Marcela+Garcia-Final+Project

Marcela P. Garcia  Drexel University Department of Chemistry Drexel University, Philadelphia, PA 19104-2876  Submitted on December 4, 2010
 * Understanding the cell biology of the EGFR family and its ligands in human cancer cells **

**Abstract** The epidermal growth factor receptor (EGFR) is part of the ErbB family of receptor tyrosine kinases (1-3). EGFR plays an important role in cell proliferation, survival, migration and differentiation. In humans, there are more than 30 ligands and the EGFR family of four receptors (EGFR/ErbB-1, HER-2/ErbB-2, HER-3/ErbB-3, and HER-4/ErbB-4) can form a complex signal-transduction network (20-21). The receptor-ligand complex that is formed varies in the strength as well as in the type of cellular response that it induces (20-21). In fact, proteins in the ErbB family have the ability to form homodimers or heterodimers, a process that is then followed by ligand binding. As a consequence, each dimer portrays a different affinity for ligands and also for signaling properties (20). In this sense, studies have shown that EGFR is involved in the pathogenesis and progression of different carcinoma types. The EGFR and its proteins are often overexpressed in carcinoma cells and evidence suggests that these proteins have the ability to induce cell transformation (6, 14). Up to date, there are two major pathways in which EGFR works, one is the traditional EGFR signal transduction pathway, which is most commonly known and it has been well studied (16-18, 20). It transmits extra-cellular mitogenic signals (EGF and TGF-α) to activate downstream signaling cascades (16). The other pathway, which has only recently been discovered, is the nuclear EGFR pathway which involves cellular transport of EGFR from the cell surface to cell nucleus (16, 20). Since EGFR is a major characteristic of many human malignancies, it is important to get a better understanding of both the traditional and the nuclear pathway of EGFR because it will help in the development of new therapeutic treatments (14, 16, 20).

**1. Introduction**

The importance of growth factor driven signaling in the pathogenesis of human cancer was first proposed by Mike Sporn and Anita Roberts in 1985 (9), they suggested that cancer cells normally need less exogenously supplied growth factors to maintain a high rate of proliferation. The decrease in growth factor dependency could be accredited to the ability of tumor cells to produce high levels of peptide growth factors (9). Different studies have shown that there are various mechanisms that could contribute to intensify the signal driven by growth factors (9,10). For example, the participation of growth factors in maintaining the survival of cancer cells and in promoting tumor-induced angiogenesis has been shown, thus suggesting that growth factors help tumor development through different mechanisms (9,10). There are different families of growth factors and growth factor receptors that have been determined to be involved in the growth of cancer cells (6,11,17). Among them, the epidermal growth factor receptor (EGFR) and the EGF-family appear to have a central role in the pathogenesis and development of various carcinoma types (6,11,17). EGFR is also a member of the ErbB family of receptor tyrosine kinases (TK) (17). It is in fact EGFR the one that plays an important role in initiating the signaling that directs the behavior of epithelial cell origin (6,11,17). When talking about the role of EGFR signaling, it is important to take into account the complex interactions that are present within the ErbB family of receptors and growth factors (11). EGFR plays such an important role in regulating cellular proliferation, survival and metastasis that it is an attractive molecular target because if any of its activities are interrupted, signaling transduction might not occur (20-21). What sets tumor cells and normal cells apart is the over-expression of EGFR, which makes it easier for the EGFR inhibitors to act more selectively on tumor cells and control their aggressive behavior. The understanding of these interactions between receptor and ligand could lead to the development of new approaches designed to block EGFR signaling in cancer patients (20-21).

