**4. Breast cancer receptors (ER, PR and HER2) and their involvement in cancer progression**

#### **4.1. Estrogen receptor (ER)**

**Subtypes ER, PgR, Her 2 Status**

108 Breast Cancer and Surgery

Luminal A ER + or PgR + or both, Her 2-

Luminal B ER + or PgR + or both, Her 2+

Her2−/+

Tumors that do not fill into any of these categories

Claudin low ER-, PR-, Her2- Mesenchymal

**Table 2.** Microarray classification of breast cancer [19].

markers

IHC, immunohistochemistry; ER, estrogen receptor; PgR, progesterone receptor, +, positive, − negative.

Basal-like ER-, PgR-,

Normal breast-like **Other IHC features Cell of origin Other characteristics**

Keratin 8/18 + ve Luminal epithelial cell Higher tumor grade

— Luminal epithelial cell 6–10% of all breast cancers

Stem cell 5–10% of all tumors

Bipotent progenitor

EGFR + ve Younger age

Keratin 5/6/17 + ve Basal/myoepithelial cell/

Her 2+ ER-, PgR-, Her2+ — Late luminal progenitor 20–25%

Best prognosis

Poorer prognosis

15%

BRCA 1

to other types

Poorer grade

Poor prognosis Frequent relapse

Small tumors Good prognosis More common in postmenopausal Associated with fibroadenomas

Typically triple negative Low expression of cell-cell junction proteins (like E-Cadherin)

Lymphocytic infiltrates

common to bones

Lymph nodes positive Early distant metastases

Low rates of recurrence Higher survival rate

Associated with hereditary

Poorer prognosis compared

Spread to axillary nodes, less

Keratin 8/18 + ve Luminal epithelial cell Younger age

Despite the fact that a large number of potentially valuable factors have been identified, only three receptors, the estrogen receptor-alpha (ER*α*), the progesterone receptor (PgR), and the HER2 are utilized in clinical practice, and their assessment is obligatory [21]. Approximately 70% of all breast cancers, which belong to the molecular subtypes luminal A or luminal B, express ER*α*. There is strong evidence demonstrating that estrogen plays an important role in the progression and development of breast cancers, although the causes behind these malignancies still remains uncertain [22]. ER alpha positive breast cancers depend on estrogen signaling for proliferation. Binding of estrogen to ERs leads to dimerization of the receptor which then translocates to the nucleus and binds estrogen response elements in the DNA sequence. This leads to cell proliferation as a result of stimulation of target genes [23]. ER*α* mediates a number of molecular signaling such as mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways which are involved in cell growth and proliferation [24] as can be seen in **Figure 2**.

**Figure 2.** The PI3K/AKT/mTOR and the RAS/RAF/MEK/MAPK pathways. Modified from Toss and Cristofanilli [25].

ER*α*-positive breast cancer depends on these signaling for proliferation. Therefore, the most effective approach to terminate or slow the growth of this type of cancer is by blocking estrogen action in the tumor using hormone therapies. For the past few decades, one of the most widely used drug for the treatment of breast cancer is tamoxifen, which is a selective ER modulator and acts as anantagonizer for ER*α* function. It has been utilized as a long-term adjuvant therapy and as preventative agent in a lot of women at increased risk for the disease. Also, fulvestrant, which acts as an anti-estrogen, downregulates ERα and has been approved for clinical use [22].

**Drug Target Indications/trials**

Tamoxifen ER Treatment of metastatic estrogen receptor positive breast cancer.

Fulvestrant ER Hormone receptor (HR)-positive, human epidermal growth factor

endocrine therapy. Anastrozole Aromatase enzyme Adjuvant treatment (treatment following surgery with or without

Adjuvant treatment of node-positive breast cancer in postmenopausal women following total mastectomy or segmental mastectomy. Adjuvant treatment of axillary node-negative breast cancer in women

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111

receptor 2 (HER2)-negative advanced breast cancer in postmenopausal women who have not been previously treated with endocrine therapy. HR-positive advanced breast cancer in postmenopausal women whose

HR-positive, HER2-negative advanced breast cancer or breast cancer that has spread to other parts of the body (metastatic), in combination with palbociclib or abemaciclib in women whose disease has progressed after

radiation) of postmenopausal women with hormone receptor positive early breast cancer approved for the initial treatment of postmenopausal women with hormone receptor positive or hormone receptor-unknown locally advanced or metastatic breast cancer and for the treatment of postmenopausal women with advanced breast cancer that has progressed

positive early breast cancer who have received 2–3 years of tamoxifen and are switched to exemestane for completion of a total of five consecutive

treatment of patients with HER2-positive early breast cancer at high risk

patients with primary inflammatory breast cancer (IBC) without HER2

metastatic breast cancer that overexpresses the HER2 receptor and for

overexpressed/amplified breast cancer, to follow adjuvant trastuzumab-

following total mastectomy or segmental mastectomy.

disease has progressed after endocrine therapy

following treatment with tamoxifen.

years of adjuvant hormonal therapy.

