib represses ERα phosphorylation and signal transduction. Western Blot analysis showed that FRAX1036 decreased phosphorylated ERα-S167 and ERα-S305 levels in both BT474 and T47D xenograft models, whereas alisertib caused similar effect but to a lesser extent, only in

BT474 tumors (Fig. 6A, B). Combined use of FRAX1036 and alisertib also inhibited phosphorylation of ERα at these serine residues in BT474 orthotopic tumors. However, the combination therapy surprisingly tended to reverse the effect in T47D xenograft.

Cytoplasmic and membrane ERα exerts non-genomic effect in breast cancer cells upon ligand binding. When ERα activation occurs, it recruits various molecules to the cell membrane such as membrane receptors (receptor tyrosine kinases and G-protein coupled receptors), adaptor proteins (Src, Shc, etc.), as well as protein kinases including PI3K, and AKT. Activated ERα induces rapid phosphorylation of adapter proteins and thus transduces signals through various pathways including PI3K/AKT (Arpino, Wiechmann et al. 2008). Activated AKT increases ERα transcriptional activity through 33 phosphorylation at S167 (Campbell, Bhat-Nakshatri et al. 2001). Interestingly, PAK1 is promotes AKT activation to enhance growth signal autonomy in cancer cells (Higuchi,

Onishi et al. 2008), while AKT, in turn, phosphorylates PAK1 and enhances PAK1 activity, even though the PAK1 activation by AKT is weaker than activation by Cdc42 or

Rac (Zhou, Zhuo et al. 2003). Therefore, we used antibodies against phosphorylated and total AKT proteins to examine activation status of PI3K/AKT signaling pathway, aiming to investigate the mechanisms underlying distinctive consequences of treatments in

BT474 and T47D xenograft models. We found that BT474 xenograft mice treated with

FRAX1036 or/and alisertib had a substantial decline of phosphorylated and total AKT levels in solid tumors. Notably, combination treatment completely depleted phosphorylation of AKT and caused a 90% reduction of total AKT proteins (Fig. 6C). In

T47D solid tumors, FRAX1036 treatment also decreased both levels of activated and total AKT. However, alisertib and combination group had a lower level of phosphorylated AKT, and both treatments exerted a reverse effect in total AKT protein expression as compared to control group, possibly representing a compensatory mechanism.

Figure 5. Concurrent use of alisertib and FRAX1036 decreases phosphorylation of PAK1 and AURKA kinase activity in tamoxifen-resistant breast cancer. Xenograft mice were dosed for 3 days before euthanasia and tumor collection. Tumors were lysed and homogenized, and the tumor lysates were subjected to Western blotting analysis and in vitro kinase assay. A. Alisertib enhanced FRAX1036 inhibitory effect on PAK1 phosphorylation in BT474 xenograft model. B. Combined treatment of FRAX1036 and alisertib cooperatively suppressed AURKA kinase activity in BT474, but not in T47D tumors. *P<0.05, ****P<0.0001 relative to vehicle treated. 35

Figure 6. Combined treatment with alisertib and FRAX1036 reduces ERα phosphorylation and signaling activity in tamoxifen-resistant breast cancer. A-B. Combination of FRAX1036 and alisertib exerted reverse effect on ERα phosphorylation in BT474 and T47D xenograft models. BT474 orthotopic tumors had decreased level of p-ERα(S167) (A) and p-ERα(S305) (B), whereas T47D tumors had an increase in ERα phosphorylation. C. Phosphorylation of AKT was downregulated upon dual inhibition of PAK1 and AURKA. *P<0.05, ****P<0.0001 relative to vehicle treated. 36

DISCUSSION

Several studies have emphasized the significant roles of AURKA and PAK1 in tumor development and progression of breast cancer. However, co-targeting AURKA and

PAK1 in breast cancer, especially in the context of tamoxifen resistance, has not previously been examined. In this study, we demonstrated that AURKA and PAK1 inhibitors cooperatively suppressed cell viability in tamoxifen-resistant and tamoxifen- sensitive breast cancer cell lines in vitro (Fig. 3A). We also observed an antitumor effect of alisertib and FRAX1036 in both xenograft models.

When alisertib and FRAX1036 were used as single agents, breast cancer cells generally were more sensitive to FRAX1036 in vitro (Fig. 3A). In contrast, alisertib decreased tumor volume to a greater degree than FRAX1036 in both xenograft models (Fig. 3B), suggesting that inhibition of tumor growth by alisertib is predominant in vivo. The diverse responses might result from differences in various aspects such as duration of the treatments (3 days for in vitro study versus 21 days for in vivo study), tumor microenvironment, efficacy of drug delivery, and the biostability of these drugs in different experimental systems. The results of tumor volume measurement (Fig. 3B) and

Ki-67 staining (Fig. 4B, D) reveal that alisertib effectively limited the growth of breast tumors. However, alisertib reduced proliferation in BT474 to a greater extent compared to T47D orthotopic tumors, which might result from the higher dosing concentration of alisertib for BT474 xenograft mice (15mg/kg BID for BT474 cohorts versus 7.5mg/kg

BID for T47D cohorts). 37

BT474 xenograft mice had a greater response to FRAX1036 alone compared to T47D cohorts. FRAX1036 enhanced the antitumor effect of alisertib in the BT474 cohorts with combination treatment, whereas there was no significant difference in tumor volume between T47D xenograft mice with alisertib alone and combination treatments (Fig. 3B).

These results indicate that FRAX1036 as a single agent or in combination with alisertib might be more beneficial to tamoxifen-resistant tumors, as represented by BT474 cancer cell line. One of the potential explanations is that T47D tumors had a lower growth rate compared to BT474 tumors, which led to a small effect size shown in tumor volume measurement. BT474 tumors generally grow faster than T47D, partially due to HER2 amplification/ overexpression, a major difference between two cell lines in molecular phenotypes (Table 2). HER2 is overexpressed in BT474 cells, while T47D cells are identified as HER2 negative (Neve, Chin et al. 2006). HER2 overexpression has been correlated with more aggressive phenotype in breast cancer. HER2 hyperactivation caused by amplification or overexpression provides an additional stimulus for proliferation of HR-positive cancer cells. It has been previously shown that protein level and function of PAK1 are associated with HER2 overexpression in ER-positive breast cancer, but not in ER-negative breast cancer. Suppression of PAK1 activity hampers

HER2-driven tumorigenicity in vivo, which implicates that PAK1 is an important mediator in HER2 signaling in tumor development (Arias-Romero, Villamar-Cruz et al.

