adjuvant therapies for node-negative breast cancer: predictive value of centrally reviewed expression of estrogen and progesterone receptors – International Breast

different association to clinicopathological parameters and their co-expression might predict a better response to endocrine treatment in breast cancer. Clin Pathol.

breast and prostate cancer cells: targeting the association of Src with steroid

and relationships with their receptors the bio-pathological features and prognosis.

disease of the vulva and the breast: results of a preliminary study. Arch Gynecol

race on response to primary systemic treatment and survival outcomes. J Clin

estrogen and progesterone receptor status - a meta-analysis.  Biochem Biophys Res Commun.

human (CG-5) breast cancer cells. Pharmacol Res. 1992; 26:377-384.

cancer treatment. Cancer Biol Ther. 2008; 7: 28-35.

p300 exchange. J Biol Chem. 2008; 283: 3433-3444.

Cancer Study Group. J Clin Oncol. 2008; 26: 1404-1410.

2000; 60:153-166.

2008; 61: 197-203.

receptors. Steroids. 2008; 73: 880-884.

Ann Oncol. 2008;19:73-80.

Obstet. 2008; 277: 135-138.

Oncol. 2009; 27:220-226.

2009; 378: 174-178.

698-712.

Treat. 2008; 110:153-159.

Breast cancer is the most frequent malignancy and one of the leading causes to cancer related deaths in women. Most human breast cancers express the estrogen receptor (ER) which belongs to the family of nuclear receptors and is a ligand-regulated transcription factor. It is well established that the natural ligand of ER, estrogen, has pro-carcinogenic and growth promoting effects in the mammary epithelium by stimulating proliferation and leaving the cells prone to mutations during cell cycle progression (Foster et al., 2001). Endocrine treatment of hormone sensitive breast cancer targets the estrogen activity in breast cancer cells by blocking the ER with a selective ER modulator (SERM) such as tamoxifen or inhibiting estrogen synthesis using aromatase inhibitors such as anastrozole or letrozole. Endocrine treatment decreases mortality, prolongs disease-free survival and can even reduce the incidence of breast cancer in women at increased risk (Cuzick et al., 2003). Approximately 70 % of women with ER positive tumors respond to endocrine therapy, but resistance do occur, either *de novo* or develop over time. The molecular mechanism involved in endocrine resistance is one of the central areas of breast cancer research.

The trancriptional activity of the ER is not only regulated by its ligands, but also by the level and activity of coregulator proteins. Nuclear receptor coactivators serve as adapters between the receptor and the trancriptional machinery. They possess diverse enzymatic acitivities such as histone acetyltransferase, histone methyltransferase, chromatin remodeling and ubiquitin-conjugation activity and are involved in every step of ER regulated transcription, from chromatin remodeling to transcriptional termination. The members of the p160 family of coactivators are some of the best studied coactivators. These steroid receptor coactivators (SRCs) are small proteins of 160 kDa with similar structural and functional properties, and include SRC-1, SRC-2/transcription intermediary factor-2 (TIF-2) and SRC-3/amplified in breast cancer 1 (AIB1). The SRCs are not only crucial to ER mediated effects in normal tissue. They have also been shown to be involved in the carcinogenic process and are

Steroid Receptor Coactivators and Their Expression, Regulation

**2.1 SRC-1**

**2.2 SRC-2/TIF-2**

**2.3 SRC-3/AIB1**

studied of the coactivators in breast cancer.

such as *c-MYC* and *cyclin D1* (Dubik & Shiu, 1992; Sabbah et al., 1999).

recruit macrophages to mammary tumors in mice (Wang et al., 2009).

and Functional Role in Endocrine Responsive and Resistant Breast Cancer 717

several types of human cancers, including breast cancer, where their overexpression is due to enhanced cellular amplification of their genes, and/or by a decrease in the intracellular degradation process of the coactivators (Xu et al., 2009). The SRCs are known to contribute to proliferation and development of breast cancers by mechanisms such as stimulation of the G1 to S phase transition during cell cycle by regulation of expression of mitogenic genes

SRC-1 was the first nuclear receptor coactivator to be cloned and identified (Onate et al., 1995). The gene is located on the short arm of chromosome 2 at position 2p23 (Carapeti et al., 1998). SRC-1-/- mice show partial steroid hormone resistance and hepatic insufficiency (Xu et al., 1998; Louet et al., 2010). Noteworthy, ovariectomized female SRC-1-/- mice have decreased uterine growth and reduced mammary gland ductal side branching and alveolar formation in response to estrogen compared with wild-type mice, indicating a role of SRC-1 in estrogen-regulated breast development (Xu et al., 1998; Xu & Li, 2003). We found higher SRC-1 mRNA levels by real-time RT-PCR in human breast cancer samples compared to normal breast tissue (Haugan Moi et al., 2010), and the same has been observed at the protein level (Hudelist et al., 2003; Fleming et al., 2004b; Myers et al., 2004). SRC-1 protein expression in breast cancer has been reported to associate positively with human epidermal growth factor receptor 2/neu (HER-2/neu) and negatively with ERβ (Fleming et al., 2004b). *In vitro* studies indicate that SRC-1 plays an important role in ER-mediated growth of breast cancer cells. Overexpression of SRC-1 potentiates E2-stimulated growth of MCF-7 breast cancer cells in accordance with an increase in the expression of estrogen-responsive genes (Tai et al., 2000). SRC-1 has also been reported to specifically promote breast cancer metastasis, possibly by promoting migration and invasion by enhancing PEA3 mediated transcriptional activation of Twist, a master regulator of metastasis (Qin et al., 2009). Furthermore, SRC-1 may promote metastasis in mammary tumors by facilitating Ets-2 mediated HER-2/neu expression and activating colony stimulating factor-1 expression to

The human SRC-2/TIF-2 gene is located on the long arm of chromosome 8 at position 8q21 (Kalkhoven et al., 1998).  Increased levels of SRC-2/TIF-2 mRNA have been observed in intraductal and invasive carcinoma compared to normal breast tissue (Kurebayashi et al., 2000; Haugan Moi et al., 2010). Knockdown of SRC-2/TIF-2 has been shown to inhibit growth of MCF-7 breast cancer cells due to decreased cell proliferation, increased apoptosis and reduced ER mediated transcriptional activity (Cavarretta et al., 2002; Karmakar et al., 2009). However, overall SRC-2/TIF-2 is the least

The third member of the SRC family, SRC-3/AIB1, is located on the long arm of chromosome 20 at position 20q12 in humans and was first identified as a gene amplified and

overexpressed in breast cancer. In addition, the SRCs are of relevance to the tissue-specific effects of tamoxifen and data suggest that they may be important to the cellular sensitivity to endocrine treatment. The SRCs and their expression, regulation and functional role during endocrine treatment in breast cancer *in vitro* and *in vivo* are the focus of this chapter.

## **2. Expression and functional role of SRCs in breast tissue**

The SRCs are genetically distinct, but have structural similarities with 40 - 55 % sequence homology (Xu & Li, 2003). Although the SRCs have similar functional properties, experimental evidence indicates different physiological functions for the SRCs, which in part can be explained by tissue-specific expression levels, different affinities for the various nuclear receptors and variations in post-translational modifications (Chauchereau et al., 2003; Fenne et al., 2008; Hoang et al., 2004; Wu et al., 2004; Xu et al., 2009). In the classical activation of ER-mediated gene transcription, the ligand estradiol (E2) binds to the ER and promotes binding of the ER-E2 complex to the estrogen receptor element (ERE) of the target gene promoter. Here they recruit coregulatory proteins to a multi-subunit complex for gene transcription (Mangelsdorf et al., 1995; Pearce & Jordan, 2004). The ER has an activation function-1 (AF-1) domain in the N-terminus whereas the centrally located DNA-binding domain (DBD) is responsible for specific binding of the ER to EREs on target genes. The dimerization domain contains the ligand binding domain (LBD) and the AF-2. The AF-1 contributes to the constitutive estrogen-independent activation by the receptor and is separated by a hinge region from the AF-2. The SRCs have a basic helix-loop-helix (bHLH) domain, a Per-Arnt-Sim (PAS) domain and a nuclear hormone interaction domain (NID) including three LXXLL-sequences where L is leucine and X any amino acid (Fig. 1).

Fig. 1. Schematic presentation of the functional domains of the ER and the SRCs.

Upon ligand binding, the helix 12 of the ER is positioned across the LBD, and together with helices 3-5 form a hydrophobic groove where the LXXLL motifs of coactivators can bind (Heery et al., 1997). The coactivators in the multi-subunit protein complex alter the chromatin structure and facilitate recruitment of the RNA polymerase II and the basal transcriptional machinery in a programmed cyclic manner to initiate transcription of the target gene (Metivier et al., 2003).

The SRC family members are widely expressed and are detected in tissues such as placenta, testis, pancreas, lung, kidney, liver and brain (Xu & O'Malley, 2002; Xu & Li, 2003). The expression levels of the three SRC proteins in normal human breast epithelial cells are variable, but usually very low (Xu et al., 2009). SRCs are known to be overexpressed in several types of human cancers, including breast cancer, where their overexpression is due to enhanced cellular amplification of their genes, and/or by a decrease in the intracellular degradation process of the coactivators (Xu et al., 2009). The SRCs are known to contribute to proliferation and development of breast cancers by mechanisms such as stimulation of the G1 to S phase transition during cell cycle by regulation of expression of mitogenic genes such as *c-MYC* and *cyclin D1* (Dubik & Shiu, 1992; Sabbah et al., 1999).

## **2.1 SRC-1**

716 Breast Cancer – Carcinogenesis, Cell Growth and Signaling Pathways

overexpressed in breast cancer. In addition, the SRCs are of relevance to the tissue-specific effects of tamoxifen and data suggest that they may be important to the cellular sensitivity to endocrine treatment. The SRCs and their expression, regulation and functional role during endocrine treatment in breast cancer *in vitro* and *in vivo* are the focus of this chapter.

The SRCs are genetically distinct, but have structural similarities with 40 - 55 % sequence homology (Xu & Li, 2003). Although the SRCs have similar functional properties, experimental evidence indicates different physiological functions for the SRCs, which in part can be explained by tissue-specific expression levels, different affinities for the various nuclear receptors and variations in post-translational modifications (Chauchereau et al., 2003; Fenne et al., 2008; Hoang et al., 2004; Wu et al., 2004; Xu et al., 2009). In the classical activation of ER-mediated gene transcription, the ligand estradiol (E2) binds to the ER and promotes binding of the ER-E2 complex to the estrogen receptor element (ERE) of the target gene promoter. Here they recruit coregulatory proteins to a multi-subunit complex for gene transcription (Mangelsdorf et al., 1995; Pearce & Jordan, 2004). The ER has an activation function-1 (AF-1) domain in the N-terminus whereas the centrally located DNA-binding domain (DBD) is responsible for specific binding of the ER to EREs on target genes. The dimerization domain contains the ligand binding domain (LBD) and the AF-2. The AF-1 contributes to the constitutive estrogen-independent activation by the receptor and is separated by a hinge region from the AF-2. The SRCs have a basic helix-loop-helix (bHLH) domain, a Per-Arnt-Sim (PAS) domain and a nuclear hormone interaction domain (NID)

including three LXXLL-sequences where L is leucine and X any amino acid (Fig. 1).

