The ErbB Signaling Network and Its Potential Role in Endometrial Cancer

Endometrial cancer (EC) is the second most common malignancy of the female reproductive system worldwide. The updated EC classification emphasizes the significant role of various signaling pathways such as PIK3CA-PIK3R1-PTEN and RTK/RAS/β-catenin in EC pathogenesis. Some of these pathways are part of the EGF system signaling network, which becomes hyperactivated by various mechanisms and participates in cancer pathogenesis. In EC, the expression of ErbB receptors is significantly different, compared with the premenopausal and postmenopausal endometrium, mainly because of the increased transcriptional activity of ErbB encoding genes in EC cells. Moreover, there are some differences in ErbB-2 receptor profile among EC subgroups that could be explained by the alterations in pathophysiology and clinical behavior of various EC histologic subtypes. The fact that ErbB-2 receptor expression is more common in aggressive EC histologic subtypes (papillary serous and clear cell) could indicate a future role of ErbB-targeted therapies in well-defined EC subgroups with overexpression of ErbB receptors.

Current evidence does not support any screening methodology for early detection of EC, as cervical cytology performs poorly [23].However, the fluorescence in situ hybridization test (FISH) in vaginal swab specimens has shown promising results in EC detection [24].Artificial intelligence and machine learning algorithms have been proposed to assist in the discrimination between benign and malignant endometrial nuclei, obtained via image analysis and measured from liquid-based cytology slides and lesions so far.These show promising results, as their performance appears to be similar to that of traditional regression models in EC [25][26][27][28].Moreover, biospectroscopy has several applications in biomedical science, from detecting toxins and pollutants in the human body to identifying areas of stem cells in human tissue.It can effectively identify biomarkers of disease states at many organ sites without the need for staining or isotopic labeling [29,30].Apart from that, pelvic ultrasound scans or saline infusion sonograms can be offered on a 1-2-yearly basis in the context of routine gynecological examination, except for individuals undergoing close follow-up for hereditary, non-polyposis colon cancer (HNPCC) [31].
In the past, the sporadic classification of EC cases was based on clinical, metabolic, endocrine and pathological features [32,33].More recently, genomic data including somatic mutation rates, frequency of copy number alterations and MSI status have been used to create an updated EC classification, reflecting the increased impact of molecular biology in disease progression and patients' outcome [34,35].Moreover, the updated EC classification emphasizes the significant role of various signaling pathways, such as PIK3CA-PIK3R1-PTEN and RTK/RAS/β-catenin, in EC pathogenesis [34][35][36].
Our aim is to provide an update on current knowledge of the signaling network of ErbB receptors and their participation in cancer pathogenesis, as well as their potential clinical role in EC cases.

Physiology of ErbB Receptors
The EGF system is present in various human organs and plays a significant role in cell proliferation, differentiation, migration and apoptosis during embryogenesis and postnatal development [39,43,44].

Receptor Homodimerization and Heterodimerization
There are two distinct conformations of the extracellular ligand-binding domain, based on the activation status of EGFR, ErbB-3 and ErbB-4 receptors: 1. Closed conformation.When ErbB receptors are inactive, there are intramolecular interactions between the cysteine-rich subdomains CR1 and CR2, causing closed conformation of the extracellular ligand-binding domain [44][45][46]51,52].
2. Open conformation.When ErbB receptors become active, the leucine-rich subdomains L1 and L2 create a ligand-binding pocket, allowing interactions with a single ligand, while the extracellular ligand-binding domain takes an open conformation and the β-hairpin loop dimerization arm of subdomain CR1 is exposed [44][45][46]51,52].

Intracellular Tyrosine Kinase Activation
Following homodimerization and heterodimerization of ErbB receptors, conformational changes of the intracellular tyrosine kinase domain take place, which in turn cause tyrosine kinase activation and phosphorylation of the tyrosine-containing C-terminal tail [44][45][46]55,56].
The interaction between Sos and Ras causes conformational changes and allosteric activation of Sos through a rotation of its REM domain [72][73][74][75].The allosteric activation of Sos allows Ras binding and promotes replacement of GDP with GTP in Ras that leads to Ras activation (Ras-GTP) and initiation of the Ras pathway [72,73,75,76].
More specifically, Ras-GTP recruits and dimerizes Raf-1 protein kinase on the inner side of the cell membrane, in order to activate it through tyrosine phosphorylation [72,77,78].Subsequently, activated Raf-1 interacts and activates MAPK/ERK kinase (MEK1 and MEK2), which in turn phosphorylates, activates and anchors to the cytoplasm downstream proteins such as extracellular signal-regulated kinases (ERK1 and ERK2) [64,72].Then, activated ERK1 and ERK2 translocate to the nucleus in order to phosphorylate and activate various nuclear transcription factors involved in cell proliferation, differentiation and migration [63,64,72].
Overall, the Ras/Raf/MAPK pathway is implicated in a wide variety of cellular biological functions, but is also related to tumorigenesis [64,72].