2010). Better response to FRAX1036 in BT474 xenograft mice might be also due to the fact that chromosomal region 11q13 is amplified in BT474, but not T47D breast cancer cells. As previously discussed, PAK1 gene is clustered with other oncogenes within the 38 chromosomal region 11q13, and 11q13 amplification is one of the mechanisms explaining PAK1 dysfunction in ovarian and breast malignancy (Lundgren, Holm et al.

2008). The Chernoff group has demonstrated that ovarian cancer cells with 11q13 amplification have higher sensitivity to FRAX1036 compared to cells without PAK1 overexpression (Prudnikova and Chernoff 2016). Therefore, it is possible that BT474 xenograft mice had a greater response to FRAX1036 as a single agent or in combination with alisertib than T47D cohorts due to the presence of 11q13 amplicon and HER2 amplification/overexpression.

We confirmed the inhibitory efficacy of FRAX1036 in vitro by detecting the levels of phosphorylated and total PAK1 (Fig. 5A). The results of Western Blot analysis suggest that FRAX1036 significantly suppresses PAK1 activation in both xenograft models, showing its potency against PAK1 (Chow, Dong et al. 2015). Interestingly, alisertib also inhibited PAK1 phosphorylation, which might be due to the fact that alisertib indirectly suppresses PI3K/AKT/mTOR pathway and decreases the level of phosphorylated AKT

(Fig. 6C) that normally phosphorylates and promotes PAK1 activity (Wang, Li et al.

2015). We found a decrease in the protein level of total PAK1 in BT474 cohorts dosed with FRAX1036 alone or combination. A similar effect has been reported in a Kras- driven skin cancer model treated with another group I-specific PAK inhibitor, FRAX597

(Chow, Jubb et al. 2012). Downregulation of total PAK1 level by FRAX597 is disrupted when cells are treated with proteasome inhibitor MG132, which indicates that FRAX597 might serve as a destabilizing agent, in addition to its function as an ATP-competitive inhibitor (Radu, Semenova et al. 2014). Since FRAX1036 is optimized on the basis of 39 arylamino pyridopyrimidinone PAK inhibitors including FRAX597, FRAX1036 might also have a dual inhibitory effect on PAK1 (Ong, Gierke et al. 2015). However, such inhibition was not seen in the T47D xenograft model, suggesting that the additional effect might be cell-specific, or other compensatory mechanisms might render T47D cells overcome depletion of total PAK1 protein by FRAX1036.

In intro kinase assays demonstrated that alisertib is able to target AURKA and downregulate its kinase activity of AUKRA to phosphorylate histone H3 (Fig. 5B).

FRAX1036 also led to a decrease in histone H3 phosphorylation when used as a single agent or in combination with alisertib, in support of previous studies that PAK1 can induce AURKA activation by directly phosphorylating AURKA as well as its activators

(Edwards, Sanders et al. 1999, Vadlamudi, Li et al. 2004, Zhao, Lim et al. 2005).

FRAX1036 and alisertib cooperatively suppressed AURKA kinase activity in BT474 tumors, however, did not exert a synergistic effect in T47D cohort. Notably, one specimen in this cohort displayed a high level of phosphorylated histone H3, whereas the remaining samples exhibited low level of AURKA kinase activity, implicating that heterogeneity existed in this cohort and it might disrupt the consistency of the results. It would be important to analyze additional animals in the future.

We assessed ERα phosphorylation at S167 and S305 in both xenograft models (Fig. 6A,

B). In BT474 xenograft tumors, p-ERα(S167) level in the tumors treated with alisertib is similar to that with FRAX1036 or combination treatment. Since PAK1 does not directly phosphorylate ERα at S167, reduced level of p-ERα(S167) caused by FRAX1036 may 40 mainly arise from the decreased AURKA kinase activity upon PAK1 inhibition (Fig. 5B).

A reduced level of p-ERα(S305) following alisertib treatment is consistent with the results from the Cheng group, showing that AURKA promotes phosphorylation of ERα at

S305 (Zheng, Guo et al. 2014). FRAX1036 or combination treatment caused a larger reduction than alisertib, which is compatible with our hypothesis that FRAX1036 reduces

ERα phosphorylation at S305 by inhibiting PAK1 activity to phosphorylate this residue directly, and also indirectly (by impairing AURKA kinase activity).

Similar to the results in BT474 xenograft mice, FRAX1036 downregulated ERα phosphorylation at both S167 and S305 in T47D tumors. However, alisertib didn’t suppress ERα phosphorylation on these residues, and combination treatment even elevated the levels of p-ERα(S167) and p-ERα(S305). These significant differences between BT474 and T47D tumor cell lines may potentially result from the fact that

BT474 is a tamoxifen-resistant cell line, while T47D is sensitive to tamoxifen. In BT474 cells, pathway of ligand-independent activation of ER may be very active, while ligand- dependent activation of ER may be less critical for cell survival. In this circumstance, inhibition of PAK1 and AURKA may result in significant suppression of ligand- independent phosphorylation of ERα. T47D cells are not tamoxifen-resistant and possibly rely much more on ligand-dependent ERα signaling with only some contribution of ligand-independent activation of ER. When ligand-independent signaling is suppressed, cells continue using the main pathway of ligand-dependent ER activation, and significant amount of phosphorylated ERα may be present as a result of normal signaling through

E2. All mice in the xenograft experiment were supplemented with estrogen pellets and 41 therefore had a substantial level of estradiol in the blood, making this hypothesis feasible.

Another possible explanation is that alisertib might upregulate expression of ERα in some cell lines, and some protein kinases that can also phosphorylate S167 and S305 residues of ERα. To further investigate the suppression of cancer cell growth by FRAX1036 and alisertib, we might generate BT474 and T47D cells that express ERα with phosphomimetic mutation at S167 or S305, or utilize CRISPR to deplete ERα in both cell lines, and perform cell viability assay to explore the influence of combination treatment when ERα is constitutively active or absent in breast cancer cells.

AKT activity supports cell survival and prevents apoptosis. AKT activation can be modulated by PAK1, AURKA, and ERα. PAK1 serves as a scaffold protein to facilitate interaction between PDK1 and AKT and thus promotes activation of PI3K/AKT pathway

(Higuchi, Onishi et al. 2008). AKT can phosphorylate PAK1 and thus enhances its activity in cell migration (Zhou, Zhuo et al. 2003). AURKA promotes phosphorylation of

AKT, which activates MDM2-mediated degradation of p53 and prevents cell cycle arrest and apoptosis (Yang, He et al. 2006). ERα promotes PI3K/AKT activation through non- genomic signaling, and AKT, in turn, upregulates ERα activity by phosphorylating S167

(Campbell, Bhat-Nakshatri et al. 2001, Arpino, Wiechmann et al. 2008). Western Blot analysis demonstrated a striking effect of FRAX1036 or/and alisertib on inhibition of both AKT protein level and activation in BT474 xenograft models (Fig. 6C). However, such inhibition only moderately influenced the level of phosphorylated AKT in T47D orthotopic tumors. Results of cleaved caspase-3 staining in both xenograft tumors (Fig.

