The biology of Epidermal Growth Factor Receptor (EGFR) from regulating cell cycle to promoting carcinogenesis: the state of art including treatment options

Receptor Tyrosine Kinases (RTKs) are cell surface receptors for many ligands such as polypeptide growth factors, cytokines, and hormones for which they have a high affi nity [1]. So far 58 RTKs have been identifi ed which are distributed in 20 families and amongst those, the Epidermal Growth Factor Receptor (EGFR) and its other family members (erbB2/ HER2, erbB3/ HER3, erbB4/HER4) have been discovered to play an important role in signalling and cancerogenesis [1]. These receptors can initiate intracellular signalling regulating cell proliferation and survival [2]. The mechanisms through which this happens and can be targeted by specifi c drugs is related to the structure and biology of the receptor itself [3]. The EGFR gene is located on the short arm of chromosome 7 (7p11.2) and encodes a 170-kDa type I transmembrane growth factor receptor with tyrosine kinase activity. All these trans-membrane proteins are composed of an extracellular ligand-binding domain, a transmembrane lipophilic domain, and an intracellular tyrosine kinase domain and all bind to receptor-specifi c ligands except HER2 [4].

term portion creates a docking site for proteins containing Src homology2 and phosphotyrosine-binding domains [5,6].
These proteins activate intracellular signalling by many different pathways: the RAS/RAF/MEK/MAPK pathway, the Phospatidylinositol3-kinase (PI3K)/PTEN/AKT pathway, Phospholipase C and the Signal Transducers and Activators of Transcription (STAT) pathway. Depending on which of these pathways is activated, the fi nal effect is cell proliferation or inhibition of apoptosis [7,8] The RAS/RAF/MEK/MAPK pathway regulates cell proliferation and survival through the activation of mitogenactivated protein kinases (MAPKs) which migrate in the nucleus and phosphorylate transcription factors involved in cell proliferation [8,9] MAPKs are activated through phosphorylation by Raf-1 which is activated by Ras-GTP after recruitment by Sos [10,11]. Sos is capable to recruit Ras-GDP and activate it to Ras-GTP because of a conformational change following EGFR phosphorylation which creates a docking site for Sos and the adaptor protein Grb2 [12,13].
The PI3K/PTEN/AKT pathway has been linked to cell growth, apoptosis resistance, migration and invasion [8,14]. PI3K is a dimeric enzyme containing a subunit (p85) of STAT proteins has been found in numerous primary cancers [15].

The Role of egfr mutations in cancerogenesis
All these effects are initiated by EGFR activation which can happen in three ways: ligand-dependent activation, ligandindependent activation and overexpression. Six EGFR ligands have been identifi ed which causes the dimerization of the receptor and its autophosphorylation [2,16]. On the contrary, ligand-independent activation is caused by stress such as radiation and fi nally overexpression of the EGFR which is mediated with interaction with integrins [2].
EGFR has long been studied with important discoveries regarding its role in cell signalling and potential oncogenic role dated back in the 1980s [17,18].
Through the years and with a better understanding of those pathways [19], it has been recognised that EGFR plays an important role in tumorigenesis with at least three different mechanisms: overexpression of EGFR ligands, receptor amplifi cation or activating mutation of EGFR [20]. The overall effect is the acquired ability of cells to proliferate continuously and avoid apoptosis which is one of the hallmarks of cancer described by Hanahan and Weinberg [21,22]. This involves bypassing a series of check-point that control the main phases of the replication cycle such as cell size control, DNA damage responses or monitoring DNA replication [23]. Besides playing an important role in initiating tumorigenesis in many solid tumours, genetic alteration of growth factor receptors has also clinical relevance in particularly in a subtype of carcinoma of the lung were is considered a predictive marker [24].

