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

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 to endocrine therapy in breast cancer patients.

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

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

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

Pre-clinical and early clinical trial.

Pre-clinical studies in animal models

receptors

early stage breast cancer prior to surgery.

Pre-clinical studies in animal models.

locally recurrent or metastatic breast cancer.

Phase I study in patients with advanced solid tumors.

A pilot study in combination with tamoxifen or letrozole in patients with

A phase 1b dose escalation in combination with paclitaxel in patients with

Yttrium 90-radiolabeled OTSA101 in phase I trial in patients with relapsed or refractory non-resectable synovial sarcomas

Pre-clinical studies and phase 2 trial in patients with advanced triplenegative breast cancer with or without genomic alterations in notch

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

is correlated with higher hormone therapy response rate independent of ER

the clinical management of this disease.

**Drug Target Indications/trials**

(NOTCH Pathway)

(NOTCH Pathway)

(NOTCH Pathway)

(NOTCH Pathway)

catenin pathway)

catenin pathway)

catenin pathway)

catenin pathway)

RO4929097 γ-secretase inhibitor

112 Breast Cancer and Surgery

MRK-003 γ-secretase inhibitor

MK-0752 γ-secretase inhibitor

PF-03084014 γ-secretase inhibitor

Salinomycin LRP6 (Wnt/β-

OMP-18R5 FZD7 (Wnt/β-

OMP-54F28 FZD8 (Wnt/β-

OTSA101 FZD10 (Wnt/β-

**4.2. Progesterone receptor (PR)**

noticed that PR+

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

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

Jagged2, Delta-like1 (Dll1, Dll3 and Dll4). The activation of Notch signaling pathway occurs by the interaction of DSL ligands with Notch receptors on adjacent cells. This interaction induces a proteolytic cleavage of the Notch protein at the S2 cleavage site mediated by two enzymes, Disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and Disintegrin and metalloproteinase domain-containing protein 17 (ADAM17). After this cleavage, the remaining part of the Notch protein will be cleaved by the γ-secretase enzyme complex and releases the Notch intracellular domain (NICD). Finally, NICD translocates to the nucleus and forms a complex with DNA-binding protein recombination signal binding protein for immunoglobulin Kappa J region (RBPj) and a member of the mastermindlike (MAML) family transcriptional coactivators (**Figure 4**). This complex activates various target genes of Notch pathway that are associated with tumorigenesis [42], cell growth,

Endocrine and Cell Surface Receptor Signaling in Breast Carcinogenesis

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A number of studies indicate that aberrant activation of Notch signaling has been observed in breast cancer. The high expression of Notch signaling pathway components including Dll1, Dll3 and Dll4, Jagged1–2 and Notch receptors has been observed in invasive breast cancer. This expression is associated with poor prognosis in breast cancer patients [43, 44]. Due to the important role of Notch signaling pathway in breast cancer, it becomes a very attractive therapeutic target. At present, several classes of Notch inhibitors have been developed and are under various clinical trials including γ-secretase inhibitor (GSI), monoclonal antibodies against Notch receptors or ligands and small interfering RNA (siRNA).GSI effectively represses cancer stem cells (CSCs) in *in vitro* studies and triggers apoptosis via inhibiting proteasome activity and enhancing endoplasmic reticulum (ER) stress in tumor cells [45]. Several GSIs have completed pre-clinical and early clinical trials including RO4929097 which significantly sensitizes putative breast cancer stem cells to ionizing radiation and may be effective in treating inflammatory breast cancer (IBC), MRK-003 in combination with trastuzumab induced tumor regression and prevented tumor recurrence post-trastuzumab

differentiation, and cell survival [43].

**Figure 4.** Basics of notch signaling pathway. Modified from Acar et al. [42].

**Figure 3.** TKIs and MAbs target HERs for the treatment of breast, lung and several other types of cancer. Modified from Alanazi and Khan [16].

transducer and activator of transcription protein 3 (STAT3) has been associated with trastuzumab resistance. This suggests that STAT3 acts as a prognostic indicator of trastuzumab resistance in primary HER2-positive breast cancer [39]. More than 50% of breast cancer patients with aggressive and chemotherapeutic resistant condition have been detected with the phosphorylation of STAT3. Abnormal STAT3 activation due to alterations in HER2, HER1, BRCA1, and ER leads to deregulation of cell proliferation, migration, survival and angiogenesis. Several up-regulated genes including hypoxia inducible factor 1 alpha (HIF-1α) as a result of unexpected STAT3 activation have been implicated in trastuzumab resistance. During their study, Aghazadeh and Yazdanparast found that HIF-1α is an essential signaling element required to downregulate phosphatase and tensin homolog (PTEN) via Hes Family BHLH Transcription Factor 1 (HES-1) repressor during the induction of trastuzumab resistance [39]. Similarly, Sonnenblick et al. provide convincing evidence for a link between phosphorylation of STAT3 and trastuzumab resistance in HER2 positive primary breast cancers. Patients with activated STAT3 may suggest novel approaches to block the STAT3 pathway in combination with trastuzumab treatment particularly in PTEN-deleted breast cancer [40]. Additionally, it has been observed that inhibition of IL6-STAT3 pathway in PTEN-deleted HER2 positive breast cancer leads to decrease in the population of cancer stem cells and inhibits the development of distance metastasis using IL6R antibody alone or in combination with trastuzumab [41].