**2. ErbB family of receptors and their ligands**

The ErbB family of receptor tyrosine kinases is made up of four different receptors: the EGFR (also referred to as HER-1/ErbB1), HER-2 (also termed aErbB2 or Neu) HER-3/ErbB3 and HER-4/ErbB4 (Fig. 1) (16,20). HER-3 does not have much tyrosine kinase activity when compared to other receptors, while HER-2 displays a strong tyrosine kinase activity, but it does not have an evident ligand. Therefore, the main function of HER-2 is to serve as a co-receptor, it forms heterodimers with other types of EGFRs resulting in increased signal transduction on ligand binding (16,20). These transmembrane receptors contain an extracellular ligand-binding domain and a cytoplasmic region with enzymatic activity. This structure allows signals to be transmitted across the plasma membrane, where they can activate gene expression and also induce cellular responses, such as proliferation, survival, migration and differentiation (16,20-21).

Over-expression of HER-2 could be observed in breast cancer cells, it tends to be associated with a poor diagnosis and short survival rates (14-16). The expression of EGFR in normal cells has a range of 40,000 to 100,000 EGF receptors per cell, instead over-expression of EGFR is observed in a vast majority of tumors, some of which include breast cancer, head-and-neck cancer, renal cancer, non-small-cell lung cancer and ovarian cancer. Certain breast cancers could express as much as 2,000,000 EGF receptor molecules per cell (6). The result of this over-expression is an intense signal generation and activation of downstream signaling pathway, which in turn causes cells to have a more aggressive growth and invasivenesscaracteristics(6,9). Multiple ligands bind to and activate EGFR, for example, ErbB receptors are activated by binding to growth factors of the EGF-family which are expressed by the same cells that express ErbB receptors (6,16). The ErbB receptors can be divided into three different groups, the first group includes EGF, transforming growth factor α (TGF- α ) and Amphiregulin (AR), they all bind specifically to EGFR (16-18). Betacellulin (BTC), Heparin-Binding Growth Factor (HB-EGF) and Epiregulin (EPR) make part of the second group, they all portray dual specificity because can they bind to both EGFR and ErbB-4 (16-18). The third group is made of the Neuregulins (NRGs), which can in turn be divided into two subgroups based on their ability to bind to ErbB-3 and ErbB-4 or only to ErbB-4 (16-28). Out of all the ligands mentioned before, the most important stimulatory ones are EGF and TGF-α. This is because after ligand binding takes place, the receptor undergoes dimerisation, forming either homodimers or heterodimers, this is then followed by the internalization of the receptor-ligand complex and tyrosine autophosphorylation (Fig. 2) (16). Together, all of these events trigger a cascade of responses that mainly affect cell proliferation, survival, angiogenesis and even metastasis.



**3. EGFR Pathway**

The EGF pathway has two major routes. The first one is the traditional one, which involves the transduction of mitogenic signals via activating cascades of signaling molecules. The second one, involves the direct shuttling of EGFR by EGF stimulation, from the cell surface to the nucleus without any obvious intermediates (18,20-21).

**3.1. Traditional EGFR signal transduction pathway**

The transmembrane receptors that make up the EGFR family are composed of an extracelluar ligand-binding domain and also of a cytoplasmic region with enzymatic activity (17-18, 20). This structure is what permits signals to be transmitted across the plasma membrane of the cell where they can activate gene expression and induce proliferation, survival, and apoptosis (20-21). When the receptors are in isolation, the signal-transduction tyrosine kinase activity of the EGFR is inactive. This receptor is activated when ligands such as TGF-α, EGF and neuregulins bind to the extracellular domain, thus inducing the formation of homodimers or heterodimers (Fig. 3) (20). The tyrosine residues found on one of the receptors is cross-phosphorylated by another member of the receptor pair. Once this takes place, docking sites are formed for signaling complexes that are made up of enzymes and proteins (17-18,20). Then a continuous signaling complex composed of effector and adaptor proteins is assembled into the cytoplasm, where they can stimulate many different signaling transduction cascades. The inactivation of the EGFR is done by endocytosis of the receptor ligand-complex; the resulting endosomes are then degraded and sometimes even recycled to the cell surface (16-18). Finally, the dissociation of this complex leads to gene activation as well as cellular activation, such as malignant transformation, increase proliferation rate, tumor progression and/or resistance to chemotherapy (11,17-18).