LXXLL

Fig. 1. Schematic presentation of the functional domains of the ER and the SRCs.

NID

target gene (Metivier et al., 2003).

bHLH PAS

**ER**

**SRC**

Upon ligand binding, the helix 12 of the ER is positioned across the LBD, and together with helices 3-5 form a hydrophobic groove where the LXXLL motifs of coactivators can bind (Heery et al., 1997). The coactivators in the multi-subunit protein complex alter the chromatin structure and facilitate recruitment of the RNA polymerase II and the basal transcriptional machinery in a programmed cyclic manner to initiate transcription of the

H2N AF-1 COOH DBD HINGE LBD AF-2

p300 and CBP interaction domain

The SRC family members are widely expressed and are detected in tissues such as placenta, testis, pancreas, lung, kidney, liver and brain (Xu & O'Malley, 2002; Xu & Li, 2003). The expression levels of the three SRC proteins in normal human breast epithelial cells are variable, but usually very low (Xu et al., 2009). SRCs are known to be overexpressed in

**2. Expression and functional role of SRCs in breast tissue**

SRC-1 was the first nuclear receptor coactivator to be cloned and identified (Onate et al., 1995). The gene is located on the short arm of chromosome 2 at position 2p23 (Carapeti et al., 1998). SRC-1-/- mice show partial steroid hormone resistance and hepatic insufficiency (Xu et al., 1998; Louet et al., 2010). Noteworthy, ovariectomized female SRC-1-/- mice have decreased uterine growth and reduced mammary gland ductal side branching and alveolar formation in response to estrogen compared with wild-type mice, indicating a role of SRC-1 in estrogen-regulated breast development (Xu et al., 1998; Xu & Li, 2003). We found higher SRC-1 mRNA levels by real-time RT-PCR in human breast cancer samples compared to normal breast tissue (Haugan Moi et al., 2010), and the same has been observed at the protein level (Hudelist et al., 2003; Fleming et al., 2004b; Myers et al., 2004). SRC-1 protein expression in breast cancer has been reported to associate positively with human epidermal growth factor receptor 2/neu (HER-2/neu) and negatively with ERβ (Fleming et al., 2004b). *In vitro* studies indicate that SRC-1 plays an important role in ER-mediated growth of breast cancer cells. Overexpression of SRC-1 potentiates E2-stimulated growth of MCF-7 breast cancer cells in accordance with an increase in the expression of estrogen-responsive genes (Tai et al., 2000). SRC-1 has also been reported to specifically promote breast cancer metastasis, possibly by promoting migration and invasion by enhancing PEA3 mediated transcriptional activation of Twist, a master regulator of metastasis (Qin et al., 2009). Furthermore, SRC-1 may promote metastasis in mammary tumors by facilitating Ets-2 mediated HER-2/neu expression and activating colony stimulating factor-1 expression to recruit macrophages to mammary tumors in mice (Wang et al., 2009).

## **2.2 SRC-2/TIF-2**

The human SRC-2/TIF-2 gene is located on the long arm of chromosome 8 at position 8q21 (Kalkhoven et al., 1998).  Increased levels of SRC-2/TIF-2 mRNA have been observed in intraductal and invasive carcinoma compared to normal breast tissue (Kurebayashi et al., 2000; Haugan Moi et al., 2010). Knockdown of SRC-2/TIF-2 has been shown to inhibit growth of MCF-7 breast cancer cells due to decreased cell proliferation, increased apoptosis and reduced ER mediated transcriptional activity (Cavarretta et al., 2002; Karmakar et al., 2009). However, overall SRC-2/TIF-2 is the least studied of the coactivators in breast cancer.

## **2.3 SRC-3/AIB1**

The third member of the SRC family, SRC-3/AIB1, is located on the long arm of chromosome 20 at position 20q12 in humans and was first identified as a gene amplified and

Steroid Receptor Coactivators and Their Expression, Regulation

and Functional Role in Endocrine Responsive and Resistant Breast Cancer 719

**3. SRCs and endocrine treatment in breast cancer: Molecular mechanisms**  The regulation of SRCs during endocrine treatment is especially interesting since the coactivators are directly involved in the molecular mechanisms underlying the antiestrogenic effects. The natural ligand of ER, estrogen, is converted from androgens by the enzyme aromatase. Endocrine treatment of ER positive breast cancer includes aromatase inhibitors or the SERM tamoxifen. Aromatase inhibitors block the synthesis of estrogens by binding to and suppressing the aromatase enzyme that converts androgens to estrogens. Tamoxifen binds to the ER and functions as an antagonist in breast tissue and prevents estrogen from binding to the ER. The net effect of both therapeutic regiments is to block ER-

dependent transcriptional regulation of genes and prevent proliferation (Fig. 2).

**Androgens**

Fig. 2. Schematic presentation of the mechanisms of action of endocrine treatment in breast

ERE ER target genes

**X**

ER ER

Tamoxifen is a synthetic estrogen antagonist which has been in clinical use for over 30 years. While the success of tamoxifen in breast cancer therapy is based on its ER antagonistic effects in malignant breast tissue, tamoxifen demonstrates ER agonistic effects in other organ systems such as bone and liver. ER appears to bind to corepressors in the presence of SERMs in breast tissue, while coactivator recruitment is favored when E2 is bound to ER. Upon binding to ER, SERMs inhibit ER transcriptional activity by competing with E2 for the binding site and by blocking the AF-2 activity of ER (Shiau et al., 1998; Brzozowski et al., 1997). The potent ER antagonistic metabolite 4-hydroxytamoxifen induces a displacement and rotation of the receptor's helix 12. The helix 12 then binds to the hydrophobic pocket via a sequence resembling the NR box of the coactivators, and thereby inhibits coactivator recruitment (Brzozowski et al., 1997; Shiau et al., 1998). The binding of 4-hydroxytamoxifen instead favors recruitment of the two corepressors silencing mediator for retinoid and thyroid hormone receptor (SMRT) and nuclear receptor corepressor (NCoR).   Female SRC-3/AIB1-/- mice have significantly lower levels of estrogen and delayed mammary gland development, indicating a proliferative role of this coactivator in breast tissue (Xu et al., 2000). In transgenic mice, overexpression of SRC-3/AIB1 leads to development of tumors in several organs including breast, in addition to increased expression of the insulin-like growth factor-1 (IGF-1) and activation of intracellular pathways suggesting that SRC-3/AIB1 is acting as an oncogene (Torres-Arzayus et al., 2004). The oncogenic potential of SRC-3/AIB1 has been ascribed to mechanisms such as enhanced interaction between ER and the *cyclin D1* promoter, hence leading to increased levels of cyclin D1 and stimulation of cell cycle progression (Planas-Silva et al., 2001). Conversely, cyclin D1 expression has been shown to be reduced in SRC-3/AIB1 knock-out cells (Karmakar et al., 2009), and mice with reduced SRC-3/AIB1 expression have a decrease in epithelial proliferation associated with a reduction in cyclin expression (Fereshteh et al., 2008). Overexpression of SRC-3/AIB1 also stimulates the Akt signaling pathway which promotes cell growth (Torres-Arzayus et al., 2004; Zhou et al., 2003). Matrix metalloproteinases (MMPs) are zink-dependent enzymes involved in the degradation of extracellular matrix and are essential to the metastatic process. Experimental evidence suggests that SRC-3/AIB1 promotes breast cancer metastasis by stimulating the transcription factor PEA3 to enhance expression of MMP2 and MMP9 (Qin et al., 2008).

#### **2.4 Regulation of SRCs expression during endocrine treatment**

Most studies seem to indicate higher levels of the SRCs in malignant breast tumors compared to normal breast tissue. However, the expression levels of the SRCs have been shown to change during endocrine treatment in breast cancer. In a clinical study of preoperative tamoxifen treatment for 4 weeks using tamoxifen doses from 1 to 20 mg/daily, we found the mRNA levels of all three SRCs to be significantly upregulated in tamoxifen treated normal and malignant breast tissue compared to samples from untreated patients. The increase in coactivator mRNA expression was especially evident for SRC-3/AIB1 (Haugan Moi et al., 2010). In a clinical study on neoadjuvant treatment with aromatase inhibitors in locally advanced breast cancer, we also found the mRNA levels of coactivators in tumors to increase during treatment, especially for SRC-1 (Flågeng et al., 2009). This is in line with *in vitro* studies. E2 has been shown to repress SRC-3/AIB1 mRNA and protein expression in MCF-7 human breast cancer cells primarily by suppressing SRC-3/AIB1 gene transcription (Lauritsen et al., 2002).  We also found an increase in the mRNA levels of HER-2/neu and a positive correlation between SRC-1 and HER-2/neu in human breast tissue treated with aromatase inhibitors. This finding is interesting in light of *in vitro* assays suggesting that ER and HER-2/neu compete for the coactivator SRC-1. Under antiestrogenic conditions, SRC-1 will be released from the ER and may instead bind to the HER-2/neu enhancer and facilitate transcription of HER-2/neu, leading to increased expression of HER-2/neu under estrogen deprived conditions (Newman et al., 2000).

## **3. SRCs and endocrine treatment in breast cancer: Molecular mechanisms**

The regulation of SRCs during endocrine treatment is especially interesting since the coactivators are directly involved in the molecular mechanisms underlying the antiestrogenic effects. The natural ligand of ER, estrogen, is converted from androgens by the enzyme aromatase. Endocrine treatment of ER positive breast cancer includes aromatase inhibitors or the SERM tamoxifen. Aromatase inhibitors block the synthesis of estrogens by binding to and suppressing the aromatase enzyme that converts androgens to estrogens. Tamoxifen binds to the ER and functions as an antagonist in breast tissue and prevents estrogen from binding to the ER. The net effect of both therapeutic regiments is to block ERdependent transcriptional regulation of genes and prevent proliferation (Fig. 2).

Fig. 2. Schematic presentation of the mechanisms of action of endocrine treatment in breast cancer using tamoxifen or aromatase inhibitors.

#### **3.1 The SERM tamoxifen**

718 Breast Cancer – Carcinogenesis, Cell Growth and Signaling Pathways

overexpressed in several human breast cancer cell lines (Anzick et al., 1997).  Female SRC-3/AIB1-/- mice have significantly lower levels of estrogen and delayed mammary gland development, indicating a proliferative role of this coactivator in breast tissue (Xu et al., 2000). In transgenic mice, overexpression of SRC-3/AIB1 leads to development of tumors in several organs including breast, in addition to increased expression of the insulin-like growth factor-1 (IGF-1) and activation of intracellular pathways suggesting that SRC-3/AIB1 is acting as an oncogene (Torres-Arzayus et al., 2004). The oncogenic potential of SRC-3/AIB1 has been ascribed to mechanisms such as enhanced interaction between ER and the *cyclin D1* promoter, hence leading to increased levels of cyclin D1 and stimulation of cell cycle progression (Planas-Silva et al., 2001). Conversely, cyclin D1 expression has been shown to be reduced in SRC-3/AIB1 knock-out cells (Karmakar et al., 2009), and mice with reduced SRC-3/AIB1 expression have a decrease in epithelial proliferation associated with a reduction in cyclin expression (Fereshteh et al., 2008). Overexpression of SRC-3/AIB1 also stimulates the Akt signaling pathway which promotes cell growth (Torres-Arzayus et al., 2004; Zhou et al., 2003). Matrix metalloproteinases (MMPs) are zink-dependent enzymes involved in the degradation of extracellular matrix and are essential to the metastatic process. Experimental evidence suggests that SRC-3/AIB1 promotes breast cancer metastasis by stimulating the transcription factor PEA3 to enhance expression of MMP2 and MMP9 (Qin et al., 2008).