PI3K/Akt Pathway
The phosphatidylinositol 3-kinase (PI3K)/Akt pathway has an essential role in cell biology, mainly in transduction of extracellular signals to intracellular messages [79].Ιt is actively involved in cell cycle regulation (proliferation, migration and apoptosis) and cytoskeletal rearrangement [80].
Following ErbB receptor activation and phosphorylation of the tyrosine-containing C-terminal tail, the activated ErbB receptor directly recruits the PI3K (subclass IA) The interaction between Sos and Ras causes conformational changes and allosteric activation of Sos through a rotation of its REM domain [72][73][74][75].The allosteric activation of Sos allows Ras binding and promotes replacement of GDP with GTP in Ras that leads to Ras activation (Ras-GTP) and initiation of the Ras pathway [72,73,75,76].
More specifically, Ras-GTP recruits and dimerizes Raf-1 protein kinase on the inner side of the cell membrane, in order to activate it through tyrosine phosphorylation [72,77,78].Subsequently, activated Raf-1 interacts and activates MAPK/ERK kinase (MEK1 and MEK2), which in turn phosphorylates, activates and anchors to the cytoplasm downstream proteins such as extracellular signal-regulated kinases (ERK1 and ERK2) [64,72].Then, activated ERK1 and ERK2 translocate to the nucleus in order to phosphorylate and activate various nuclear transcription factors involved in cell proliferation, differentiation and migration [63,64,72].
Overall, the Ras/Raf/MAPK pathway is implicated in a wide variety of cellular biological functions, but is also related to tumorigenesis [64,72].

PI3K/Akt Pathway
The phosphatidylinositol 3-kinase (PI3K)/Akt pathway has an essential role in cell biology, mainly in transduction of extracellular signals to intracellular messages [79].It is actively involved in cell cycle regulation (proliferation, migration and apoptosis) and cytoskeletal rearrangement [80].
In particular, PIP3 directly recruits Akt to the cell membrane via its PH domain and this results in Akt conformational changes and exposure of two crucial amino-acid residues (Thr308 and Ser473) [79,80,83].Thr308 is phosphorylated by PDK1, while Ser473 is phosphorylated by PDK2 [79][80][81]83,84].Both phosphorylation events are necessary for full Akt activation, which in turn phosphorylates many cytoplasmic and nuclear proteins and regulates a wide range of cellular processes involved in protein synthesis, cell cycle progression and cell survival [79][80][81][82][83][84].
It is interesting to note that specific docking sites for the PI3K (subclass IA) regulatory subunit are present on the ErbB-3 receptor, while they are absent on the EGFR receptor [63,85].Moreover, EGFR-dependent PI3K activation occurs either through EGFR and ErbB-3 dimerization or through a Gab-1 docking protein [63,86].
Overall, the PI3K/Akt pathway is implicated in various cellular processes and plays an important role in carcinogenesis [63,80].

STAT Pathway
The signal transducers and activators of transcription (STAT) pathway has a principal role in cell biology, mainly as a transducer of extracellular cytokine signals to cellular responses [87][88][89].It is actively involved in cell cycle regulation (proliferation, differentiation, migration and apoptosis) [87][88][89].
Following ErbB receptor activation and phosphorylation of the tyrosine-containing C-terminal tail, the activated ErbB receptor can cause JAK-independent tyrosine phosphorylation of STAT proteins, probably via the Src kinase [87][88][89][90].Phosphorylated and activated STAT proteins create dimers via SH2 domain interactions and translocate to the nucleus, where they bind to specific DNA sequences in gene promoters and regulate gene transcription [87][88][89]91].

Src Kinase Pathway
The Src kinase pathway has a critical role in cell biology, especially as a transducer of extracellular signals to cellular responses [92].It is actively involved in cell cycle regulation (proliferation, adhesion, migration and apoptosis), integrin signaling and angiogenesis [91,92].
Following ErbB receptor activation and phosphorylation of the tyrosine-containing C-terminal tail, the activated ErbB receptor recruits Src kinase via its SH2 domain and causes Src activation [91,93].Subsequently, activated Src acts as signal transducer and enhancer of ErbB receptor activation [63,94,95].
Overall, the Src kinase pathway is implicated in many cellular processes and plays an important role in carcinogenesis [63,91,96,97].