4C, E) indicate that the level of apoptosis is correlated with suppression of AKT activity, 42 which also reflects that BT474 is more sensitive to AKT inhibition indirectly by alisertib and FRAX1036. This might be explained by different molecular phenotypes between two cell lines (Table 2). Breast cancer cells with HER2 amplification or PIK3CA mutation

(PI3K-encoding gene) are more dependent on AKT signaling (She, Chandarlapaty et al.

2008). Oncogenic mutation of PIK3CA is present in both BT474 and T47D cell lines

(Hollestelle, Elstrodt et al. 2007), and BT474 is HER2-positive. Therefore, HER2 amplification and PIK3CA mutation in BT474 cell lines might synergistically result in higher sensitivity to AKT suppression by FRAX1036 and alisertib.

Phosphorylation at S167 and S305 residues is associated with elevated ERα transactivation activity. Therefore, further investigation should focus on the influence of

AURKA and PAK1 inhibition on transcription of ER-targeted genes, such as VEGF and

PS2. CCND1 might be another ER-regulated gene that we should take into consideration.

CCND1 is an oncogene that encodes cyclin D1, a key regulator of G1 progression in cell cycle. Activation of PAK1, AURKA, and ERα can enhance expression of CCND1. Thus,

CCND1 might be downregulated upon treatment of alisertib or/and FRAX1036, which contributes to suppression of proliferation in HR-positive breast cancer.

Further, signaling molecules in MAPK pathway might be involved in the PAK1-

AURKA-ERα network by activating ERα, and perhaps conferring tamoxifen resistance.

PAK1 can activate ERK by phosphorylation of c-RAF at S338 and MEK1 at S298, or by its function as a scaffold protein to promote interaction between RAF and ERK (Radu,

Semenova et al. 2014). Non-genomic effect of ERα triggers activation of 43

RAF/MEK/MAPK pathway, and MAPK, in turn, modulates ERα activity by ligand- independent phosphorylation at S104, S106 and S118 (Kato, Endoh et al. 1995, Thomas,

Sarwar et al. 2008). AURKA enhances MEK/ERK pathway in colorectal cancer, and

AURKA expression is induced upon MAPK activation in pancreatic cancer, suggesting a possible positive feedback loop between AURKA and MAPK (Katsha, Belkhiri et al.

2015). Interestingly, breast cancer cells with HER2 overexpression become more dependent on ERK pathway upon PI3K/AKT/mTOR suppression, and compensatory activation of ERK pathway leads to enhanced HER2 activation. AKT inhibitors in combination with anti-HER2 monoclonal antibody trastuzumab or MEK1/2 inhibitors eradicate the compensatory activation of MAPK pathway (Serra, Scaltriti et al. 2011).

Since PAK1, AURKA, and ERα regulate activation of both PI3K/AKT and MAPK pathways, the balance between both pathways in HR-positive breast cancer would influence the antitumor effect of alisertib and FRAX1036. Future experiments should explore the long term effect of combination treatment. Our study has shown that combination of FRAX1036 and alisertib may be the most beneficial in HR-positive /

HER2-positive breast cancer. The next logical step is to combine alisertib and

FRAX1036 with HER2 targeted agents (herceptin and pertuzumab) to prevent drug resistance and tumor progression due to upregulation of HER2 signaling.

LIST OF REFERENCES

Applanat, M. P., H. Buteau-Lozano, M. A. Herve and A. Corpet (2008). "Vascular endothelial growth factor is a target gene for estrogen receptor and contributes to breast cancer progression." Adv Exp Med Biol 617: 437-444.

Arias-Romero, L. E. and J. Chernoff (2008). "A tale of two Paks." Biol Cell 100(2): 97-108.

Arias-Romero, L. E., O. Villamar-Cruz, A. Pacheco, R. Kosoff, M. Huang, S. K. Muthuswamy and J. Chernoff (2010). "A Rac-Pak signaling pathway is essential for ErbB2-mediated transformation of human breast epithelial cancer cells." Oncogene 29(43): 5839-5849.

Arpino, G., L. Wiechmann, C. K. Osborne and R. Schiff (2008). "Crosstalk between the Estrogen Receptor and the HER Tyrosine Kinase Receptor Family: Molecular Mechanism and Clinical Implications for Endocrine Therapy Resistance." Endocrine Reviews 29(2): 217-233.

Balasenthil, S., C. J. Barnes, S. K. Rayala and R. Kumar (2004). "Estrogen receptor activation at serine 305 is sufficient to upregulate cyclin D1 in breast cancer cells." FEBS Lett 567(2-3): 243-247.

Balasenthil, S., A. A. Sahin, C. J. Barnes, R. A. Wang, R. G. Pestell, R. K. Vadlamudi and R. Kumar (2004). "p21-activated kinase-1 signaling mediates cyclin D1 expression in mammary epithelial and cancer cells." J Biol Chem 279(2): 1422-1428.

Barletta, F., C. W. Wong, C. McNally, B. S. Komm, B. Katzenellenbogen and B. J. Cheskis (2004). "Characterization of the interactions of estrogen receptor and MNAR in the activation of cSrc." Mol Endocrinol 18(5): 1096-1108.

Bostner, J., M. Ahnstrom Waltersson, T. Fornander, L. Skoog, B. Nordenskjold and O. Stal (2007). "Amplification of CCND1 and PAK1 as predictors of recurrence and tamoxifen resistance in postmenopausal breast cancer." Oncogene 26(49): 6997-7005.

Campbell, R. A., P. Bhat-Nakshatri, N. M. Patel, D. Constantinidou, S. Ali and H. Nakshatri (2001). "Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for antiestrogen resistance." J Biol Chem 276(13): 9817-9824.

Carmena, M. and W. C. Earnshaw (2003). "The cellular geography of aurora kinases." Nat Rev Mol Cell Biol 4(11): 842-854.