**2.4 Regulation of SRCs expression during endocrine treatment**

et al., 2000).

Most studies seem to indicate higher levels of the SRCs in malignant breast tumors compared to normal breast tissue. However, the expression levels of the SRCs have been shown to change during endocrine treatment in breast cancer. In a clinical study of preoperative tamoxifen treatment for 4 weeks using tamoxifen doses from 1 to 20 mg/daily, we found the mRNA levels of all three SRCs to be significantly upregulated in tamoxifen treated normal and malignant breast tissue compared to samples from untreated patients. The increase in coactivator mRNA expression was especially evident for SRC-3/AIB1 (Haugan Moi et al., 2010). In a clinical study on neoadjuvant treatment with aromatase inhibitors in locally advanced breast cancer, we also found the mRNA levels of coactivators in tumors to increase during treatment, especially for SRC-1 (Flågeng et al., 2009). This is in line with *in vitro* studies. E2 has been shown to repress SRC-3/AIB1 mRNA and protein expression in MCF-7 human breast cancer cells primarily by suppressing SRC-3/AIB1 gene transcription (Lauritsen et al., 2002).  We also found an increase in the mRNA levels of HER-2/neu and a positive correlation between SRC-1 and HER-2/neu in human breast tissue treated with aromatase inhibitors. This finding is interesting in light of *in vitro* assays suggesting that ER and HER-2/neu compete for the coactivator SRC-1. Under antiestrogenic conditions, SRC-1 will be released from the ER and may instead bind to the HER-2/neu enhancer and facilitate transcription of HER-2/neu, leading to increased expression of HER-2/neu under estrogen deprived conditions (Newman

Tamoxifen is a synthetic estrogen antagonist which has been in clinical use for over 30 years. While the success of tamoxifen in breast cancer therapy is based on its ER antagonistic effects in malignant breast tissue, tamoxifen demonstrates ER agonistic effects in other organ systems such as bone and liver. ER appears to bind to corepressors in the presence of SERMs in breast tissue, while coactivator recruitment is favored when E2 is bound to ER. Upon binding to ER, SERMs inhibit ER transcriptional activity by competing with E2 for the binding site and by blocking the AF-2 activity of ER (Shiau et al., 1998; Brzozowski et al., 1997). The potent ER antagonistic metabolite 4-hydroxytamoxifen induces a displacement and rotation of the receptor's helix 12. The helix 12 then binds to the hydrophobic pocket via a sequence resembling the NR box of the coactivators, and thereby inhibits coactivator recruitment (Brzozowski et al., 1997; Shiau et al., 1998). The binding of 4-hydroxytamoxifen instead favors recruitment of the two corepressors silencing mediator for retinoid and thyroid hormone receptor (SMRT) and nuclear receptor corepressor (NCoR). These corepressors are associated with histone deacetylase activity and inhibit ER regulated gene transcription (Webb et al., 2003; Fleming et al., 2004a).

Steroid Receptor Coactivators and Their Expression, Regulation

Li & Shang, 2007; Xu et al., 2009).

and other transcription

factors

(Wu et al., 2005).

and Functional Role in Endocrine Responsive and Resistant Breast Cancer 721

variety of posttranslational modifications of SRCs, including acetylation, methylation, phosphorylation, ubiquitination and sumoylation. These modifications influence the SRCs transcriptional activity and/or the SRC protein levels and stability (Baek & Rosenfeld, 2004;

**Extracellular Signals**

Growth factors Hormones Cytokines

Acetylation Methylation Phosphorylation Sumoylation Ubiquitination

**Cellular signalling**

**SRC/p160**

Subcellular localization Interactions with NRs

Protein stability Chromatin structure

Phosphorylation of coactivators modulates ER-dependent gene transcription by regulating coactivator function in various ways. Three SRC-1 phosphorylation sites with corresponding kinases have been identified (S395, T1179 and S1185), one SRC-2/TIF-2 (S736) and sixteen SRC-3/AIB1 phosphorylation sites (T24, S505, S543, S601, S857, S860, S867, S1033, S1042, S1048, T1059, S1062, T1064, T1067, T1114 and Y1357) (Bulynko & O'Malley, 2011). Comparison of these sites reveals little conservation of sequences among the SRCs, indicating that phosphorylation is a significant determinant of the specificity of the SRCs

Phosphorylation may influence the function and acitivity of the SRCs. It is shown *in vitro* using COS-1 cells that positions S395 and T1179/S1185 of SRC-1 are phosphorylated by the MAPK family members ERK1 and ERK2 (Rowan et al., 2000b) where MAPK-mediated phosphorylation on T1179 and S1185 has been shown to increase the affinity of SRC-1 for androgen receptor (AR) in prostate cancer cells (Ueda et al., 2002; Gregory et al., 2004). ERK2 may also phosphorylate SRC-3/AIB1 *in vitro* which stimulates the recruitment of p300 and associated histone acetyltransferase activity (Font de Mora & Brown, 2000). cAMP regulated phosphorylation of SRC-1 occurs through an indirect pathway in which protein kinase A (PKA) induces the activity of ERK1 and ERK2 (Rowan et al., 2000a). SRC-3/AIB1 phosphorylation-defective mutants exhibit reduced ability to interact with ER compared to wild type SRC-3/AIB1, both in the absence and presence of E2 (Wu et al., 2004). Epidermal growth factor (EGF)-induced activation of ER-, progesterone receptor (PR)- and AR-

Fig. 3. Functional aspects of posttranslational modifications of the SRCs.

However, tamoxifen may exert ER agonistic effects depending on the coactivator context. For example, it has been shown that overexpression of SRC-3/AIB1 and the growth factor HER-2/neu increases the ER agonistic properties of tamoxifen (Shou et al., 2004) and that tamoxifen resistance develops when SRC-3/AIB1 is high and the transcriptional repressor paired box 2 (PAX2) is low in breast cancer cells (Hurtado et al., 2008). Elevated expression of SRC-1 in the uterine derived Ishikawa cell line increases the agonist behavior of 4 hydroxytamoxifen, whereas lower expression of SRC-1 in MCF-7 cells contributed to an ERantagonistic behavior of tamoxifen (Shang & Brown., 2002). Studies have shown that the estrogenic effects of tamoxifen can be mediated by the constitutive active AF-1 domain of ER which can be stimulated by several mechanisms, including high levels of coactivators (Webb et al., 1998). Hence, the levels of SRCs may determine the response to tamoxifen treatment, at least *in vitro*.

#### **3.2 Aromatase inhibitors**

Aromatase inhibitors work by blocking the estrogen synthesis and depriving the breast cancer cells of this important growth factor. In premenopausal women, estrogens are primarily synthesized by the granulose cells in the ovaries, but aromatase activity and conversion of androgens to estrogens also take place in tissues such as subcutaneous fat, breast tissue and bone which are the primary sources of estrogens after menopause. Aromatase is a cytochrome P450 enzyme where the haem protein binds the androgen and catalyzes the formation of the phenolic A-ring which is characteristic for estrogens. Type 1 aromatase inhibitors such as formestane and exemestane, also known as steroidal inhibitors, are analogues to androstenedione and work by competitive binding to the active site of aromatase. Type 2 aromatase inhibitors include the first generation compound aminoglutethimide, the second generation drug fadrozole and the third generation compounds anastrozole and letrozole, which are widely used clinically. These non-steroidal aromatase inhibitors work by binding to an iron atom in the haem group of aromatase and have proved very effective in inhibiting aromatase activity. In the absence of agonist, the ER will be locacted in the cytoplasm associated with heat shock protein (hsp), and dimerization, conformational changes and coactivator recruitment will be inhibited, hence leading to reduced transcription of ERregulated genes. However, resistance to aromatase inhibitors does occur. In the frequently used cellular model system for resistance to aromatase inhibitors, breast cancer cells are grown in estrogen-deprived conditions for 1-6 months. These long-term estrogen deprived cells (LTED) develop enhanced sensitivity to E2 (Masamura et al., 1995; Santen et al., 2005). This hypersensitivity is associated with upregulation of ERα and the mitogen activated protein kinases (MAPKs) (Jeng et al., 1998; Jeng et al., 2000). The MAPKs are found downstream of several growth factor receptors including HER-2/neu and could phosphorylate and influence the activity of the SRCs, but also the ER. Accumulated evidence points to an important crosstalk between ER and growth factor pathways where posttranslational modifications of the SRCs are involved. These modifications could influence not only SRC activity, but also the effect of endocrine treatment in breast cancer over time.

#### **4. SRCs and growth factor signaling**

#### **4.1 Posttranslational modifications of SRCs with functional aspects**

The SRCs are components and targets of multiple cell signaling pathways that modulate their activity. Extracellular stimuli such as hormones, growth factors and cytokines induce a

However, tamoxifen may exert ER agonistic effects depending on the coactivator context. For example, it has been shown that overexpression of SRC-3/AIB1 and the growth factor HER-2/neu increases the ER agonistic properties of tamoxifen (Shou et al., 2004) and that tamoxifen resistance develops when SRC-3/AIB1 is high and the transcriptional repressor paired box 2 (PAX2) is low in breast cancer cells (Hurtado et al., 2008). Elevated expression of SRC-1 in the uterine derived Ishikawa cell line increases the agonist behavior of 4 hydroxytamoxifen, whereas lower expression of SRC-1 in MCF-7 cells contributed to an ERantagonistic behavior of tamoxifen (Shang & Brown., 2002). Studies have shown that the estrogenic effects of tamoxifen can be mediated by the constitutive active AF-1 domain of ER which can be stimulated by several mechanisms, including high levels of coactivators (Webb et al., 1998). Hence, the levels of SRCs may determine the response to tamoxifen

Aromatase inhibitors work by blocking the estrogen synthesis and depriving the breast cancer cells of this important growth factor. In premenopausal women, estrogens are primarily synthesized by the granulose cells in the ovaries, but aromatase activity and conversion of androgens to estrogens also take place in tissues such as subcutaneous fat, breast tissue and bone which are the primary sources of estrogens after menopause. Aromatase is a cytochrome P450 enzyme where the haem protein binds the androgen and catalyzes the formation of the phenolic A-ring which is characteristic for estrogens. Type 1 aromatase inhibitors such as formestane and exemestane, also known as steroidal inhibitors, are analogues to androstenedione and work by competitive binding to the active site of aromatase. Type 2 aromatase inhibitors include the first generation compound aminoglutethimide, the second generation drug fadrozole and the third generation compounds anastrozole and letrozole, which are widely used clinically. These non-steroidal aromatase inhibitors work by binding to an iron atom in the haem group of aromatase and have proved very effective in inhibiting aromatase activity. In the absence of agonist, the ER will be locacted in the cytoplasm associated with heat shock protein (hsp), and dimerization, conformational changes and coactivator recruitment will be inhibited, hence leading to reduced transcription of ERregulated genes. However, resistance to aromatase inhibitors does occur. In the frequently used cellular model system for resistance to aromatase inhibitors, breast cancer cells are grown in estrogen-deprived conditions for 1-6 months. These long-term estrogen deprived cells (LTED) develop enhanced sensitivity to E2 (Masamura et al., 1995; Santen et al., 2005). This hypersensitivity is associated with upregulation of ERα and the mitogen activated protein kinases (MAPKs) (Jeng et al., 1998; Jeng et al., 2000). The MAPKs are found downstream of several growth factor receptors including HER-2/neu and could phosphorylate and influence the activity of the SRCs, but also the ER. Accumulated evidence points to an important crosstalk between ER and growth factor pathways where posttranslational modifications of the SRCs are involved. These modifications could influence not only SRC activity, but also the

treatment, at least *in vitro*.