PLCγ/PKC Pathway
The phospholipase Cγ (PLCγ)/protein kinase C (PKC) pathway has an essential role in cell biology, mainly in transduction of extracellular signals to intracellular messages [46,98].It is actively involved in cell cycle regulation (proliferation, differentiation and migration) and angiogenesis [46,98].
Overall, the PLCγ/PKC pathway is implicated in many cellular processes and plays an important role in carcinogenesis [46,98].
However, heritable changes in gene function without alterations in the DNA sequence (epigenetic changes) could possibly affect ErbB-mediated signal transduction and gene transcription via several mechanisms [104,105]: DNA methylation is an extensively studied mechanism of epigenetic alterations [106].DNA methylation patterns (methylation and demethylation) are regulated by specific enzymes and subsequently affect gene transcription [106,107].
More specifically, DNA methylation is catalyzed by the family of DNA methyltransferase (DNMT) enzymes, which transfer methyl groups from S-adenosyl-L-methionine (SAM) to cytosine residues and form 5-methylcytosine (5-mC) [106,108].The majority of DNA methylation occurs in CpG islands, in which cytosine is followed by a guanine [106].Most CpG islands are present in promoters and their methylation leads to transcriptional silencing [106,108].Especially in ErbB signaling, PTEN promoter hypermethylation suppresses PTEN expression and activity, with a direct effect on PI3K/Akt pathway signaling [104,109].
Likewise, DNA demethylation is achieved either by active enzymatic demethylation or by passive replication-dependent on the dilution of methylation [108].Particularly in active enzymatic demethylation, 5-mC undergoes a series of oxidation reactions catalyzed by the methylcytosine dioxygenases Ten-Eleven-Translocation (TET) enzymes [108,110].The 5-hydroxymethylcytosine (5hmC) is the first intermediate of active DNA demethylation [108].Enrichment of 5hmc in promoter regions is often associated with activation of gene expression [108].In this way, DNA demethylation links to genomic instability [106,108].Especially in ErbB signaling, Ras promoter hypomethylation enhances Ras expression and activity, with a direct effect on signaling of the Ras/Raf/MAPK and PI3K/Akt pathways [104].

Histone Modification
Histone modifications represent another mechanism of epigenetic alterations [106].They affect lysine and arginine residues on histone tails, which are targets of covalent post-transcriptional modifications (acetylation, methylation, phosphorylation and ubiquitylation) [106,108].
More specifically, histone acetylation occurs through the addition of an acetyl group to the lysine residues in histone tails [106].Histone acetyltransferases (HATs) add acetyl groups and are associated with active gene transcription at promoter and enhancer sites [106].In contrast, histone deacetylases (HDACs) remove acetyl groups and are associated with gene silencing and transcriptional repression [106].Especially in ErbB signaling, EGFR acetylation by CREB-binding protein (CBP) acetyltransferase affects receptor phosphorylation and subsequent activation [104,111].
Likewise, histone methylation occurs through the addition of methyl groups to the arginine or lysine residues in histone tails [106].Histone methyltransferases (HMTs) add methyl groups and are associated with both active gene transcription and gene repression [106].In contrast, histone demethylases (HDMs) remove methyl groups [106].
More specifically, in malignant transformation, the EGF system becomes hyperactivated with the following four main mechanisms: gain of function mutations, genomic amplification, chromosomal rearrangements and autocrine activation [40][41][42].

Gain of Function Mutations
The gain of function (GOF) mutations may have a crucial role in carcinogenesis, as they generate novel protein isoforms with new and important functions [125].Based on their consequences for cancer development, GOF mutations could be further subclassified into driver and passenger mutations [125,126].Driver mutations provide a selective cell growth advantage and promote cancer development, while passenger mutations do not confer any cell growth advantage and do not contribute to carcinogenesis [126,127].
Especially in the EGF system, GOF mutations could possibly affect most domains of an ErbB receptor and lead to aberrant downstream signaling [42].More specifically, a GOF mutation usually involves the bilobed tyrosine kinase domain of an ErbB receptor and causes tyrosine kinase hyperactivation and aberrant downstream signaling, as well as conferring oncogenic properties [42].However, GOF mutations could also affect various ErbB receptor domains (the extracellular ligand-binding domain, transmembrane domain and short juxtamembrane section) and cause receptor activation using alternative mechanisms [42].

Genomic Amplification
Genomic amplification is the copy number increase in a specific region of the genome and is associated with overexpression of the amplified genes [128,129].It usually occurs during development and carcinogenesis and may be promoted by common chromosomal fragile sites, errors in DNA replication or telomere dysfunction [129,130].Amplified sequences can be organized as extrachromosomal elements, repeated units at a single locus or interspersed throughout the genome [128,129].
Especially in the EGF system, genomic amplification and subsequent ErbB receptor overexpression leads to increased receptor local concentration, constitutive receptor activation, avoidance of receptor regulatory mechanisms and aberrant downstream signaling [42,131,132].More specifically, ErbB-2 overexpression causes constitutive ErbB-2 activation as well as EGFR ligand-independent activation [131].Moreover, ErbB-2 overexpression inhibits down-regulation mechanisms of ErbB-2 and EGFR [131].