Carroll, J. S., C. A. Meyer, J. Song, W. Li, T. R. Geistlinger, J. Eeckhoute, A. S. Brodsky, E. K. Keeton, K. C. Fertuck, G. F. Hall, Q. Wang, S. Bekiranov, V. Sementchenko, E. A. Fox, P. A. Silver, T. R. Gingeras, X. S. Liu and M. Brown (2006). "Genome-wide analysis of estrogen receptor binding sites." Nat Genet 38(11): 1289-1297.

Cervantes, A., E. Elez, D. Roda, J. Ecsedy, T. Macarulla, K. Venkatakrishnan, S. Rosello, J. Andreu, J. Jung, J. M. Sanchis-Garcia, A. Piera, I. Blasco, L. Manos, J. A. Perez-Fidalgo, H. Fingert, J. Baselga and J. Tabernero (2012). "Phase I pharmacokinetic/pharmacodynamic study of MLN8237, an investigational, oral, selective aurora a kinase inhibitor, in patients with advanced solid tumors." Clin Cancer Res 18(17): 4764- 4774.

Chen, D., P. E. Pace, R. C. Coombes and S. Ali (1999). "Phosphorylation of human estrogen receptor alpha by protein kinase A regulates dimerization." Mol Cell Biol 19(2): 1002-1015. 45

Chou, T. C. and P. Talalay (1984). "Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors." Adv Enzyme Regul 22: 27-55.

Chow, H. Y., B. Dong, S. G. Duron, D. A. Campbell, C. C. Ong, K. P. Hoeflich, L. S. Chang, D. B. Welling, Z. J. Yang and J. Chernoff (2015). "Group I Paks as therapeutic targets in NF2-deficient meningioma." Oncotarget 6(4): 1981-1994.

Chow, H. Y., A. M. Jubb, J. N. Koch, Z. M. Jaffer, D. Stepanova, D. A. Campbell, S. G. Duron, M. O'Farrell, K. Q. Cai, A. J. Klein-Szanto, J. S. Gutkind, K. P. Hoeflich and J. Chernoff (2012). "p21- Activated kinase 1 is required for efficient tumor formation and progression in a Ras-mediated skin cancer model." Cancer Res 72(22): 5966-5975.

Creighton, C. J., X. Li, M. Landis, J. M. Dixon, V. M. Neumeister, A. Sjolund, D. L. Rimm, H. Wong, A. Rodriguez, J. I. Herschkowitz, C. Fan, X. Zhang, X. He, A. Pavlick, M. C. Gutierrez, L. Renshaw, A. A. Larionov, D. Faratian, S. G. Hilsenbeck, C. M. Perou, M. T. Lewis, J. M. Rosen and J. C. Chang (2009). "Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features." Proc Natl Acad Sci U S A 106(33): 13820-13825.

Crosio, C., G. M. Fimia, R. Loury, M. Kimura, Y. Okano, H. Zhou, S. Sen, C. D. Allis and P. Sassone- Corsi (2002). "Mitotic phosphorylation of histone H3: spatio-temporal regulation by mammalian Aurora kinases." Mol Cell Biol 22(3): 874-885.

D'Assoro, A. B., T. Liu, C. Quatraro, A. Amato, M. Opyrchal, A. Leontovich, Y. Ikeda, S. Ohmine, W. Lingle, V. Suman, J. Ecsedy, I. Iankov, A. Di Leonardo, J. Ayers-Inglers, A. Degnim, D. Billadeau, J. McCubrey, J. Ingle, J. L. Salisbury and E. Galanis (2014). "The mitotic kinase Aurora--a promotes distant metastases by inducing epithelial-to-mesenchymal transition in ERalpha(+) breast cancer cells." Oncogene 33(5): 599-610.

Deroo, B. J. and K. S. Korach (2006). "Estrogen receptors and human disease." J Clin Invest 116(3): 561- 570.

DeRose, Y. S., K. M. Gligorich, G. Wang, A. Georgelas, P. Bowman, S. J. Courdy, A. L. Welm and B. E. Welm (2013). "Patient-derived models of human breast cancer: protocols for in vitro and in vivo applications in tumor biology and translational medicine." Curr Protoc Pharmacol Chapter 14: Unit14 23.

Dong, L., W. Wang, F. Wang, M. Stoner, J. C. Reed, M. Harigai, I. Samudio, M. P. Kladde, C. Vyhlidal and S. Safe (1999). "Mechanisms of transcriptional activation of bcl-2 gene expression by 17beta-estradiol in breast cancer cells." J Biol Chem 274(45): 32099-32107.

Edwards, D. C., L. C. Sanders, G. M. Bokoch and G. N. Gill (1999). "Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics." Nat Cell Biol 1(5): 253-259.

Enmark, E., M. Pelto-Huikko, K. Grandien, S. Lagercrantz, J. Lagercrantz, G. Fried, M. Nordenskjold and J. A. Gustafsson (1997). "Human estrogen receptor beta-gene structure, chromosomal localization, and expression pattern." J Clin Endocrinol Metab 82(12): 4258-4265.

Eyers, P. A., E. Erikson, L. G. Chen and J. L. Maller (2003). "A novel mechanism for activation of the protein kinase Aurora A." Curr Biol 13(8): 691-697.

Ferlay, J., I. Soerjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, D. M. Parkin, D. Forman and F. Bray (2015). "Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012." Int J Cancer 136(5): E359-386. 46

Gatti, A., Z. Huang, P. T. Tuazon and J. A. Traugh (1999). "Multisite autophosphorylation of p21-activated protein kinase gamma-PAK as a function of activation." J Biol Chem 274(12): 8022-8028.

Giancotti, F. G. (2013). "Mechanisms governing metastatic dormancy and reactivation." Cell 155(4): 750- 764.

Glidewell-Kenney, C., J. Weiss, E. J. Lee, S. Pillai, T. Ishikawa, E. A. Ariazi and J. L. Jameson (2005). "ERE-independent ERalpha target genes differentially expressed in human breast tumors." Mol Cell Endocrinol 245(1-2): 53-59.

Hammond, M. E., D. F. Hayes, M. Dowsett, D. C. Allred, K. L. Hagerty, S. Badve, P. L. Fitzgibbons, G. Francis, N. S. Goldstein, M. Hayes, D. G. Hicks, S. Lester, R. Love, P. B. Mangu, L. McShane, K. Miller, C. K. Osborne, S. Paik, J. Perlmutter, A. Rhodes, H. Sasano, J. N. Schwartz, F. C. Sweep, S. Taube, E. E. Torlakovic, P. Valenstein, G. Viale, D. Visscher, T. Wheeler, R. B. Williams, J. L. Wittliff, A. C. Wolff, O. American Society of Clinical and P. College of American (2010). "American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version)." Arch Pathol Lab Med 134(7): e48-72.