**3.2 Aromatase inhibitors**

effect of endocrine treatment in breast cancer over time.

**4.1 Posttranslational modifications of SRCs with functional aspects**

The SRCs are components and targets of multiple cell signaling pathways that modulate their activity. Extracellular stimuli such as hormones, growth factors and cytokines induce a

**4. SRCs and growth factor signaling**

variety of posttranslational modifications of SRCs, including acetylation, methylation, phosphorylation, ubiquitination and sumoylation. These modifications influence the SRCs transcriptional activity and/or the SRC protein levels and stability (Baek & Rosenfeld, 2004; Li & Shang, 2007; Xu et al., 2009).

Fig. 3. Functional aspects of posttranslational modifications of the SRCs.

Phosphorylation of coactivators modulates ER-dependent gene transcription by regulating coactivator function in various ways. Three SRC-1 phosphorylation sites with corresponding kinases have been identified (S395, T1179 and S1185), one SRC-2/TIF-2 (S736) and sixteen SRC-3/AIB1 phosphorylation sites (T24, S505, S543, S601, S857, S860, S867, S1033, S1042, S1048, T1059, S1062, T1064, T1067, T1114 and Y1357) (Bulynko & O'Malley, 2011). Comparison of these sites reveals little conservation of sequences among the SRCs, indicating that phosphorylation is a significant determinant of the specificity of the SRCs (Wu et al., 2005).

Phosphorylation may influence the function and acitivity of the SRCs. It is shown *in vitro* using COS-1 cells that positions S395 and T1179/S1185 of SRC-1 are phosphorylated by the MAPK family members ERK1 and ERK2 (Rowan et al., 2000b) where MAPK-mediated phosphorylation on T1179 and S1185 has been shown to increase the affinity of SRC-1 for androgen receptor (AR) in prostate cancer cells (Ueda et al., 2002; Gregory et al., 2004). ERK2 may also phosphorylate SRC-3/AIB1 *in vitro* which stimulates the recruitment of p300 and associated histone acetyltransferase activity (Font de Mora & Brown, 2000). cAMP regulated phosphorylation of SRC-1 occurs through an indirect pathway in which protein kinase A (PKA) induces the activity of ERK1 and ERK2 (Rowan et al., 2000a). SRC-3/AIB1 phosphorylation-defective mutants exhibit reduced ability to interact with ER compared to wild type SRC-3/AIB1, both in the absence and presence of E2 (Wu et al., 2004). Epidermal growth factor (EGF)-induced activation of ER-, progesterone receptor (PR)- and AR-

Steroid Receptor Coactivators and Their Expression, Regulation

GPCR

Ligand

A

B

and Functional Role in Endocrine Responsive and Resistant Breast Cancer 723

Fig. 4. cAMP-PKA signaling influence SRC-2/TIF-2 function in a time-dependent manner.

cAMP

AC

ATP

PKA SRC-2/TIF-2

1-4 hours ↑ activity

4-24 hours ↓ activity

The SRCs are regulated by post-translational modifications by kinases found downstream in growth factor signaling pathways often activated in cancers, such as the MAPKs operating downstream of HER-2/neu. Posttranslational modification can stabilize and functionally activate the SRC proteins, a mechanism which has been shown to contribute not only to ERagonstic effects of tamoxifen, but also to estrogen hypersensitivity and resistance to aromatase inhibitors. *In vitro* it has been shown that tamoxifen resistance with loss of ER antagonistic effects develops when SRC-3/AIB1 is high and the transcriptional repressor PAX2 is low in breast cancer cells (Hurtado et al., 2008). Conversely, dissociation of SRC-3/AIB1 from ER restores tamoxifen's antagonistic effect in resistant breast cancer cells and inhibits further

Clinically, studies have shown an association between SRC-1 and reduced disease-free survival in breast cancer patients with locally advanced disease treated with endocrine therapy (Al-azawi et al., 2008; Redmond et al., 2009). During neoadjuvant treatment with aromatase inhibitors, we found higher levels of SRC-1 mRNA levels during treatment, especially in tumors that responded to treatment (Flågeng et al., 2009). Low expression of SRC-1 combined with high ERβ expression has been found to be a good prognostic indicator to endocrine treatment in breast cancers (Myers et al., 2004). However, the clearest association between high levels of SRCs and poor clinical outcome has been found in tumors also overexpressing HER-2/neu. Patients with tumors overexpressing HER-2/neu in

**4.2 SRCs, growth factor signaling and response to endocrine therapy**

breast cancer cell growth (Planas-Silva et al., 2001; List et al., 2001).

dependent transcription is shown to be regulated through phosphorylation of SRC-2/TIF-2 at S736 by the EGF-activated ERK MAPK and p38MAPK which stimulate SRC-2/TIF-2 coactivator function (Lopez et al., 2001; Gregory et al., 2004; Frigo et al., 2006).

Phosphorylation and dephosphorylation of proteins also regulate the nuclear import and export by modifying the nuclear localization signals (NLS) and nuclear export signals (NES) of the proteins (Whitmarsh & Davis, 2000). The sequence of the bHLH domain of the SRCs has been shown to be important for their nuclear localization. SRC-1 and SRC-3/AIB1 contain a conserved bipartite NLS in their bHLH-PAS domain (Amazit et al., 2003; Li et al., 2007). Furthermore, specific residues in the NLS of SRC-3/AIB1 are identified to signal proteasome-dependent turnover of SRC-3/AIB1 in the nucleus (Li et al., 2007). SRC-1 also contains a non-conserved sequence localized in its C-terminal region that is suggested to serve as a NES. The return of SRC-1 to the cytoplasm is proposed to be involved in termination of hormone action (Amazit et al., 2003).

Phosphorylation does not only influence the activation and subcellular localization of the SRCs, but also regulate the ubiquitination and degradation of the coactivators. Phosphorylated SRCs are suggested to be targets for enzymes in the ubiquitin-proteasome pathway. The ubiquitin-proteasome degradation pathway is regarded as an important mechanism to control the steady state levels of SRCs, thereby modulating growth responses to various growth-promoting factors (Lonard & O'Malley, 2005). Retinoic acid-induced phosphorylation of SRC-3/AIB1 by p38MAPK at S860, and phosphorylation at S505 by Akt/protein kinase B (PKB)-activated glycogen synthase kinase-3 (GSK3) have been shown to mediate SRC-3/AIB1 degradation (Gianni et al., 2006; Wu et al., 2007). On the other hand, atypical PKC-induced phosphorylation of the C-terminal region of SRC-3/AIB1 was reported to increase its stabilization by protecting it from proteasome-mediated degradation leading to an increased estrogen-induced breast cancer cell growth (Yi et al., 2008).

Growth factor pathways regulate SRC function not only through phosphorylation. We found activation of the cAMP/PKA pathway to stimulate association of SRC-2/TIF-2 with an ER-transcription complex prior to its degradation by the ubiquitin-proteasome system (Fenne et al., 2008). MCF-7 breast cancer cells were transfected with an expression plasmid encoding HA-GRIP1, the rodent homologue to SRC-2/TIF-2, along with the luciferase reporter construct ERE-TATA-luc. Cells were treated with cAMP analog and cAMPelevating agents for different time-lengths and after 48 hours the cells where lysed and subjected to luciferase assay. A time-dependent regulation of cAMP/PKA on SRC-2/TIF-2 coactivator function was observed (Fig. 4A). PKA is activated when hormones bind to a Gprotein coupled receptor (GPCR). The activated receptor interacts with adenylyl cyclase (AC) which catalyses the conversion of ATP to cAMP, further activating the cAMP dependent PKA (Fig. 4b). PKA can regulate SRC-2/TIF-2 coactivator function in a timedependent matter. Short-term treatment stimulated SRC-2/TIF-2 coactivator function, whereas long-term treatments inhibited SRC-2/TIF-2 function due to ubiquitin-proteasomemediated degradation (Hoang et al., 2004; Fenne et al., 2008).

All three SRCs can also be modified by site-specific sumoylation of lysine residues in their respective NIDs (Kotaja et al., 2002; Chauchereau et al., 2003). Sumoylation of SRC-2/TIF-2 has been shown to increase its coactivation of AR by enhancing their interaction (Kotaja et al., 2002). Conversely, sumoylation of SRC-1 increases its interaction with the PR and leads to prolonged retention of SRC-1 in the nucleus (Chauchereau et al., 2003). In contrast to SRC-1 and SRC-2/TIF-2, sumoylation of SRC-3/AIB1 seems to attenuate its coactivation function (Wu et al., 2006).

dependent transcription is shown to be regulated through phosphorylation of SRC-2/TIF-2 at S736 by the EGF-activated ERK MAPK and p38MAPK which stimulate SRC-2/TIF-2

Phosphorylation and dephosphorylation of proteins also regulate the nuclear import and export by modifying the nuclear localization signals (NLS) and nuclear export signals (NES) of the proteins (Whitmarsh & Davis, 2000). The sequence of the bHLH domain of the SRCs has been shown to be important for their nuclear localization. SRC-1 and SRC-3/AIB1 contain a conserved bipartite NLS in their bHLH-PAS domain (Amazit et al., 2003; Li et al., 2007). Furthermore, specific residues in the NLS of SRC-3/AIB1 are identified to signal proteasome-dependent turnover of SRC-3/AIB1 in the nucleus (Li et al., 2007). SRC-1 also contains a non-conserved sequence localized in its C-terminal region that is suggested to serve as a NES. The return of SRC-1 to the cytoplasm is proposed to be involved in

Phosphorylation does not only influence the activation and subcellular localization of the SRCs, but also regulate the ubiquitination and degradation of the coactivators. Phosphorylated SRCs are suggested to be targets for enzymes in the ubiquitin-proteasome pathway. The ubiquitin-proteasome degradation pathway is regarded as an important mechanism to control the steady state levels of SRCs, thereby modulating growth responses to various growth-promoting factors (Lonard & O'Malley, 2005). Retinoic acid-induced phosphorylation of SRC-3/AIB1 by p38MAPK at S860, and phosphorylation at S505 by Akt/protein kinase B (PKB)-activated glycogen synthase kinase-3 (GSK3) have been shown to mediate SRC-3/AIB1 degradation (Gianni et al., 2006; Wu et al., 2007). On the other hand, atypical PKC-induced phosphorylation of the C-terminal region of SRC-3/AIB1 was reported to increase its stabilization by protecting it from proteasome-mediated degradation

leading to an increased estrogen-induced breast cancer cell growth (Yi et al., 2008).

mediated degradation (Hoang et al., 2004; Fenne et al., 2008).

function (Wu et al., 2006).