**3.2. Nuclear EGFR pathway** The existence of EGFR in the nucleus was observed in hepatocytes that underwent regeneration almost a decade ago (9,16-17). Around that time, it was also observed that EGF and TGF-α were found to translocate into the nucleus of proliferating hepatocytes (9,16-17). Years later, nuclear EGFR was also detected in other cells and tissues, such as epithelial cells from the ovaries and the kidneys, thyroid and pregnant uterus (9). Very high levels of EGFR were also found in the nucleus of certain cancer cells, some of which include breast, bladder, cervix, thyroid and oral cavity (9,16). In addition to the EGFR, other receptors of the ErbB family have been found in the cell nucleus; among them are HER-2 and HER-3. There are also many tyrosine kinases that undergo nuclear transport, some of which are fibroblast growth factor receptor (FGFR), insulin receptor and TGF-β type I receptor (9). The main function of nuclear EGFR is to work as a transcription co-factor, that has the ability to interact as well as activate the Cyclin D1 gene, a positive regulator of proliferation (9). Another significant gene that works as a second transcriptional target of nuclear EGFR is the iNOS gene. Both genes respond positively to nuclear EGFR in cell culture and in certain tumor specimens (9,18). The mechanism of the nuclear EGFR-mediated gene regulation includes its own DNA-binding cofactor STAT3 and other transcription factors that have not been identified (9,18). Nuclear EGFR physically and functionally interacts with STAT3, which leads to activation of the iNOS promoter. This association of nuclear EGFR/STAT3 and iNOS promoter yields an increased expression of iNOS and nitric oxide (18). These results were further confirmed when an increased expression of iNOS and nitric oxide were reported in human cancers (9,18). Over-expression of iNOS is related to tumor growth advantage and angiogenesis. These observations indicate that the deregulation of iNOS expression may contribute to the malignant nature of tumors with a deregulated EGFR/STAT3 pathway (9,18).

**4. EGFR inhibitors** Some of the approaches that have been developed to understand the EGFR-family and their ligands have led to the development of therapeutic strategies to target EGFR levels in EGFR-mediated cellular effects; two of these strategies include monoclonal antibodies and tyrosine kinase inhibitors (6, 16, 20).

**4.1. Monoclonal Antibodies** Monoclonal antibodies have been developed to target different members of the EGRF-family; they are directed against the extracellular receptor domain. They exhibit greater specificity for EGFR when compared to the tyrosine kinase inhibitors (6,16). In addition, lower concentration of monoclonal antibodies is needed to achieve receptor inhibition, unlike the tyrosine kinase inhibitors, which require a higher dose. One of the problems that could arise from working with monoclonal antibodies is that they could induce an immune-antibody response (2,4). Monoclonal antibodies are also less effective against altered forms of EGFR, such as mutant types found in certain cells (2). Some of the agents that make part of this antibody group include antibodies to EGFR and monoclonal antibodies against HER-2, truncated monoclonal antibodies (scFv), and fusion ligands, which could be conjugated with toxins and antisense oligonucleotides (2,4,6).

**4.1.1. Antibodies to EGFR** Cetuximab (IMC-C225) is a human-murine chimeric IgG monoclonal antibody that binds to the extracellular domain of EGFR, thus preventing tyrosine kinase activation, inhibiting cell growth, and sometimes inducing apoptosis (6,7,10). A few of the preclinical studies that have been performed using this antibody indicate that the cetuximab does inhibit the proliferation of cell lines expressing EGFR, and in turn it increases the cytotoxic activity of chemotherapy and radiation (7, 8, 10). ABX-EGF is a humanized IgG2 monoclonal antibody with higher affinity for EGFR than cetuximab (7,19). It inhibits tyrosine phosphorylation in a dose-dependent manner because it blocks the EGF binding site on the receptor and it causes fast internalization of EGFR (19). Studies have shown it prevents solid tumor formation as well as eliminates large tumors in humans. ABX-EGF also appears to be synergistic with chemotherapy (7, 9, 10,19). EMD-7200 is another type of humanized monoclonal antibody that selectively binds to EGFR. It has revealed antiproliferative effects against squamous carcinoma cells in head and neck cancer (6). It behaves very similarly to MAvb-ICR62, which is a rat monoclonal antibody that blocks binding of EGF and TGF-α to EGFR. Studies done in vitro on this monoclonal antibody have demonstrated that it inhibits the growth of tumor cells that over-express EGFR and it also appears to remove EGFR-expressing tumors (6,7).