Higuchi, M., K. Onishi, C. Kikuchi and Y. Gotoh (2008). "Scaffolding function of PAK in the PDK1-Akt pathway." Nat Cell Biol 10(11): 1356-1364.

Hirokawa, Y., M. Arnold, H. Nakajima, J. Zalcberg and H. Maruta (2005). "Signal therapy of breast cancers by the HDAC inhibitor FK228 that blocks the activation of PAK1 and abrogates the tamoxifen- resistance." Cancer Biol Ther 4(9): 956-960.

Hollestelle, A., F. Elstrodt, J. H. Nagel, W. W. Kallemeijn and M. Schutte (2007). "Phosphatidylinositol-3- OH kinase or RAS pathway mutations in human breast cancer cell lines." Mol Cancer Res 5(2): 195-201.

Holm, C., S. Rayala, K. Jirstrom, O. Stal, R. Kumar and G. Landberg (2006). "Association between Pak1 expression and subcellular localization and tamoxifen resistance in breast cancer patients." J Natl Cancer Inst 98(10): 671-680.

Horn, V., J. Thelu, A. Garcia, C. Albiges-Rizo, M. R. Block and J. Viallet (2007). "Functional interaction of Aurora-A and PP2A during mitosis." Mol Biol Cell 18(4): 1233-1241.

Howlader, N., S. F. Altekruse, C. I. Li, V. W. Chen, C. A. Clarke, L. A. Ries and K. A. Cronin (2014). "US incidence of breast cancer subtypes defined by joint hormone receptor and HER2 status." J Natl Cancer Inst 106(5).

Howlader, N., A. M. Noone, M. M. Krapcho, D., K. Bishop, S. F. Altekruse, C. L. Kosary, M. Yu, J. Ruhl, Z. Tatalovich, A. Mariotto, D. R. Lewis, H. S. Chen, E. J. Feuer and K. A. Cronin. (2016, April 2016). "SEER Cancer Statistics Review, 1975-2013." from http://seer.cancer.gov/csr/1975_2013/.

Huang, B., Y. Omoto, H. Iwase, H. Yamashita, T. Toyama, R. C. Coombes, A. Filipovic, M. Warner and J. A. Gustafsson (2014). "Differential expression of estrogen receptor alpha, beta1, and beta2 in lobular and ductal breast cancer." Proc Natl Acad Sci U S A 111(5): 1933-1938.

Hutterer, A., D. Berdnik, F. Wirtz-Peitz, M. Zigman, A. Schleiffer and J. A. Knoblich (2006). "Mitotic activation of the kinase Aurora-A requires its binding partner Bora." Dev Cell 11(2): 147-157.

Jiang, S., H. Katayama, J. Wang, S. A. Li, Y. Hong, L. Radvanyi, J. J. Li and S. Sen (2010). "Estrogen- induced aurora kinase-A (AURKA) gene expression is activated by GATA-3 in estrogen receptor-positive breast cancer cells." Horm Cancer 1(1): 11-20. 47

Joel, P. B., J. Smith, T. W. Sturgill, T. L. Fisher, J. Blenis and D. A. Lannigan (1998). "pp90rsk1 regulates estrogen receptor-mediated transcription through phosphorylation of Ser-167." Mol Cell Biol 18(4): 1978- 1984.

Kato, S., H. Endoh, Y. Masuhiro, T. Kitamoto, S. Uchiyama, H. Sasaki, S. Masushige, Y. Gotoh, E. Nishida, H. Kawashima, D. Metzger and P. Chambon (1995). "Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase." Science 270(5241): 1491-1494.

Katsha, A., A. Belkhiri, L. Goff and W. El-Rifai (2015). "Aurora kinase A in gastrointestinal cancers: time to target." Mol Cancer 14: 106.

Kok, M., W. Zwart, C. Holm, R. Fles, M. Hauptmann, L. J. Van't Veer, L. F. Wessels, J. Neefjes, O. Stal, S. C. Linn, G. Landberg and R. Michalides (2011). "PKA-induced phosphorylation of ERalpha at serine 305 and high PAK1 levels is associated with sensitivity to tamoxifen in ER-positive breast cancer." Breast Cancer Res Treat 125(1): 1-12.

Kufer, T. A., H. H. Sillje, R. Korner, O. J. Gruss, P. Meraldi and E. A. Nigg (2002). "Human TPX2 is required for targeting Aurora-A kinase to the spindle." J Cell Biol 158(4): 617-623.

Kwon, Y. S., I. Garcia-Bassets, K. R. Hutt, C. S. Cheng, M. Jin, D. Liu, C. Benner, D. Wang, Z. Ye, M. Bibikova, J. B. Fan, L. Duan, C. K. Glass, M. G. Rosenfeld and X. D. Fu (2007). "Sensitive ChIP-DSL technology reveals an extensive estrogen receptor alpha-binding program on human gene promoters." Proc Natl Acad Sci U S A 104(12): 4852-4857.

Lapidus, R. G., S. J. Nass and N. E. Davidson (1998). "The loss of estrogen and progesterone receptor gene expression in human breast cancer." J Mammary Gland Biol Neoplasia 3(1): 85-94.

Lei, M., W. Lu, W. Meng, M. C. Parrini, M. J. Eck, B. J. Mayer and S. C. Harrison (2000). "Structure of PAK1 in an autoinhibited conformation reveals a multistage activation switch." Cell 102(3): 387-397.

Li, F., L. Adam, R. K. Vadlamudi, H. Zhou, S. Sen, J. Chernoff, M. Mandal and R. Kumar (2002). "p21- activated kinase 1 interacts with and phosphorylates histone H3 in breast cancer cells." EMBO Rep 3(8): 767-773.

Li, L., M. P. Haynes and J. R. Bender (2003). "Plasma membrane localization and function of the estrogen receptor alpha variant (ER46) in human endothelial cells." Proc Natl Acad Sci U S A 100(8): 4807-4812.

Lim, S. K. and G. Gopalan (2007). "Antizyme1 mediates AURKAIP1-dependent degradation of Aurora- A." Oncogene 26(46): 6593-6603.

Littlepage, L. E. and J. V. Ruderman (2002). "Identification of a new APC/C recognition domain, the A box, which is required for the Cdh1-dependent destruction of the kinase Aurora-A during mitotic exit." Genes Dev 16(17): 2274-2285.

Lundgren, K., K. Holm, B. Nordenskjold, A. Borg and G. Landberg (2008). "Gene products of chromosome 11q and their association with CCND1 gene amplification and tamoxifen resistance in premenopausal breast cancer." Breast Cancer Res 10(5): R81.