Growth factor pathways regulate SRC function not only through phosphorylation. We found activation of the cAMP/PKA pathway to stimulate association of SRC-2/TIF-2 with an ER-transcription complex prior to its degradation by the ubiquitin-proteasome system (Fenne et al., 2008). MCF-7 breast cancer cells were transfected with an expression plasmid encoding HA-GRIP1, the rodent homologue to SRC-2/TIF-2, along with the luciferase reporter construct ERE-TATA-luc. Cells were treated with cAMP analog and cAMPelevating agents for different time-lengths and after 48 hours the cells where lysed and subjected to luciferase assay. A time-dependent regulation of cAMP/PKA on SRC-2/TIF-2 coactivator function was observed (Fig. 4A). PKA is activated when hormones bind to a Gprotein coupled receptor (GPCR). The activated receptor interacts with adenylyl cyclase (AC) which catalyses the conversion of ATP to cAMP, further activating the cAMP dependent PKA (Fig. 4b). PKA can regulate SRC-2/TIF-2 coactivator function in a timedependent matter. Short-term treatment stimulated SRC-2/TIF-2 coactivator function, whereas long-term treatments inhibited SRC-2/TIF-2 function due to ubiquitin-proteasome-

All three SRCs can also be modified by site-specific sumoylation of lysine residues in their respective NIDs (Kotaja et al., 2002; Chauchereau et al., 2003). Sumoylation of SRC-2/TIF-2 has been shown to increase its coactivation of AR by enhancing their interaction (Kotaja et al., 2002). Conversely, sumoylation of SRC-1 increases its interaction with the PR and leads to prolonged retention of SRC-1 in the nucleus (Chauchereau et al., 2003). In contrast to SRC-1 and SRC-2/TIF-2, sumoylation of SRC-3/AIB1 seems to attenuate its coactivation

coactivator function (Lopez et al., 2001; Gregory et al., 2004; Frigo et al., 2006).

termination of hormone action (Amazit et al., 2003).

Fig. 4. cAMP-PKA signaling influence SRC-2/TIF-2 function in a time-dependent manner.

### **4.2 SRCs, growth factor signaling and response to endocrine therapy**

The SRCs are regulated by post-translational modifications by kinases found downstream in growth factor signaling pathways often activated in cancers, such as the MAPKs operating downstream of HER-2/neu. Posttranslational modification can stabilize and functionally activate the SRC proteins, a mechanism which has been shown to contribute not only to ERagonstic effects of tamoxifen, but also to estrogen hypersensitivity and resistance to aromatase inhibitors. *In vitro* it has been shown that tamoxifen resistance with loss of ER antagonistic effects develops when SRC-3/AIB1 is high and the transcriptional repressor PAX2 is low in breast cancer cells (Hurtado et al., 2008).

Clinically, studies have shown an association between SRC-1 and reduced disease-free survival in breast cancer patients with locally advanced disease treated with endocrine therapy (Al-azawi et al., 2008; Redmond et al., 2009). During neoadjuvant treatment with aromatase inhibitors, we found higher levels of SRC-1 mRNA levels during treatment, especially in tumors that responded to treatment (Flågeng et al., 2009). Low expression of SRC-1 combined with high ERβ expression has been found to be a good prognostic indicator to endocrine treatment in breast cancers (Myers et al., 2004). However, the clearest association between high levels of SRCs and poor clinical outcome has been found in tumors also overexpressing HER-2/neu. Patients with tumors overexpressing HER-2/neu in

Steroid Receptor Coactivators and Their Expression, Regulation

**6. References**

and Functional Role in Endocrine Responsive and Resistant Breast Cancer 725

aromatase inhibitors. The SRCs are crucial to ER mediated effects and their expression level and activity have been shown to dictate the effect of ER on gene expression to a large extent. SRC-1, SRC-2/TIF-2 and SRC-3/AIB1 are expressed in normal and malignant breast tissue where SRC-3/AIB1 is now considered to be an oncogene. SRC-1 and SRC-3/AIB1 may promote metastasis in mammary tumors by enhancing the transcriptional activation of regulators of metastasis such as Twist and MMPs. The expression levels of the SRCs are influenced by endocrine treatment, an observation which may be of relevance to the treatment response to endocrine therapy over time. We found the mRNA levels of the SRCs, especially SRC-3/AIB1, to be significantly upregulated in both normal and malignant breast tissue after 4 weeks of tamoxifen in the 1-20 mg dose range. The mRNA expression of SRC-1 has also been shown to increase significantly in a clinical study of neoadjuvant treatment with aromatase inhibitors for 14-16 weeks, especially in the subgroup of patients achieving an objective treatment response. This is in line with *in vitro* studies in MCF-7 cells showing that estrogens suppress the mRNA levels of SRC-3/AIB1 by suppressing SRC-3/AIB1 gene transcription whereas 4-hydroxytamoxifen increases the SRC-3/AIB1 mRNA expression level. The importance of the expression level and functional activation of the SRCs during endocrine treatment is evident from cellular assays on tamoxifen treatment. High levels of coactivators relative to corepressors may lead to ER agonistic effects by 4 hydroxytamoxifen. Further, posttranslational modification of both coactivators and ER can lead to altered molecular conformations, intracellular relocation, stabilization and ubiquitination which would influence the activity and stability of the SRCs, as shown for the PKA-mediated regulation of SRC-2/TIF-2. In several clinical trials the levels of coactivators have been found of relevance, not only to the response to endocrine treatment, but also to long term clinical outcome. High protein levels of SRC-1 have been shown to be associated with reduced disease-free survival, both in untreated and tamoxifen treated patients, whereas elevated mRNA expression levels of SRC-3/AIB1 have been associated with high tumor grade and shorter disease-free and overall survival. Tumors undergoing tamoxifen therapy and overexpressing HER-2/neu in combination with SRC-3/AIB1 are more likely to be tamoxifen resistant and are associated with reduced disease-free survival. High expression of HER-2/neu in combination with SRC-1 has also been associated with a greater risk of recurrence on endocrine treatment. In summary, SRCs are expressed in normal and malignant breast tissue and they have a crucial role in mediating the effect of endocrine treatment in breast cancer. The expression levels of SRCs are regulated by endocrine treatment and their functional role is modified by posttranslational modifications mediated by growth factor pathways involved in breast cancer development and endocrine resistance. Further research on SRCs and their role in the crosstalk between ER and growth factor pathways during endocrine treatment is important to improve breast cancer therapy.

Al-azawi, D., McIlroy, M., Kelly, G., Redmond, A.M., Bane, F.T., Cocchiglia, S., Hill, A.D.K.

endocrine resistant breast cancer. *Oncogene*, Vol. 27, No. 21, pp. 3021-3031. Amazit, L., Alj, Y., Tyagi, R.K., Chauchereau, A., Loosfelt, H., Pichon, C., Pantel, J., Foulon-

coactivator-1. *J Biol Chem,* Vol. 278, No. 34, pp. 32195-32203.

& Young, L.S. (2008). Ets-2 and p160 proteins collaborate to regulate c-Myc in

Guinchard, E., Leclerc, P., Milgrom, E. & Guiochon-Mantel, A. (2003). Subcellular localization and mechanisms of nucleocytoplasmic trafficking of steroid receptor

combination with SRC-3/AIB1 or SRC-1 undergoing tamoxifen treatment show reduced sensitivity to endocrine therapy, greater risk of disease recurrence and reduced disease-free survival (Fleming et al., 2004b; Osborne et al., 2003). Overexpression of SRC-3/AIB1 and HER-2/neu in breast tumors is associated with disease recurrences and poor prognosis. This could be linked to the HER-2/neu-mediated activation of MAPK and Akt which causes phosphorylation of SRC-3/AIB1 and ER, resulting in transcriptional activation and cell proliferation. Activation of Akt has also been shown to stabilize SRC-3/AIB1 by inhibiting GSK3 (Wu et al., 2007) whereas PKA-induced resistance to tamoxifen is associated with an altered orientation between ER and SRC-1 (Zwart et al., 2007). Overall, the SRCs can be targeted by central growth factor pathways mediating pro-survival signals and stimulating proliferation. The SRCs close functional relationship with the ER makes posttranslational modifications of the SRCs important points of crosstalk between ER and growth factor signaling pathways during endocrine treatment in breast cancer (Fig. 5).

Fig. 5. Cross-talk between growth-factor signaling pathways and SRCs in breast cancer. Ligand-activated growth factor receptor dimers including the human epidermal growth factor receptor-2 and -3 (HER-2/3) and the insulin-like growth factor-1 receptor (IGF-1R) are phosphorylated at intracellular domains and signal both through the MAPK and the phosphatidyl-inositol 3-kinase (PI3K) signaling pathway. ERK 1 and 2 may phosphorylate SRC-1, SRC-2/TIF-2 and SRC-3/AIB1. Akt may phosporylate SRC-2/TIF-2 and SRC-3/AIB1. SRC-3/AIB1 is a modulator increasing the activity and signaling both through HER-2 and IGF-1R leading to cell growth.

## **5. Conclusion**

Most human breast cancers express ER which belongs to the family of nuclear receptors and is a ligand-regulated transcription factor. Endocrine treatment involves blocking the ER with a selective ER modulator such as tamoxifen or inhibiting estrogen synthesis using aromatase inhibitors. The SRCs are crucial to ER mediated effects and their expression level and activity have been shown to dictate the effect of ER on gene expression to a large extent. SRC-1, SRC-2/TIF-2 and SRC-3/AIB1 are expressed in normal and malignant breast tissue where SRC-3/AIB1 is now considered to be an oncogene. SRC-1 and SRC-3/AIB1 may promote metastasis in mammary tumors by enhancing the transcriptional activation of regulators of metastasis such as Twist and MMPs. The expression levels of the SRCs are influenced by endocrine treatment, an observation which may be of relevance to the treatment response to endocrine therapy over time. We found the mRNA levels of the SRCs, especially SRC-3/AIB1, to be significantly upregulated in both normal and malignant breast tissue after 4 weeks of tamoxifen in the 1-20 mg dose range. The mRNA expression of SRC-1 has also been shown to increase significantly in a clinical study of neoadjuvant treatment with aromatase inhibitors for 14-16 weeks, especially in the subgroup of patients achieving an objective treatment response. This is in line with *in vitro* studies in MCF-7 cells showing that estrogens suppress the mRNA levels of SRC-3/AIB1 by suppressing SRC-3/AIB1 gene transcription whereas 4-hydroxytamoxifen increases the SRC-3/AIB1 mRNA expression level. The importance of the expression level and functional activation of the SRCs during endocrine treatment is evident from cellular assays on tamoxifen treatment. High levels of coactivators relative to corepressors may lead to ER agonistic effects by 4 hydroxytamoxifen. Further, posttranslational modification of both coactivators and ER can lead to altered molecular conformations, intracellular relocation, stabilization and ubiquitination which would influence the activity and stability of the SRCs, as shown for the PKA-mediated regulation of SRC-2/TIF-2. In several clinical trials the levels of coactivators have been found of relevance, not only to the response to endocrine treatment, but also to long term clinical outcome. High protein levels of SRC-1 have been shown to be associated with reduced disease-free survival, both in untreated and tamoxifen treated patients, whereas elevated mRNA expression levels of SRC-3/AIB1 have been associated with high tumor grade and shorter disease-free and overall survival. Tumors undergoing tamoxifen therapy and overexpressing HER-2/neu in combination with SRC-3/AIB1 are more likely to be tamoxifen resistant and are associated with reduced disease-free survival. High expression of HER-2/neu in combination with SRC-1 has also been associated with a greater risk of recurrence on endocrine treatment. In summary, SRCs are expressed in normal and malignant breast tissue and they have a crucial role in mediating the effect of endocrine treatment in breast cancer. The expression levels of SRCs are regulated by endocrine treatment and their functional role is modified by posttranslational modifications mediated by growth factor pathways involved in breast cancer development and endocrine resistance. Further research on SRCs and their role in the crosstalk between ER and growth factor pathways during endocrine treatment is important to improve breast cancer therapy.