Letrozole Aromatase enzyme Adjuvant treatment of postmenopausal women with estrogen receptor positive early breast cancer. Exemestane Aromatase enzyme Adjuvant treatment of postmenopausal women with estrogen receptor

Trastuzumab HER2 Treatment of patients with HER2-overexpressing breast cancer.

of recurrence.

overexpression.

Neratinib HER2/HER1 Adjuvant treatment of adult patients with early stage HER2-

based therapy.

cancer.

Pertuzumab HER2 Used in combination with trastuzumab and chemotherapy as adjuvant

T-DM1 HER2 Used for the treatment of patients with metastatic HER2-positive breast

Cetuximab HER1 Pre-clinical and clinical studies especially in combination therapy to treat triple-negative breast cancer. Panitumumab HER1 Phase II study of Panitumumab, Nab-paclitaxel, and Carboplatin for

Erlotinib HER1 Phase I study of Erlotinib and Metformin in triple-negative breast cancer.

Lapatinib HER2/HER1 Treatment of postmenopausal women with hormone receptor positive

whom hormonal therapy is indicated.

Pre-clinical studies in triple-negative breast cancer.

Another class of breast cancer treatment drugs that have evolved are called aromatase inhibitors (AIs). These inhibitors include anastrozole, letrozole and exemestane as detailed in **Table 3**. AIs are able to inhibit the aromatase enzyme (a cytochrome P450 heme-containing protein), which is required for estrogen synthesis [26] During menopause, the level of estrogen decreases due to cessation of estrogen production by the ovaries. Hence, locally synthesized estrogen via breast adipose tissue plays crucial role in the survival and growth of ERα-positive breast tumors [27].Unfortunately, majority of patients treated with endocrine therapy develop resistance. This leads to the progression of disease and fatality. Several signal transduction pathways such as MAPK and PI3K are involved in tamoxifen resistance. These pathways are activated by growth factors including human epidermal growth factor receptor 2 (HER2). It has been noticed that MCF-7/HER2–18 tamoxifen-resistant model system that overexpress HER2 shows increased growth when cells are treated with tamoxifen. Several studies indicate that there exists a molecular cross-talk between the ER and HER2 pathways [28, 29]. Also, the mechanisms associated with AIs resistance share similarities with tamoxifen resistance, particularly in the upregulation of growth factor pathway such as HER2 and its dimerization partner epidermal growth factor receptor (EGFR/HER1) [30].

Retinoblastoma (RB) is the tumor suppressor protein that plays an important role in regulating the progression of cell cycle. This occurs by the RB inhibitory action on E2Fs which are a family of transcription factors that are crucial for the expression of S-phase genes. It has been noticed that deregulation in the RB pathway occurs in various cancers including breast cancer 2. About 50% of breast cancers overexpress cyclin D1 that lead to an aberrant phosphorylation of RB facilitating cell cycle progression [31]. Adjuvant tamoxifen-treated ER positive breast cancer patients having functional RB pathway have fewer breast cancer recurrences, while those with RB non-functional tumors have no benefit of tamoxifen. Therefore, knowing the RB status in breast cancer can be utilized as predictive factor to identify patients who will benefit from tamoxifen therapy [32]. Further, it has been observed that histone demethylase retinoblastoma-binding protein 2 (RBP2) has a potential to develop endocrine therapy resistance in breast cancer. Choi et al. demonstrated that tamoxifen resistance in vitro and in vivo occurs as a result of RBP2 overexpression, while knocking down RBP2 imparted tamoxifen sensitivity. The cooperation between RBP2 and ER coactivators and corepressors regulates a number of tamoxifen resistance-associated genes. Moreover, RBP2 increased IGF1R-HER2 cross-talk that lead to PI3K-AKT activation through demethylase activity-independent HER2 protein stabilization. Therefore, RBP2-mediated tamoxifen resistance might be overcome using combinational treatment with PI3K inhibitor and tamoxifen. ER-IGF1R-HER2 signaling cascade can be activated in various ways by RBP2 to induce tamoxifen resistance, thus making RBP2