Chromosomal Rearrangements
Chromosomal rearrangements have important roles in carcinogenesis and include deletions, duplications, inversions and translocations [133].They are mainly caused by either defective DNA double strand break repair or faulty DNA replication [134].Based on their effect on chromosomes, they could be further subclassified into simple and complex [134].Simple chromosomal rearrangement results from a single fusion that preserves genetic information but sometimes disrupts regulation of the genes involved [134].In contrast, complex chromosomal rearrangement results from multiple fusions at a single locus that cause changes in genetic content and in chromosomal linear structure [134].Overall, chromosomal rearrangements lead to either hybrid gene formation or gene dysregulation [133,134].
Especially in the EGF system, chromosomal rearrangements cause the formation of fusion oncoproteins, consisting partly of the ErbB receptor and partly of the fusion partner [42,135].These fusion oncoproteins have remarkable structural similarities, can be membrane bound or cytoplasmic, and contain an activated tyrosine kinase domain [42,135].

Autocrine Activation
Autocrine activation is a type of self-stimulation in which a cell secretes a hormone-like factor that binds functional receptors on the same cell [136].This type of cell signaling has a significant role in carcinogenesis, particularly in cases of constitutive autocrine activation [136][137][138].
Especially in the EGF system, autocrine activation of ErbB receptors is a well described phenomenon that leads to downstream signaling via several pathways and may confer oncogenic properties [42,139,140].

ErbB Receptors in Endometrial Cancer
During the menstrual cycle, there is a wide variation in the profile of ErbB receptors, indicating a central role of the EGF system in the regulation of endometrial cyclical growth and shedding [141,142].
In EC, the expression of ErbB receptors is significantly different, compared with the premenopausal and postmenopausal endometrium [141,143,144].This is mainly because of the increased transcriptional activity of ErbB encoding genes in EC cells [144].
Moreover, the exact frequency of ErbB-2 overexpression and ErbB-2 gene amplification in type II EC remains controversial, as there are many racial differences [149,151,157,164,165].More specifically, ErbB-2 overexpression and ErbB-2 gene amplification are more common in African-American patients with type II EC, when compared with Caucasian individuals [164,165].
ErbB-3 overexpression is reported in 30% of unselected EC cases [141,153].More specifically, ErbB-3 overexpression is more common in well differentiated tumors when compared with moderately and poorly differentiated ones [141].
Overall, there are some differences in ErbB-2 receptor profile in selected EC patients (EC histologic subtypes and racial-ethnic subgroups) [143,150,157,164].ErbB-2 receptor expression is more common in papillary serous and clear cell EC cases [143,150,157].This is mainly based on differences in the pathophysiology and clinical behavior of various EC histologic subtypes [143,150,157].

Clinical Role in Endometrial Cancer
The relationship of the ErbB receptors profile with disease stage, tumor grade and response to treatment remains controversial in EC cases [149,153].
In particular, the clinical role of EGFR overexpression has not been studied thoroughly in EC patients [149,153].Some studies demonstrate an association between EGFR overexpression and poor clinical outcome, while others report otherwise [145][146][147][148].It seems that EGFR overexpression may have a dual role in EC cases [149].EGFR overexpression in type I EC is associated with less aggressive disease and more favorable outcomes [149,151,153,157].In contrast, EGFR overexpression in type II EC is associated with more aggressive disease and adverse clinical outcomes [149,151,153,157].
It becomes apparent that ErbB-2 receptor expression is more common in aggressive EC histologic subtypes (papillary serous and clear cell) [143,150,157].This possibly indicates a future role of ErbB-targeted therapies in well-defined EC subgroups with overexpression of ErbB receptors [150,157,172].

Conclusions
Overall, the EGF system signaling network becomes hyperactivated with various mechanisms and possibly participates in EC pathogenesis via several signaling pathways [37][38][39][40][41][42].There are some differences in ErbB-2 receptor profile among EC subgroups that could be explained by the differences in pathophysiology and clinical behavior of various EC histologic subtypes [143,150,157].
The fact that ErbB-2 receptor expression is more common in aggressive EC histologic subtypes (papillary serous and clear cell), might indicate a future role of ErbB-targeted therapies in well-defined EC subgroups with overexpression of ErbB receptors [150,157,172].In this context, future studies are needed in order to evaluate thoroughly the effectiveness of ErbB-targeted therapies as single agents or adjuvant treatment in well-defined EC subgroups with overexpression of ErbB receptors [7,21,[172][173][174][175][176][177][178].

Table 1 .
ErbB ligands and their affinity for ErbB receptors.