## **6. References**

724 Breast Cancer – Carcinogenesis, Cell Growth and Signaling Pathways

combination with SRC-3/AIB1 or SRC-1 undergoing tamoxifen treatment show reduced sensitivity to endocrine therapy, greater risk of disease recurrence and reduced disease-free survival (Fleming et al., 2004b; Osborne et al., 2003). Overexpression of SRC-3/AIB1 and HER-2/neu in breast tumors is associated with disease recurrences and poor prognosis. This could be linked to the HER-2/neu-mediated activation of MAPK and Akt which causes phosphorylation of SRC-3/AIB1 and ER, resulting in transcriptional activation and cell proliferation. Activation of Akt has also been shown to stabilize SRC-3/AIB1 by inhibiting GSK3 (Wu et al., 2007) whereas PKA-induced resistance to tamoxifen is associated with an altered orientation between ER and SRC-1 (Zwart et al., 2007). Overall, the SRCs can be targeted by central growth factor pathways mediating pro-survival signals and stimulating proliferation. The SRCs close functional relationship with the ER makes posttranslational modifications of the SRCs important points of crosstalk between ER and growth factor

signaling pathways during endocrine treatment in breast cancer (Fig. 5).

 Cross-talk between growth-factor signaling pathways and SRCs in breast cancer. Ligand-activated growth factor receptor dimers including the human epidermal growth factor receptor-2 and -3 (HER-2/3) and the insulin-like growth factor-1 receptor (IGF-1R) are

MAPK

**Proliferation Survival**

phosphorylated at intracellular domains and signal both through the MAPK and the phosphatidyl-inositol 3-kinase (PI3K) signaling pathway. ERK 1 and 2 may phosphorylate SRC-1, SRC-2/TIF-2 and SRC-3/AIB1. Akt may phosporylate SRC-2/TIF-2 and SRC-3/AIB1. SRC-3/AIB1 is a modulator increasing the activity and signaling both through

Most human breast cancers express ER which belongs to the family of nuclear receptors and is a ligand-regulated transcription factor. Endocrine treatment involves blocking the ER with a selective ER modulator such as tamoxifen or inhibiting estrogen synthesis using

HER-2 and IGF-1R leading to cell growth.

**5. Conclusion**

Steroid Receptor Coactivators and Their Expression, Regulation

No. 10, pp. 1069-1074.

983.

739-751.

2176-2186.

387, No. 6634, pp. 733-736.

*J Biol Chem,* Vol. 279, No. 47, pp. 49120-49130.

and Functional Role in Endocrine Responsive and Resistant Breast Cancer 727

Fleming, F. J., Hill, A. D., McDermott, E.W., O'Higgins, N. J. & Young, L. S. (2004a).

Fleming, F. J., Myers, E., Kelly, G., Crotty, T. B., McDermott, E.W., O'Higgins, N. J., Hill, A.

Flågeng, M.H., Haugan Moi, L.L., Dixon, J.M., Geisler, J., Lien, E.A., Miller, W.R., Lønning,

Font de Mora, J. & Brown, M. (2000). AIB1 is a conduit for kinase-mediated growth factor signaling to the estrogen receptor. *Mol Cell Biol*, Vol. 20, No. 14, pp. 5041-5047. Foster, J. S., Henley, D. C., Ahamed, S.& Wimalasena, J. (2001). Estrogens and cell-cycle regulation in breast cancer. *Trends Endocrinol Metab,* Vol. 12, No. 7, pp. 320-327. Frigo, D.E., Basu, A., Nierth-Simpson, E.N., Weldon, C.B., Dugan, C.M., Elliott, S., Collins-

Gehin, M., Mark, M., Dennefeld, C., Dierich, A., Gronemeyer H. & Chambon, P. (2002). The

Gianni, M., Parrella, E., Raska, I. Jr, Gaillard, E., Nigro, E.A., Gaudon, C., Garattini, E.

Gregory C.W., Fei, X., Ponguta, L.A., He, B., Bill, H.M., French, F.S. & Wilson, E.M. (2004).

Haugan Moi, L.L., Flågeng, M.H., Gandini, S., Guerrieri-Gonzaga, A., Bonanni, B.,

Heery, D. M., Kalkhoven, E., Hoare S. & Parker, M.G. (1997). A signature motif in

Hoang, T., Fenne, I. S., Cook, C., Borud, B., Bakke, M., Lien, E.A. & Mellgren, G. (2004).

recurrent prostate cancer. *J Biol Chem,* Vol. 279, No. 8, pp. 7119-30.

p/CIP. *Mol Cell Biol,* Vol. 22, No. 16, pp. 5923-5937.

breast cancer. *J Clin Endocrinol Metab,* Vol. 89, No. 1, pp. 375-383.

aromatase inhibitors. *Br J Cancer*, Vol. 101, No. 8, pp. 1253-1260.

Differential recruitment of coregulator proteins steroid receptor coactivator-1 and silencing mediator for retinoid and thyroid receptors to the estrogen receptorestrogen response element by beta-estradiol and 4-hydroxytamoxifen in human

D. & Young, L. S. (2004b). Expression of SRC-1, AIB1, and PEA3 in HER2 mediated endocrine resistant breast cancer; a predictive role for SRC-1. *J Clin Pathol*, Vol. 57,

P.E. & Mellgren, G. (2009). Nuclear receptor co-activators and HER-2/neu are upregulated in breast cancer patients during neo-adjuvant treatment with

Burow, B.M., Salvo, V.A., Zhu, Y., Melnik, L.I., Lopez, G.N., Kushner, P.J., Curiel, T.J., Rowan, B.G., McLachlan, J.A. & Burow M.E. (2006). p38 mitogen-activated protein kinase stimulates estrogen-mediated transcription and proliferation through the phosphorylation and potentiation of the p160 coactivator glucocorticoid receptor-interacting protein 1. *Mol Endocrinol,* Vol. 20, No. 5, pp. 971-

function of TIF2/GRIP1 in mouse reproduction is distinct from those of SRC-1 and

&Rochette-Egly, C. (2006). P38MAPK-dependent phosphorylation and degradation of SRC-3/AIB1 and RARalpha-mediated transcription. *EMBO J*, Vol. 25, No. 4, pp.

Epidermal growth factor increases coactivation of the androgen receptor in

Lazzeroni, M., Gjerde, J., Lien, E.A., DeCensi, A. & Mellgren, G. (2010). Effect of low dose tamoxifen on Steroid Receptor Coactivator 3/Amplified in Breast Cancer 1 in normal and malignant human breast tissue. *Clin Cancer Res*, Vol. 16, No. 7, pp.

transcriptional co-activators mediates binding to nuclear receptors. *Nature,* Vol.

cAMP-dependent protein kinase regulates ubiquitin-proteasome-mediated degradation and subcellular localization of the nuclear receptor coactivator GRIP1.

Anzick, S. L., Kononen, J., Walker, R. L., Azorsa, D. O., Tanner, M. M., Guan, X. Y., Sauter,

Baek, S.H. & Rosenfeld, M.G. (2004). Nuclear receptor coregulators: their modification codes

Bautista, S., Valles, H., Walker, R. L., Anzick, S., Zeillinger, R., Meltzer, P. & Theillet, C.

Bouras, T., Southey, M. C. & Venter, D.J. (2001). Overexpression of the steroid receptor

Brzozowski, A. M., Pike, A. C., Dauter, Z., Hubbard, R. E., Bonn, T., Engstrom, Ohman, O.,

Bulynko, Y.A. & O'Malley, B.W. (2011). Nuclear receptor coactivators: structural and

Carapeti, M., Agular, R.C., Chase, A., Goldman, J.M. & Cross, N.C. (1998). Assignment of

Cavarretta, I.T., Mukopadhyay, R., Lonard, D.M., Cowsert, L.M., Bennett, C.F., O'Malley,

Chauchereau, A., Amazit, L., Quesne, M., Guiochon-Mantel, A. & Milgrom, E. (2003).

Cuzick, J., Powles, T., Veronesi, U., Forbes, J., Edwards, R., Ashley, S. & Boyle, P. (2003).

Dubik, D. & Shiu, R.P. (1992). Mechanism of estrogen activation of c-myc oncogene

Fenne, I.S., Hoang, T., Hauglid, M., Sagen, J.V., Lien, E.A. & Mellgren, G. (2008).

Fereshteh, M. P., Tilli, M.T., Kim, S.E., Xu, J., O'Malley, B.W., Wellstein, A., Furth, P.A. &

tumorigenesis in mice. *Cancer Res,* Vol. 68, No. 10, pp. 3697-3706.

functional biochemistry. *Biochemistry*, Vol. 50, No. 3, pp. 313-328.

proliferation. *Mol Endocrinol,* Vol. 16, No. 2, pp. 253-270.

SRC-1. *J Biol Chem*, Vol. 278, No. 14, pp. 12335-12343.

expression. *Oncogene*, Vol. 7, No. 8, pp. 1587-1594.

965-968.

No. 3, pp. 707-714.

No. 3, pp. 903-907.

No. 9354, pp. 296-300.

4336-4345.

753-758.

Vol. 4, No. 12, pp. 2925-2929.

*Genomics*, Vol. 52, No. 2, pp. 242-244.

G., Kallioniemi, O. P., Trent ,J. M. & Meltzer, P.S. (1997). AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. *Science* , Vol. 277, No. 5328, pp.

and regulatory mechanism by translocation. *Biochem Biophys Res Commun,* Vol. 319,

(1998). In breast cancer, amplification of the steroid receptor coactivator gene AIB1 is correlated with estrogen and progesterone receptor positivity. *Clin Cancer Res*,

coactivator AIB1 in breast cancer correlates with the absence of estrogen and progesterone receptors and positivity for p53 and HER2/neu. *Cancer Res,* Vol. 61,

L., Greene, G. L., Gustafsson, J. A. & Carlquist, M. (1997). Molecular basis of agonism and antagonism in the oestrogen receptor. *Nature,* Vol. 389, No. 6652, pp.

the steroid receptor coactivator-1 (SRC-1) gene to human chromosome band 2p23.