ER*α*-positive breast cancer depends on these signaling for proliferation. Therefore, the most effective approach to terminate or slow the growth of this type of cancer is by blocking estrogen action in the tumor using hormone therapies. For the past few decades, one of the most widely used drug for the treatment of breast cancer is tamoxifen, which is a selective ER modulator and acts as anantagonizer for ER*α* function. It has been utilized as a long-term adjuvant therapy and as preventative agent in a lot of women at increased risk for the disease. Also, fulvestrant, which acts as an anti-estrogen, downregulates ERα and has been approved

Another class of breast cancer treatment drugs that have evolved are called aromatase inhibitors (AIs). These inhibitors include anastrozole, letrozole and exemestane as detailed in **Table 3**. AIs are able to inhibit the aromatase enzyme (a cytochrome P450 heme-containing protein), which is required for estrogen synthesis [26] During menopause, the level of estrogen decreases due to cessation of estrogen production by the ovaries. Hence, locally synthesized estrogen via breast adipose tissue plays crucial role in the survival and growth of ERα-positive breast tumors [27].Unfortunately, majority of patients treated with endocrine therapy develop resistance. This leads to the progression of disease and fatality. Several signal transduction pathways such as MAPK and PI3K are involved in tamoxifen resistance. These pathways are activated by growth factors including human epidermal growth factor receptor 2 (HER2). It has been noticed that MCF-7/HER2–18 tamoxifen-resistant model system that overexpress HER2 shows increased growth when cells are treated with tamoxifen. Several studies indicate that there exists a molecular cross-talk between the ER and HER2 pathways [28, 29]. Also, the mechanisms associated with AIs resistance share similarities with tamoxifen resistance, particularly in the upregulation of growth factor pathway such as HER2 and its

dimerization partner epidermal growth factor receptor (EGFR/HER1) [30].

Retinoblastoma (RB) is the tumor suppressor protein that plays an important role in regulating the progression of cell cycle. This occurs by the RB inhibitory action on E2Fs which are a family of transcription factors that are crucial for the expression of S-phase genes. It has been noticed that deregulation in the RB pathway occurs in various cancers including breast cancer 2. About 50% of breast cancers overexpress cyclin D1 that lead to an aberrant phosphorylation of RB facilitating cell cycle progression [31]. Adjuvant tamoxifen-treated ER positive breast cancer patients having functional RB pathway have fewer breast cancer recurrences, while those with RB non-functional tumors have no benefit of tamoxifen. Therefore, knowing the RB status in breast cancer can be utilized as predictive factor to identify patients who will benefit from tamoxifen therapy [32]. Further, it has been observed that histone demethylase retinoblastoma-binding protein 2 (RBP2) has a potential to develop endocrine therapy resistance in breast cancer. Choi et al. demonstrated that tamoxifen resistance in vitro and in vivo occurs as a result of RBP2 overexpression, while knocking down RBP2 imparted tamoxifen sensitivity. The cooperation between RBP2 and ER coactivators and corepressors regulates a number of tamoxifen resistance-associated genes. Moreover, RBP2 increased IGF1R-HER2 cross-talk that lead to PI3K-AKT activation through demethylase activity-independent HER2 protein stabilization. Therefore, RBP2-mediated tamoxifen resistance might be overcome using combinational treatment with PI3K inhibitor and tamoxifen. ER-IGF1R-HER2 signaling cascade can be activated in various ways by RBP2 to induce tamoxifen resistance, thus making RBP2

for clinical use [22].