Márquez, D. C., J. Lee, T. Lin and R. J. Pietras (2001). "Epidermal growth factor receptor and tyrosine phosphorylation of estrogen receptor." Endocrine 16(2): 73-81.

Maroto, B., M. B. Ye, K. von Lohneysen, A. Schnelzer and U. G. Knaus (2008). "P21-activated kinase is required for mitotic progression and regulates Plk1." Oncogene 27(36): 4900-4908. 48

Marquez, D. C., H. W. Chen, E. M. Curran, W. V. Welshons and R. J. Pietras (2006). "Estrogen receptors in membrane lipid rafts and signal transduction in breast cancer." Mol Cell Endocrinol 246(1-2): 91-100.

Marumoto, T., D. Zhang and H. Saya (2005). "Aurora-A - a guardian of poles." Nat Rev Cancer 5(1): 42- 50.

Melichar, B., A. Adenis, A. C. Lockhart, J. Bennouna, E. C. Dees, O. Kayaleh, R. Obermannova, A. DeMichele, P. Zatloukal, B. Zhang, C. D. Ullmann and C. Schusterbauer (2015). "Safety and activity of alisertib, an investigational aurora kinase A inhibitor, in patients with breast cancer, small-cell lung cancer, non-small-cell lung cancer, head and neck squamous-cell carcinoma, and gastro-oesophageal adenocarcinoma: a five-arm phase 2 study." Lancet Oncol 16(4): 395-405.

Menasce, L. P., G. R. White, C. J. Harrison and J. M. Boyle (1993). "Localization of the estrogen receptor locus (ESR) to chromosome 6q25.1 by FISH and a simple post-FISH banding technique." Genomics 17(1): 263-265.

Michalides, R., A. Griekspoor, A. Balkenende, D. Verwoerd, L. Janssen, K. Jalink, A. Floore, A. Velds, L. van't Veer and J. Neefjes (2004). "Tamoxifen resistance by a conformational arrest of the estrogen receptor alpha after PKA activation in breast cancer." Cancer Cell 5(6): 597-605.

Murphy, L. C., S. V. Seekallu and P. H. Watson (2011). "Clinical significance of estrogen receptor phosphorylation." Endocr Relat Cancer 18(1): R1-14.

Nadler, Y., R. L. Camp, C. Schwartz, D. L. Rimm, H. M. Kluger and Y. Kluger (2008). "Expression of Aurora A (but not Aurora B) is predictive of survival in breast cancer." Clin Cancer Res 14(14): 4455-4462.

Nardone, A., C. De Angelis, M. V. Trivedi, C. K. Osborne and R. Schiff (2015). "The changing role of ER in endocrine resistance." Breast 24 Suppl 2: S60-66.

Neve, R. M., K. Chin, J. Fridlyand, J. Yeh, F. L. Baehner, T. Fevr, L. Clark, N. Bayani, J. P. Coppe, F. Tong, T. Speed, P. T. Spellman, S. DeVries, A. Lapuk, N. J. Wang, W. L. Kuo, J. L. Stilwell, D. Pinkel, D. G. Albertson, F. M. Waldman, F. McCormick, R. B. Dickson, M. D. Johnson, M. Lippman, S. Ethier, A. Gazdar and J. W. Gray (2006). "A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes." Cancer Cell 10(6): 515-527.

Nikonova, A. S., I. Astsaturov, I. G. Serebriiskii, R. L. Dunbrack, Jr. and E. A. Golemis (2013). "Aurora A kinase (AURKA) in normal and pathological cell division." Cell Mol Life Sci 70(4): 661-687.

Nowakowski, J., C. N. Cronin, D. E. McRee, M. W. Knuth, C. G. Nelson, N. P. Pavletich, J. Rogers, B. C. Sang, D. N. Scheibe, R. V. Swanson and D. A. Thompson (2002). "Structures of the cancer-related Aurora- A, FAK, and EphA2 protein kinases from nanovolume crystallography." Structure 10(12): 1659-1667.

Ong, C. C., S. Gierke, C. Pitt, M. Sagolla, C. K. Cheng, W. Zhou, A. M. Jubb, L. Strickland, M. Schmidt, S. G. Duron, D. A. Campbell, W. Zheng, S. Dehdashti, M. Shen, N. Yang, M. L. Behnke, W. Huang, J. C. McKew, J. Chernoff, W. F. Forrest, P. M. Haverty, S. F. Chin, E. A. Rakha, A. R. Green, I. O. Ellis, C. Caldas, T. O'Brien, L. S. Friedman, H. Koeppen, J. Rudolph and K. P. Hoeflich (2015). "Small molecule inhibition of group I p21-activated kinases in breast cancer induces apoptosis and potentiates the activity of microtubule stabilizing agents." Breast Cancer Res 17: 59.

Osborne, C. K., R. Schiff, S. A. Fuqua and J. Shou (2001). "Estrogen receptor: current understanding of its activation and modulation." Clin Cancer Res 7(12 Suppl): 4338s-4342s; discussion 4411s-4412s. 49

Paech, K., P. Webb, G. G. Kuiper, S. Nilsson, J. Gustafsson, P. J. Kushner and T. S. Scanlan (1997). "Differential ligand activation of estrogen receptors ERalpha and ERbeta at AP1 sites." Science 277(5331): 1508-1510.

Pedram, A., M. Razandi, R. C. Sainson, J. K. Kim, C. C. Hughes and E. R. Levin (2007). "A conserved mechanism for steroid receptor translocation to the plasma membrane." J Biol Chem 282(31): 22278-22288.

Pirruccello, M., H. Sondermann, J. G. Pelton, P. Pellicena, A. Hoelz, J. Chernoff, D. E. Wemmer and J. Kuriyan (2006). "A dimeric kinase assembly underlying autophosphorylation in the p21 activated kinases." J Mol Biol 361(2): 312-326.

Piva, M., G. Domenici, O. Iriondo, M. Rabano, B. M. Simoes, V. Comaills, I. Barredo, J. A. Lopez-Ruiz, I. Zabalza, R. Kypta and M. Vivanco (2014). "Sox2 promotes tamoxifen resistance in breast cancer cells." EMBO Mol Med 6(1): 66-79.

Porter, W., F. Wang, W. Wang, R. Duan and S. Safe (1996). "Role of estrogen receptor/Sp1 complexes in estrogen-induced heat shock protein 27 gene expression." Mol Endocrinol 10(11): 1371-1378.