B.W. & Smith, C.L. (2002). Reduction of coactivator expression by antisense oligodeoxynucleotides inhibits ERalpha transcriptional activity and MCF-7

Sumoylation of the progesterone receptor and of the steroid receptor coactivator

Overview of the main outcomes in breast-cancer prevention trials. *Lancet*, Vol. 361,

Recruitment of coactivator glucocorticoid receptor interacting protein 1 to an estrogen receptor transcription complex is regulated by the 3`,5`-cyclic adenosine 5`-monophosphate-dependent protein kinase. *Endocrinology,* Vol. 148, No. 9, pp.

Riegel, A.T. (2008). The nuclear receptor coactivator amplified in breast cancer-1 is required for Neu (ErbB2/HER2) activation, signaling, and mammary

Steroid Receptor Coactivators and Their Expression, Regulation

80, No. 10, pp. 2918-2925.

6266-6271.

19, No. 4, 490-497.

5240, pp. 1354-1357.

61, No. 10, pp. 3858-3862.

*Cell Biol,* Vol. 28, No. 19, pp. 5937-5950.

1693.

22182.

and Functional Role in Endocrine Responsive and Resistant Breast Cancer 729

Louet, J. F., Chopra, A. R., Sagen, J. V., An, J., York, B., Tannour-Louet, M.,., Saha, P. K.,

Masamura, S., Santner, S. J., Heitjan, D. F. & Santen, R. J. (1995). Estrogen deprivation causes

Metivier, R., Penot, G., Hubner, M. R., Reid, G., Brand, H., Kos M. & Gannon, F. (2003).

Myers, E., Fleming, F. J., Crotty, G., Kelly, E.W., McDermott, J., O'Higgins N., Hill, A. D. &

Newman, S. P., Bates, N. P. , Vernimmen, D., Parker, M. G. & Hurst, H. C. (2000). Cofactor

Onate, S.A., Tsai, ., S. Y., Tsai M. J. & O'Malley, B. W. (1995). Sequence and characterization

Osborne, C. K., Bardou, V., Hopp, T. A., Chamness, G. C., Hilsenbeck, S. G., Fuqua, S. A.,

Pearce, S. T. & Jordan, V.C. (2004). The biological role of estrogen receptors alpha and beta

Picard, F., Gehin, M., Annicotte, J., Rocchi, S., Champy, M. F., O'Malley, B. W., Chambon, P.

Planas-Silva, M. D., Shang, Y., Donaher, J. L., Brown, M. &. Weinberg, R. A. (2001). AIB1

Qin, L., Liao, L., Redmond, A., Young, L., Yuan, Y., Chen, H., O'Malley, B. W. & Xu, J.

cancer. *J Natl Cancer Inst,* Vol. 95, No. 5, pp. 353-361.

in cancer. *Crit Rev Oncol Hematol*, Vol. 50, No. 1, pp. 3-22.

brown adipose tissues. *Cell,* Vol. 111, No. 7, pp. 931-941.

cofactors on a natural target promoter. *Cell,* Vol. 115, No. 6, pp. 751-763. Murphy, L. C., Simon, S. L., Parkes, A., Leygue, E., Dotzlaw, H., Snell, L., Troup, S.,

superfamily: the second decade. *Cell,* Vol. 83, No. 6, pp. 835-839.

regulation of p160 coactivator activity. *J Biol Chem,* Vol. 276, No. 25, pp. 22177-

Stevens, R. D., Wenner, B. R., Ilkayeva, O. R., Bain, J. R., Zhou, S., DeMayo, F., Xu, J., Newgard , C. B. & O'Malley, B.W. (2010). The coactivator SRC-1 is an essential coordinator of hepatic glucose production. *Cell Metab,* Vol. 12, No. 6, pp. 606-618. Mangelsdorf, D. J., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K., Blumberg,

B., Kastner, P., Mark, M., Chambon, P. & Evans, R.M. (1995). The nuclear receptor

estradiol hypersensitivity in human breast cancer cells. *J Clin Endocrinol Metab,* Vol.

Estrogen receptor-alpha directs ordered, cyclical, and combinatorial recruitment of

Adeyinka, A. & Watson. P.H. (2000). Altered expression of estrogen receptor coregulators during human breast tumorigenesis. *Cancer Res,* Vol. 60, No. 22, pp.

Young, L. S. (2004). Inverse relationship between ER-beta and SRC-1 predicts outcome in endocrine-resistant breast cancer. *Br J Cancer,* Vol. 91, No. 9, pp. 1687-

competition between the ligand-bound oestrogen receptor and an intron 1 enhancer leads to oestrogen repression of ERBB2 expression in breast cancer. *Oncogene*, Vol.

of a coactivator for the steroid hormone receptor superfamily. *Science,* Vol. 270, No.

Wong, J., Allred, D. C., Clark, G. M. & Schiff, R. (2003). Role of the estrogen receptor coactivator AIB1 (SRC-3) and HER-2/neu in tamoxifen resistance in breast

& Auwerx, J. (2002). SRC-1 and TIF2 control energy balance between white and

enhances estrogen-dependent induction of cyclin D1 expression. *Cancer Res,* Vol.

(2008). The AIB1 oncogene promotes breast cancer metastasis by activation of PEA3-mediated matrix metalloproteinase 2 (MMP2) and MMP9 expression. *Mol*

Hudelist, G., Czerwenka, K., Kubista, E., Marton, E., Pischinger, K. & Singer, C. F. (2003).

Hurtado, A., Holmes, K. A., Geistlinger, T. R., Hutcheson, I. R., Nicholson, R. I., Brown, M.,

Jeng, M. H., Shupnik, M. A., Bender, T. P., Westin, E. H., Bandyopadhyay, D., Kumar, R.,

Jeng, M. H., Yue, W., Eischeid, A., Wang, J. P. & Santen. R.J. (2000). Role of MAP kinase in

Kalkhoven, E., Valentine, J. E., Heery,D. M. & Parker, M.G. (1998). Isoforms of steroid

Karmakar, S., Foster, E. A. & Smith, C.L. (2009). Unique roles of p160 coactivators for

Kurebayashi, J., Otsuki, T., Kunisue, H., Tanaka, K., Yamamoto, S. & Sonoo, H. (2000).

and corepressors in breast cancer. *Clin Cancer Res*, Vol. 6, No. 2, pp. 512-518. Lauritsen, K. J., List, H. J., Reiter, R., Wellstein, A. & Riegel, A.T. (2002). A role for TGF-beta

AIB1 in MCF-7 breast cancer cells. *Oncogene,* Vol. 21, No. 47, pp. 7147-7155. Li, C., Wu, R.C., Amazit, L., Tsai, S.Y., Tsai, M.J. & O'Malley, B.W. (2007). Specific amino

Li, S. & Shang, Y. (2007). Regulation of SRC family coactivators by post-translational

List, H. J., Reiter, R., Singh, B., Wellstein, A. & Riegel, A. T. (2001). Expression of the nuclear

Lonard, D. M. & O'Malley, B.W. (2005). Expanding functional diversity of the coactivators.

Lonard, D.M., Tsai, S.Y. & O'Malley, B.W. (2004). Selective estrogen receptor modulators 4-

Lopez, G. N., Turck, C. W., Schaufele, F., Stallcup, M.R. & Kushner, P.J. (2001). Growth

SRC-3 coactivator proteins. *Mol Cell Biol*, Vol. 24, No. 1, pp. 14-24.

modifications. *Cell Signal,* Vol. 19, No. 6, pp. 1101-12.

*Trends Biochem Sci,* Vol. 30, No. 3, pp. 126-132.

transcriptional activity. *Endocrinology*, Vol. 150, No. 4, pp. 1588-1596. Kotaja, N., Karvonen, U., Jänne, O.A. & Palvimo, J.J. (2002). The nuclear receptor interaction

cells. *Breast Cancer Res Treat,* Vol. 62, No. 3, pp. 167-175.

oestrogen receptor. *EMBO J*, Vol. 17, No. 1, pp. 232-243.

Vol. 277, No. 33, pp. 30283-30288.

78, No. 2, pp. 193-204.

7222, pp. 663-666.

10, pp. 4164-4174.

4, pp. 1296-1308.

68, No. 1, pp. 21-28.

 *Breast Cancer Res Treat,* Vol.

Jiang, J., Howat, W. J., Ali, S. & Carroll, J. S. (2008). Regulation of ERBB2 by oestrogen receptor-PAX2 determines response to tamoxifen. *Nature,* Vol. 456, No.

Masamura, S. & Santen, R.J. (1998). Estrogen receptor expression and function in long-term estrogen-deprived human breast cancer cells. *Endocrinoloy,* Vol. *1*39, No..

the enhanced cell proliferation of long term estrogen deprived human breast cancer

receptor co-activator 1 differ in their ability to potentiate transcription by the

regulation of breast cancer cell proliferation and estrogen receptor-alpha

domain of GRIP1 is modulated by covalent attachment of SUMO-1. *J Biol Chem,*

Expression levels of estrogen receptor-alpha, estrogen receptor-beta, coactivators,

in estrogen and retinoid mediated regulation of the nuclear receptor coactivator

acid residues in the basic helix-loop-helix domain of SRC-3 are essential for its nuclear localization and proteasome-dependent turnover. *Mol Cell Bio*l, Vol. 27, No.

coactivator AIB1 in normal and malignant breast tissue. *Breast Cancer Res Treat,* Vol.

hydroxytamoxifen and raloxifene impact the stability and function of SRC-1 and

factors signal to steroid receptors through mitogen-activated protein kinase

regulation of p160 coactivator activity. *J Biol Chem,* Vol. 276, No. 25, pp. 22177- 22182.

Steroid Receptor Coactivators and Their Expression, Regulation

151-156.

21848-21856.

630.

No. 6, pp. 1125-1140.

and Functional Role in Endocrine Responsive and Resistant Breast Cancer 731

Wang, S., Yuan, Y., Liao, L., Kuang, S. Q., Tien, J. C., O'Malley, B. W. & Xu, J. (2009).

Webb, P., Nguyen P. & Kushner, P. J. (2003). Differential SERM effects on corepressor

Webb, P., Nguyen, P., Shinsako, J., Anderson, C., Feng, W., Nguyen, M.P., Chen, D., Huang,

Whitmarsh, A.J. & Davis, R.J. (2000). Regulation of transcription factor function by

Wu, H., Sun, L., Zhang, Y., Chen, Y., Shi, B., Li, R., Wang, Y., Liang, J., Fan, D., Wu, G.,

Wu, R.C., Feng, Q., Lonard, D.M. & O'Malley, B.W. (2007). SRC-3 coactivator functional

Wu, R. C., Qin, J., Yi, P., Wong, J.S., Tsai, Y., Tsai, M. J. & O'Malley, B.W. (2004). Selective

multiple cellular signaling pathways. *Mol Cell,* Vol. 15, No. 6, pp. 937-949. Wu, R. C., Smith, C.L. & O'Malley, B.W. (2005). Transcriptional regulation by steroid receptor coactivator phosphorylation. *Endocr Rev,* Vol. 26, No. 3, pp. 393-399. Xu, J. & Li, Q. (2003). Review of the in vivo functions of the p160 steroid receptor coactivator

Xu, J., Liao, L., Ning, G., Yoshida-Komiya, H., Deng, C. & O'Malley, B.W. (2000). The steroid

Xu, J., Wu, R. C. & O'Malley, B.W. (2009). Normal and cancer-related functions of the p160

Xu, J., Qiu, Y., DeMayo, F. J., Tsai, S. Y., Tsai, M. J. & O'Malley, B.W. (1998). Partial hormone

Yi, P., Feng, Q., Amazit, L., Lonard, D.M., Tsai, S.Y., Tsai, M.J. & O'Malley, B.W. (2008).

oncogenic coactivator SRC-3/AIB1. *Mol Cell*, Vol. 29, No. 4, pp. 465-476. Zhao, C., Yasui, K., Lee, C. J., Kurioka, H., Hosokawa, Y., Oka, T. & Inazawa, J. (2003).

predictors of poor prognosis. *Cancer*, Vol. 98, No. 1, pp. 18-23.

development. *Proc Natl Acad Sci U S A,*Vol. 97, No. 12, pp. 6379-6384. Xu, J. & O'Malley, B.W. (2002). Molecular mechanisms and cellular biology of the steroid

family. *Mol Endocrinol*, Vol. 17, No. 9, pp. 1681-1692.

gene. *Science,* Vol. 279, No. 5358, pp. 1922-1925.