110 Breast Cancer and Surgery


despite the fact that the expression levels of ER and PR are correlated. Tumors with ER<sup>+</sup>

/PR−

Transmembrane receptor tyrosine kinases family (RTKs) consists of HER1 (epidermal growth factor receptor [EGFR]), HER2, HER3 and HER4. These receptors are involved in regulating a range of cellular processes that controls cell growth, differentiation, survival and migration. HER2 gene overexpression has been reported in 20–30% of patients with breast cancer [16, 36]. HER receptors are located at the plasma membrane and get activated by ligand binding to the extracellular domain. This binding induces the formation of homodimers or heterodimers [36]. While HER2 does not have a known ligand, HER3 is kinase-inactive and thus both these receptors signal by heterodimerization. Dimerization of these receptors leads to the phosphorylation of tyrosine residues within the receptor intracellular tyrosine kinase domain (cytoplasmic domain). These residues work as docking sites for adaptor proteins containing Src homology 2 and phosphotyrosine binding domains (PTB). These proteins activate a large number of signal transduction molecules such as stress-activated protein kinase and signal transducer and activator of transcription (STATs) and protein kinase B (PKB or AKT) and subsequent stimulation of downstream signaling pathways including STAT, PI3K and MAPK pathways. These signaling pathways are able to activate a wide range of cellular responses such as survival, proliferation, cell motility and differentiation [16]. Overexpression of HER2 and HER1 is responsible for poor clinical prognosis including unfavorable response

There are two types of therapeutic strategies that have been utilized against breast cancers overexpressing HER1 and HER2 receptors. The first includes monoclonal antibodies (MAbs) that bind to the extracellular domain of the receptor interfering with the binding of endogenous ligands that activates these receptors. In the second strategy, small molecule inhibitors (tyrosine kinase inhibitors [TKIs]) bind to the tyrosine kinase domain and inhibit its kinase

Anti-HER2 antibodies have been successfully utilized in the treatment of various cancers overexpressing HER2 including breast cancer. These antibodies target the extracellular domain of HER2 and prevent receptor activation. Several monoclonal antibodies have been used including trastuzumab and Pertuzumab. They bind to extracellular domain of HER2 in order to suppress its activity by preventing receptor dimerization and subsequent phosphorylation of the tyrosine kinase domain. As a result, the initiation of downstream signaling pathways gets precluded [37].In addition, a number of TKIs such as gefitinib, lapatinib and neratinib that are approved for treatment of breast cancers binds to HER1 and HER2 tyrosine kinase domains and inhibits their downstream signaling pathways [38]. A major clinical challenge in the treatment of HER2-positive breast cancer is due to resistance to the HER2-targeted antibody trastuzumab. Aghazadeh and Yazdanparast indicated that the over-activation of signal

activity and subsequent downstream signaling (**Figure 3** and **Table 3**) [16].

tial information about how tumors will respond to hormone therapies [35].

have higher response rate compared to ER<sup>+</sup>

to endocrine therapy in breast cancer patients.

**4.4. HER1 and HER2 as therapeutic targets**

**4.3. Human epidermal growth factor receptor 2 (HER2)**

/PR<sup>+</sup>

113

tumors. Thus, the status of PR provides essen-

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Endocrine and Cell Surface Receptor Signaling in Breast Carcinogenesis

**Table 3.** Approved and investigational drugs targeting estrogen receptor and other signaling pathway components.

as a potential therapeutic target for ER-driven cancer [33]. Despite these findings the mechanisms of endocrine therapy resistance are poorly understood making it a major challenge in the clinical management of this disease.

## **4.2. Progesterone receptor (PR)**

Human breast cancers rely on estrogen and/or progesterone hormones for growth, and this effect is mediated through ERs and PRs. ER or PR positive tumors represent up to two thirds of invasive breast cancers in women whose age are less than 50 years, while about 80% of tumors in women with age above 50 years are ER positive [34]. ER*α* regulates the expression of PR. Therefore, the detection of PR normally indicates that the estrogen-ER*α* pathway is intact and functional. Once PR is expressed, the hormone progesterone activates PR leading to the upregulation of several crucial cellular function such as proliferation contributing to breast cancer growth [21]. One of the most important parameter in breast cancer management is determining the response of tumor to hormone therapy as not all patients benefit from these therapies. Patients with breast cancer overexpressing ERs and PRs are more likely to respond to hormone therapy, while tumors negative for these receptors are unlikely to get benefit from them and respond better to chemotherapy [35]. Hormone therapy provides better quality of living and improves survival. Higher expression level of PR is associated with better hormone therapy response, increased survival rate and longer time for treatment failure. It has been noticed that PR+ is correlated with higher hormone therapy response rate independent of ER despite the fact that the expression levels of ER and PR are correlated. Tumors with ER<sup>+</sup> /PR<sup>+</sup> have higher response rate compared to ER<sup>+</sup> /PR− tumors. Thus, the status of PR provides essential information about how tumors will respond to hormone therapies [35].