Prudnikova, T. Y. and J. Chernoff (2016). "The Group I Pak inhibitor Frax-1036 sensitizes 11q13- amplified ovarian cancer cells to the cytotoxic effects of Rottlerin." Small GTPases: 1-6.

Pugacheva, E. N. and E. A. Golemis (2005). "The focal adhesion scaffolding protein HEF1 regulates activation of the Aurora-A and Nek2 kinases at the centrosome." Nat Cell Biol 7(10): 937-946.

Radu, M., G. Semenova, R. Kosoff and J. Chernoff (2014). "PAK signalling during the development and progression of cancer." Nat Rev Cancer 14(1): 13-25.

Rayala, S. K., A. H. Talukder, S. Balasenthil, R. Tharakan, C. J. Barnes, R. A. Wang, C. M. Aldaz, S. Khan and R. Kumar (2006). "P21-activated kinase 1 regulation of estrogen receptor-alpha activation involves serine 305 activation linked with serine 118 phosphorylation." Cancer Res 66(3): 1694-1701.

Razandi, M., A. Pedram and E. R. Levin (2000). "Plasma membrane estrogen receptors signal to antiapoptosis in breast cancer." Mol Endocrinol 14(9): 1434-1447.

Razandi, M., A. Pedram, S. T. Park and E. R. Levin (2003). "Proximal events in signaling by plasma membrane estrogen receptors." J Biol Chem 278(4): 2701-2712.

Ring, A. and M. Dowsett (2004). "Mechanisms of tamoxifen resistance." Endocr Relat Cancer 11(4): 643- 658.

Robinson, D. R., Y. M. Wu, P. Vats, F. Su, R. J. Lonigro, X. Cao, S. Kalyana-Sundaram, R. Wang, Y. Ning, L. Hodges, A. Gursky, J. Siddiqui, S. A. Tomlins, S. Roychowdhury, K. J. Pienta, S. Y. Kim, J. S. Roberts, J. M. Rae, C. H. Van Poznak, D. F. Hayes, R. Chugh, L. P. Kunju, M. Talpaz, A. F. Schott and A. M. Chinnaiyan (2013). "Activating ESR1 mutations in hormone-resistant metastatic breast cancer." Nat Genet 45(12): 1446-1451.

Rusnak, D. W., K. Lackey, K. Affleck, E. R. Wood, K. J. Alligood, N. Rhodes, B. R. Keith, D. M. Murray, W. B. Knight, R. J. Mullin and T. M. Gilmer (2001). "The effects of the novel, reversible epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor, GW2016, on the growth of human normal and tumor- derived cell lines in vitro and in vivo." Mol Cancer Ther 1(2): 85-94.

Ryerson, A. B., C. R. Eheman, S. F. Altekruse, J. W. Ward, A. Jemal, R. L. Sherman, S. J. Henley, D. Holtzman, A. Lake, A. M. Noone, R. N. Anderson, J. Ma, K. N. Ly, K. A. Cronin, L. Penberthy and B. A. 50

Kohler (2016). "Annual Report to the Nation on the Status of Cancer, 1975-2012, featuring the increasing incidence of liver cancer." Cancer 122(9): 1312-1337.

Sanders, L. C., F. Matsumura, G. M. Bokoch and P. de Lanerolle (1999). "Inhibition of myosin light chain kinase by p21-activated kinase." Science 283(5410): 2083-2085.

Schraml, P., G. Schwerdtfeger, F. Burkhalter, A. Raggi, D. Schmidt, T. Ruffalo, W. King, K. Wilber, M. J. Mihatsch and H. Moch (2003). "Combined array comparative genomic hybridization and tissue microarray analysis suggest PAK1 at 11q13.5-q14 as a critical oncogene target in ovarian carcinoma." Am J Pathol 163(3): 985-992.

Schurmann, A., A. F. Mooney, L. C. Sanders, M. A. Sells, H. G. Wang, J. C. Reed and G. M. Bokoch (2000). "p21-activated kinase 1 phosphorylates the death agonist bad and protects cells from apoptosis." Mol Cell Biol 20(2): 453-461.

Seki, A., J. A. Coppinger, C. Y. Jang, J. R. Yates and G. Fang (2008). "Bora and the kinase Aurora a cooperatively activate the kinase Plk1 and control mitotic entry." Science 320(5883): 1655-1658.

Serra, V., M. Scaltriti, L. Prudkin, P. J. Eichhorn, Y. H. Ibrahim, S. Chandarlapaty, B. Markman, O. Rodriguez, M. Guzman, S. Rodriguez, M. Gili, M. Russillo, J. L. Parra, S. Singh, J. Arribas, N. Rosen and J. Baselga (2011). "PI3K inhibition results in enhanced HER signaling and acquired ERK dependency in HER2-overexpressing breast cancer." Oncogene 30(22): 2547-2557.

Shagisultanova, E., V. Korobeynikov, A. Nikonova, J. Chernoff, V. Borges and E. Golemis (2016). "Abstract 1242: Combination of p21-activated kinase 1 (PAK1) inhibitor FRAX1036 and Aurora-A inhibitor alisertib is effective in hormone receptor-positive breast cancer." Cancer Research 76(14 Supplement): 1242-1242.

She, Q. B., S. Chandarlapaty, Q. Ye, J. Lobo, K. M. Haskell, K. R. Leander, D. DeFeo-Jones, H. E. Huber and N. Rosen (2008). "Breast tumor cells with PI3K mutation or HER2 amplification are selectively addicted to Akt signaling." PLoS One 3(8): e3065.

Skliris, G. P., Z. J. Nugent, B. G. Rowan, C. R. Penner, P. H. Watson and L. C. Murphy (2010). "A phosphorylation code for oestrogen receptor-alpha predicts clinical outcome to endocrine therapy in breast cancer." Endocr Relat Cancer 17(3): 589-597.

Song, R. X., C. J. Barnes, Z. Zhang, Y. Bao, R. Kumar and R. J. Santen (2004). "The role of Shc and insulin-like growth factor 1 receptor in mediating the translocation of estrogen receptor alpha to the plasma membrane." Proc Natl Acad Sci U S A 101(7): 2076-2081.

Stack, G., V. Kumar, S. Green, M. Ponglikitmongkol, M. Berry, M. C. Rio, A. M. Nunez, M. Roberts, C. Koehl, P. Bellocq and et al. (1988). "Structure and function of the pS2 gene and estrogen receptor in human breast cancer cells." Cancer Treat Res 40: 185-206.

Taguchi, S., K. Honda, K. Sugiura, A. Yamaguchi, K. Furukawa and T. Urano (2002). "Degradation of human Aurora-A protein kinase is mediated by hCdh1." FEBS Lett 519(1-3): 59-65.