*Disord,* Vol. 3, No. 3, pp. 185-192.

coactivator proteins. *Mol Endocrinol*, Vol. 12, No. 10, pp. 1605-1618.

phosphorylation. *Cell Mol Life Sci*, Vol. 57, No. 8-9; pp. 1172-1183.

Disruption of the SRC-1 gene in mice suppresses breast cancer metastasis without affecting primary tumor formation. *Proc Natl Acad Sci U S A*, Vol. 106, No. 1, pp.

binding dictate ERalpha activity in vivo. *J Biol Chem*, Vol. 278, No. 9, pp. 6912-6920.

S.M., Subramanian, S., McKinerney, E., Katzenellenbogen, B.S., Stallcup, M.R. & Kushner, P.J. (1998). Estrogen receptor activation function 1 works by binding p160

Wang, D., Li, S. & Shang, Y. (2006). Coordinated regulation of AIB1 transcriptional activity by sumoylation and phosphorylation. *J Biol Chem,* Vol. 281, No. 31, pp.

lifetime is regulated by a phospho-dependent ubiquitin time clock. *Cell*, Vol. 129,

phosphorylations of the SRC-3/AIB1 coactivator integrate genomic reponses to

receptor coactivator SRC-3 (p/CIP/RAC3/AIB1/ACTR/TRAM-1) is required for normal growth, puberty, female reproductive function, and mammary gland

receptor coactivator (SRC) family in steroid receptor function. *Rev Endocr Metab*

steroid receptor co-activator (SRC) family. *Nat Rev Cancer,* Vol. 9, No. 9, pp. 615-

resistance in mice with disruption of the steroid receptor coactivator-1 (SRC-1)

Atypical protein kinase C regulates dual pathways for degradation of the

Elevated expression levels of NCOA3, TOP1, and TFAP2C in breast tumors as

Qin, L., Liu, Z., Chen, H. & Xu, J. (2009). The steroid receptor coactivator-1 regulates twist

Redmond, A. M., Bane, F. T., Stafford, A. T., McIlroy, M., Dillon, M. F., Crotty, T. B., Hill, A.

Rowan, B.G., Weigel, N.L. & O'Malley, B.W. (2000b). Phosphorylation of steroid receptor

Sabbah, M., Courilleau, D., Mester, J. & Redeuilh, G. (1999). Estrogen induction of the cyclin

Santen, R. J., Song R. X., Zhang, Z., Kumar, R., Jeng, M. H., Masamura, A., Lawrence Jr., J.,

Shang, Y. & Brown, M. (2002). Molecular determinants for the tissue specificity of SERMs.

Shiau, A. K., Barstad, D., Loria, P. M., Cheng, L., Kushner, P. J., Agard, D. A. & Greene, G.L.

antagonism of this interaction by tamoxifen. *Cell*, Vol. 95, No. 7, pp. 927-937. Shou, J., Massarweh, S., Osborne, C. K., Wakeling, A. E., Ali S., Weiss, H. & Schiff, R. (2004).

Tai, H., Kubota, N. & Kato, S. (2000). Involvement of nuclear receptor coactivator SRC-1 in

Takeshita, A., Cardona, G. R., Koibuchi, N., Suen C. S. & Chin, W.W. (1997). TRAM-1, A

Torres-Arzayus, M. I., Font de Mora, J., Yuan, J., Vazquez, F. , Bronson, R., Rue, M., Sellers

Ueda, T., Mawji, N. R., Bruchovsky, N. & Sadar, M.D. (2002). Ligand-independent activation

breast cancer recurrence. *Clin Cancer Res,* Vol. 15, No. 6, pp. 2098-2106. Rowan, B.G., Garrison, N., Weigel, N.L. & O'Malley, B.W. (2000a). 8-Bromo-cyclic AMP

3819-3827.

23, pp. 8720-8730.

pp. 4475-4483.

935.

267, No. 1, pp. 311-316.

27629-27634.

pp. 263-274.

*A,* Vol. 96, No. 20, pp. 11217-11222.

*Relat Cancer,* Vol. 12, No. Suppl 1, pp. S61-73.

*Science,* Vol. 295, No. 5564, pp. 2465-2468.

expression and promotes breast cancer metastasis. *Cancer Res*, Vol. 69, No. 9, pp.

D. & Young, L.S. (2009). Coassociation of estrogen receptor and p160 proteins predicts resistance to endocrine treatment; SRC-1 is an independent predictor of

induces phosphorylation of two sites in SRC-1 that facilitate ligand-independent activation of the chicken progesterone receptor and are critical for functional cooperation between SRC-1 and CREB binding protein. *Mol Cell Biol*, Vol. 20, No.

coactivator-1. Identification of the phosphorylation sites and phosphorylation through the mitogen-activated protein kinase pathway. *J Biol Chem*, Vol. 275, No. 6,

D1 promoter: involvement of a cAMP response-like element. *Proc Natl Acad Sci U S*

Berstein, L. & Yue, W. (2005). Long-term estradiol deprivation in breast cancer cells up-regulates growth factor signaling and enhances estrogen sensitivity. *Endocr*

(1998). The structural basis of estrogen receptor/coactivator recognition and the

Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu crosstalk in ER/HER2-positive breast cancer. *J Natl Cancer Inst,* Vol. 96, No. 12, pp. 926-

estrogen-dependent cell growth of MCF-7 cells. *Biochem Biophys Res Commun*, Vol.

novel 160-kDa thyroid hormone receptor activator molecule, exhibits distinct properties from steroid receptor coactivator-1. *J Biol Chem*, Vol 272, No. 44, pp.

W. R. & Brown, M. (2004). High tumor incidence and activation of the PI3K/AKT pathway in transgenic mice define AIB1 as an oncogene. *Cancer Cell,* Vol. 6, No. 3,

of the androgen receptor by interleukin-6 and the role of steroid receptor coactivator-1 in prostate cancer cells. *J Biol Chem,* Vol. 277, No. 41, pp. 38087-38094.

Zhou, G., Hashimoto, Y., Kwak, I., Tsai, S.Y. & Tsai, M.J. (2003). Role of the steroid receptor coactivator SRC-3 in cell growth. *Mol Cell Biol*, Vol. 23, No. 21, pp. 7742-7755. Zwart, W., Griekspoor, A., Berno, V., Lakeman, K., Jalink, K., Mancini, M., Neefjes, J.,

pp.3534-3544.

Michalides, R. (2007). PKA-induced resistance to tamoxifen is associated with an altered orientation of ERalpha towards co-activator SRC-1. *EMBO J*, Vol. 26, No. 15,

## *Edited by Mehmet Gunduz and Esra Gunduz*

Cancer is the leading cause of death in most countries and its consequences result in huge economic, social and psychological burden. Breast cancer is the most frequently diagnosed cancer type and the leading cause of cancer death among females. In this book, we discussed various aspects of breast cancer carcinogenesis from clinics to its hormonebased as well as genetic-based etiologies for this deadly cancer. We hope that this book will contribute to the development of novel diagnostic as well as therapeutic approaches.

Breast Cancer - Carcinogenesis, Cell Growth and Signalling Pathways

Breast Cancer

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*Edited by Mehmet Gunduz and Esra Gunduz*

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Surgical and medical oncologists have been unable to decrease renal cell carcinoma mortality for uncertain reasons, although a lot of progress has been made in diagnosis and imaging, recognition of different genetic and pathological entities, management of localized disease and in the research on new drug treatments for advanced stages of the disease, potentially combined with surgery. The purpose of this book, which tackles a number of separate interesting topics, is to provide further insight into the disease and the management of early and advanced renal cell carcinoma. The volume is divided into different parts; the first part covers the characterization of renal masses and the second part covers rare distinct pathological entity. In the management section, active surveillance, partial nephrectomy and radiofrequency ablation are presented. A separate chapter reviews the management of Von Hippel Lindau disease, and finally, conventional and aberrant signaling pathways are explored.

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**Author details**

Rafael Bardera\*, Suthyvann Sor and Adelaida García-Magariño National Institute for Aerospace Technology, Madrid, Spain

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*Aerodynamics of Mars 2020 Rover Wind Sensors DOI: http://dx.doi.org/10.5772/intechopen.90912*


**Metamaterials and Metasurfaces**

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*Editors* Naser Ojaroudi Parchin School of Computing, Engineering & the Built Environment Edinburgh Napier University Edinburgh United Kingdom

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Raed A. Abd-Alhameed School of Engineering and Informatics University Bradford Bradford United Kingdom

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This is a reprint of articles from the Special Issue published online in the open access journal *Electronics* (ISSN 2079-9292) (available at: www.mdpi.com/journal/electronics/special issues/metamaterials metasurfaces).

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*Article* **Semantic Entropy in Language Comprehension**

#### **Noortje J. Venhuizen \*, Matthew W. Crocker and Harm Brouwer**

Department of Language Science & Technology, Saarland University, 66123 Saarbrücken, Germany<EMAIL_ADDRESS>(M.W.C<EMAIL_ADDRESS>(H.B.) **\*** Correspondence<EMAIL_ADDRESS>
Received: 30 October 2019; Accepted: 25 November 2019; Published: 27 November 2019

**Abstract:** Language is processed on a more or less word-by-word basis, and the processing difficulty induced by each word is affected by our prior linguistic experience as well as our general knowledge about the world. Surprisal and entropy reduction have been independently proposed as linking theories between word processing difficulty and probabilistic language models. Extant models, however, are typically limited to capturing linguistic experience and hence cannot account for the influence of world knowledge. A recent comprehension model by Venhuizen, Crocker, and Brouwer (2019, *Discourse Processes*) improves upon this situation by instantiating a comprehension-centric metric of surprisal that integrates linguistic experience and world knowledge at the level of interpretation and combines them in determining online expectations. Here, we extend this work by deriving a comprehension-centric metric of entropy reduction from this model. In contrast to previous work, which has found that surprisal and entropy reduction are not easily dissociated, we do find a clear dissociation in our model. While both surprisal and entropy reduction derive from the same cognitive process—the word-by-word updating of the unfolding interpretation—they reflect different aspects of this process: state-by-state expectation (surprisal) versus end-state confirmation (entropy reduction).

**Keywords:** natural language; entropy; neural networks