Types, frequency and effect of egfr mutations
In their comprehensive metanalysis, Pao, et al. [20], have summarised all type of mutations affecting EGFR tyrosine kinase domain known to date.
According to their review, 85.9% of the mutations reported happening in two "hot spots" in the EGFR gene between exon 19 and exon 21.  [20]. It has been postulated that all these mutations lead to conformational change (for example affecting the alfa C-helix or phosphate-binding loop) resulting in increased activity of the receptor and some cases to sensitivity to TK inhibitors [26][27][28]. In their study Shan et al. have gone into the depth of how mutations affect the receptor structure and function [29]. They started from the observation that the extracellular domain has a very low tendency for dimerisation in the absence of interaction with its ligand [30]. Then they described the conformational changes of the receptor, adding new data to those previously reported [31][32][33].
Amongst those, Zhang et al. described how asymmetric dimers are created upon stabilisation of one EGFR kinase (the receiver) by another EGFR kinase (the activator) through the placement of the alpha-C helix in a way that the catalytic KE salt bridge between Lys721 and Glu738 is maintained (alphaCinactive) [33]. However, EGFR can adopt also alphaC-out active conformation where the above-mentioned bridge is broken and the receptor is inactive [31,32]. Shan  supported by the higher dimerisation affi nity that the mutant receptor as compared to the wild-type counterpart. Eventually, they also discussed the possibility of lateral signal propagation involving the cytoplasmic portion of the receptor which happens during the dimerisation through the phosphorylation of a tyrosine residue (tyr845) in the activation loop which suppresses the intrinsic disorder [29].

Targeting egfr mutations: the advent of tyrosine kinase Inhibitors
The investigation of the structure of EGFR during the early 1990s, initially led to the identifi cation of a new molecule: gefi tinib, an anilinoquinazoline with antineoplastic activity targeting the EGFR-TK activity and other similar receptors [35].

Overcoming egfr tki resistance
Despite promising results, the response to these inhibitors was different amongst the patients therefore additional studies were conducted focusing on how to improve patient selection for these treatments [20]. A milestone, of course, was the discovery of somatic driver mutations in EGFR which led to a drastic shift in the clinical management of NSCLC from standard chemotherapy to precision medicine [38]. Additional studies and clinical trials (most relevant was IPASS study) were carried out and the most important fi nding was that presence of EGFR mutation is the best rationale to select patients to treat with TKI rather than clinic-pathological parameters [40]. Nevertheless, a higher frequency of those mutations was reported in Asiatic female, never smoker and with adenocarcinoma as histological type [40]. Even though most NSCLC patients harbouring TKI-sensitizing EGFR mutations show an initial pronounced response to EGFR-TKI treatment, they acquire resistance to these drugs after ~9 to 14 months of such therapy [38]. Many studies have been conducted to investigate the underlining mechanisms of acquired resistance to EGFR-TKIs: Amongst these are mutation in exon 20 of EGFR, MET amplifi cation, overexpression of hepatocyte growth factor (HGF), and activation of the Insulin-like Growth Factor 1 Receptor (IGF1R) [41].
The T790M mutation of EGFR is the most common mechanism of such an acquired resistance, having been detected in up to 50% of patients treated with either the fi rst- came out [47,48]. Although oncogenic ALK and ROS1 gene rearrangements in NSCLC are only found in respectively 4% and 2%, there are three drugs (crizotinib, ceritinib, and alectinib) available for the treatment of ALK-rearranged lung tumours, and one drug (crizotinib) is for ROS1-rearranged tumours [49]. Crizotinib became the fi rst ALK inhibitor to receive FDA approval for the treatment of patients with ALKpositive metastatic NSCLC in November 2013 [49]. Other relevant markers which have been investigated are BRAF, MET, RET, ERBB2 (HER2), and KRAS, ERCC1 but currently, no therapy is formally approved [50,51]. EGFR, KRAS, and ALK mutations in NSCLC are mutually exclusive and the presence of one or another can infl uence response to targeted therapy [52,53]. More recently the focus has been shifted towards the relationship between EGFR and immunotherapy. For example, how to better tailor the treatment of WT-EGFR lung cancer or those with T790M germline mutation. Research is still ongoing and immunotherapy seems to be a promising option, however, their role and its association with TKIs in EGFR positive NSCLC has still to be addressed.