Thomas, R. S., N. Sarwar, F. Phoenix, R. C. Coombes and S. Ali (2008). "Phosphorylation at serines 104 and 106 by Erk1/2 MAPK is important for estrogen receptor-alpha activity." J Mol Endocrinol 40(4): 173- 184.

Tran, N. H. and J. A. Frost (2003). "Phosphorylation of Raf-1 by p21-activated kinase 1 and Src regulates Raf-1 autoinhibition." J Biol Chem 278(13): 11221-11226. 51

Tsai, M. Y., C. Wiese, K. Cao, O. Martin, P. Donovan, J. Ruderman, C. Prigent and Y. Zheng (2003). "A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle assembly." Nat Cell Biol 5(3): 242-248.

Vadlamudi, R. K., F. Li, C. J. Barnes, R. Bagheri-Yarmand and R. Kumar (2004). "p41-Arc subunit of human Arp2/3 complex is a p21-activated kinase-1-interacting substrate." EMBO Rep 5(2): 154-160.

Wang, C., J. A. Mayer, A. Mazumdar, K. Fertuck, H. Kim, M. Brown and P. H. Brown (2011). "Estrogen induces c-myc gene expression via an upstream enhancer activated by the estrogen receptor and the AP-1 transcription factor." Mol Endocrinol 25(9): 1527-1538.

Wang, F., H. Li, X. G. Yan, Z. W. Zhou, Z. G. Yi, Z. X. He, S. T. Pan, Y. X. Yang, Z. Z. Wang, X. Zhang, T. Yang, J. X. Qiu and S. F. Zhou (2015). "Alisertib induces cell cycle arrest and autophagy and suppresses epithelial-to-mesenchymal transition involving PI3K/Akt/mTOR and sirtuin 1-mediated signaling pathways in human pancreatic cancer cells." Drug Des Devel Ther 9: 575-601.

Wang, R. A., A. Mazumdar, R. K. Vadlamudi and R. Kumar (2002). "P21-activated kinase-1 phosphorylates and transactivates estrogen receptor-alpha and promotes hyperplasia in mammary epithelium." EMBO J 21(20): 5437-5447.

Wang, R. A., H. Zhang, S. Balasenthil, D. Medina and R. Kumar (2006). "PAK1 hyperactivation is sufficient for mammary gland tumor formation." Oncogene 25(20): 2931-2936.

Wang, Z., X. Zhang, P. Shen, B. W. Loggie, Y. Chang and T. F. Deuel (2006). "A variant of estrogen receptor-{alpha}, hER-{alpha}36: transduction of estrogen- and antiestrogen-dependent membrane- initiated mitogenic signaling." Proc Natl Acad Sci U S A 103(24): 9063-9068.

Wertheimer, E., A. Gutierrez-Uzquiza, C. Rosemblit, C. Lopez-Haber, M. S. Sosa and M. G. Kazanietz (2012). "Rac signaling in breast cancer: a tale of GEFs and GAPs." Cell Signal 24(2): 353-362.

Wolff, A. C., M. E. Hammond, D. G. Hicks, M. Dowsett, L. M. McShane, K. H. Allison, D. C. Allred, J. M. Bartlett, M. Bilous, P. Fitzgibbons, W. Hanna, R. B. Jenkins, P. B. Mangu, S. Paik, E. A. Perez, M. F. Press, P. A. Spears, G. H. Vance, G. Viale, D. F. Hayes, O. American Society of Clinical and P. College of American (2014). "Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update." Arch Pathol Lab Med 138(2): 241-256.

Wu, W., S. Cheng, H. Deng, J. Wu, K. Mao and M. Cao (2016). "Impact of Breast Cancer Subtype Defined by Immunohistochemistry Hormone Receptor and HER2 Status on the Incidence of Immediate Postmastectomy Reconstruction." Medicine (Baltimore) 95(3): e2547.

Wyckoff, M. H., K. L. Chambliss, C. Mineo, I. S. Yuhanna, M. E. Mendelsohn, S. M. Mumby and P. W. Shaul (2001). "Plasma membrane estrogen receptors are coupled to endothelial nitric-oxide synthase through Galpha(i)." J Biol Chem 276(29): 27071-27076.

Yamashita, H., H. Iwase, T. Toyama and Y. Fujii (2003). "Naturally occurring dominant-negative Stat5 suppresses transcriptional activity of estrogen receptors and induces apoptosis in T47D breast cancer cells." Oncogene 22(11): 1638-1652.

Yang, H., L. He, P. Kruk, S. V. Nicosia and J. Q. Cheng (2006). "Aurora-A induces cell survival and chemoresistance by activation of Akt through a p53-dependent manner in ovarian cancer cells." Int J Cancer 119(10): 2304-2312. 52

Yang, N., O. Higuchi, K. Ohashi, K. Nagata, A. Wada, K. Kangawa, E. Nishida and K. Mizuno (1998). "Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization." Nature 393(6687): 809-812.

Ye, D. Z. and J. Field (2012). "PAK signaling in cancer." Cell Logist 2(2): 105-116.

Yu, X., K. Minter-Dykhouse, L. Malureanu, W.-M. Zhao, D. Zhang, C. J. Merkle, I. M. Ward, H. Saya, G. Fang, J. van Deursen and J. Chen (2005). "Chfr is required for tumor suppression and Aurora A regulation." Nat Genet 37(4): 401-406.

Zhao, Z. S., J. P. Lim, Y. W. Ng, L. Lim and E. Manser (2005). "The GIT-associated kinase PAK targets to the centrosome and regulates Aurora-A." Mol Cell 20(2): 237-249.

Zheng, X. Q., J. P. Guo, H. Yang, M. Kanai, L. L. He, Y. Y. Li, J. M. Koomen, S. Minton, M. Gao, X. B. Ren, D. Coppola and J. Q. Cheng (2014). "Aurora-A is a determinant of tamoxifen sensitivity through phosphorylation of ERα in breast cancer." Oncogene 33(42): 4985-4996.

Zhou, G. L., Y. Zhuo, C. C. King, B. H. Fryer, G. M. Bokoch and J. Field (2003). "Akt phosphorylation of serine 21 on Pak1 modulates Nck binding and cell migration." Mol Cell Biol 23(22): 8058-8069.

Zhou, H., J. Kuang, L. Zhong, W. L. Kuo, J. W. Gray, A. Sahin, B. R. Brinkley and S. Sen (1998). "Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation." Nat Genet 20(2): 189-193.