**4.1.2. Antibodies to HER-2** As stated before HER-2 is a marker for more aggressive tumor cancer cells, higher rate of recurrence and a poor diagnosis (16,20). Trastuzumab is a monoclonal antibody against the extracellular domain of HER-2. It was first developed for breast cancer cells because the over-expression of HER-2 is most commonly found in this type of cancer cells (17-18, 20). 2C4 is another antibody acting against the ectodomain of HER-, but at a different site than that of trastuzumab. It works specifically by inhibiting the association of HER-2 with other members of the EGFR-family and it does not react with trastuzumab. Studies have shown that it will inhibit the growth of both androgen dependent and androgen independent prostate tumors (16-18).

**4.1.3. Tyrosine Kinase Inhibitors** The most effective tyrosine kinase inhibitors are the small molecules that compete with and prevent the binding of adenosine triphosphate to the intracellular tyrosine kinase region (12-13, 16). These types of molecules have the ability to cause tumor regressions by increasing apoptosis and also by inhibiting cellular proliferation. There are only two compounds that are at an advanced stage of development, these are gefitinib and erlotinib, they both work by targeting EGFR (3-5). Gefitinib is highly selective for tyrosine kinase. It has the ability to prevent EGFR autophosphorylation in various EGFR-expressing human cancer cells, gefitinib does this by competing with adenosine triphosphate for its ligand binding site on the intracellular domain of EGFR (16, 18, 20). It also has the ability to interfere (noncompetitively) with signaling by EGFR ligands (16). Erlotinib is another EGFR inhibitor that can induce both the cell cycle in the G1 phase and apoptosis. This agent inhibits EGFR autophosphorylation with a much greater selectivity than other tyrosine kinase inhibitors and it also reduces EGFR-associated phosphotyrosine (3-5, 20). Another tyrosine kinase inhibitor is CI1033, it is a small molecular weight inhibitor like gefitinib and erlotinib, but unlike them it is not specific for EGFR and it can bind with other members of the ErbB family of receptor tyrosine kinase to cause inhibition (16, 18, 20). It is important to note that the binding of CI1033 is irreversible, while the binding of gefitinib and erlotinib is reversible. Studies have shown that CI1033 blocks cell growth in head and neck carcinoma cell lines (16, 18, 20).

**5. Summary** The EGFR family of growth-factor receptors is part of a complex signal transduction network which plays an important role in various cellular responses, such as cell proliferation, migration, survival, and adhesion (11). Inhibiting this signaling module could possible decrease the growth and development of cancer. Studies done on the mechanics of the EGFR signaling complex has lead to the development of many novel cancer therapies designed to inhibit EGFR signaling activity, such as monoclonal antibodies, tyrosine kinase inhibitors and even small molecules that can bind to EGFR to induce EGFR and HER-2 degradation (1,2,6,11). There is still a lot of work to be done on the nuclear EGFR signaling pathway. The research done thus far suggests the existence of this pathway, which is different from the traditional one because this new EGFR pathway involves a direct shuttling of EGFR, after it has been activated by EGF, from the cell surface into the cell nucleus (16, 20). Once inside, the main function of EGFR is to transcriptionally regulate gene expression (20). Its role in cellular responses still needs to be confirmed because it is not clear if nuclear EGFR plays a significant role in progression, metastatic growth and/or therapeutic responses to human cancers (16, 18, 20).

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