Information

8.5: Receptor Tyrosine Kinases (RTKs) - Biology


Receptor tyrosine kinases mediate responses to a large number of signals, including peptide hormones like insulin and growth factors like epidermal growth factor. Like the GPCRs, receptor tyrosine kinases bind a signal, then pass the message on through a series of intracellular molecules, the last of which acts on target proteins to change the state of the cell.

As the name suggests, a receptor tyrosine kinase is a cell surface receptor that also has a tyrosine kinase activity. The signal binding domain of the receptor tyrosine kinase is on the cell surface, while the tyrosine kinase enzymatic activity resides in the cytoplasmic part of the protein (see figure above). A transmembrane alpha helix connects these two regions of the receptor.

What happens when signal molecules bind to receptor tyrosine kinases?

Binding of signal molecules to the extracellular domains of receptor tyrosine kinase molecules causes two receptor molecules to dimerize (come together and associate). This brings the cytoplasmic tails of the receptors close to each other and causes the tyrosine kinase activity of these tails to be turned on. The activated tails then phosphorylate each other on several tyrosine residues. This is called autophosphorylation.

The phosphorylation of tyrosines on the receptor tails triggers the assembly of an intracellular signaling complex on the tails. The newly phosphorylated tyrosines serve as binding sites for signaling proteins that then pass the message on to yet other proteins. An important protein that is subsequently activated by the signaling complexes on the receptor tyrosine kinases is called Ras.

The Ras protein is a monomeric guanine nucleotide binding protein that is associated with the cytosolic face of the plasma membrane (in fact, it is a lot like the alpha subunit of trimeric G-proteins). Just like the alpha subunit of a G- protein, Ras is active when GTP is bound to it and inactive when GDP is bound to it.Also, like the alpha subunit, Ras can hydrolyze the GTP to GDP.

When a signal arrives at the receptor tyrosine kinase, the receptor monomers come together and phosphorylate each others' tyrosines, triggering the assembly of a complex of proteins on the cytoplasmic tail of the receptor. One of the proteins in this complex interacts with Ras and stimulates the exchange of the GDP bound to the inactive Ras for a GTP. This activates the Ras.


Activated Ras triggers a phosphorylation cascade of three protein kinases, which relay and distribute the signal. These protein kinases are members of a group called the MAP kinases (Mitogen Activated Protein Kinases). The final kinase in this cascade phosphorylates various target proteins, including enzymes and transcriptional activators that regulate gene expression.

The phosphorylation of various enzymes can alter their activities, and set off new chemical reactions in the cell, while the phosphorylation of transcriptional activators can change which genes are expressed. The combined effect of changes in gene expression and protein activity alter the cell's physiological state.

Once again, in following the path of signal transduction mediated by RTKs, it is possible to discern the same basic pattern of events: a signal is bound by the extracellular domains of receptor tyrosine kinases, resulting in receptor dimerization and autophosphorylation of the cytosolic tails, thus conveying the message to the interior of the cell.

The message is passed on via a signalling complex to Ras which then stimulates a series of kinases. The terminal kinase in the cascade acts on target proteins and brings about in changes in protein activities and gene expression.

The descriptions above provide a very simple sketch of some of the major classes of receptors and deal primarily with the mechanistic details of the steps by which signals received by various types of receptors bring about changes in cells. A major take-home lesson is the essential similarity of the different pathways.

Another point to keep in mind is that while we have looked at each individual pathway in isolation, a cell, at any given time receives multiple signals that set off a variety of different responses at once. The pathways described above show a considerable degree of "cross-talk" and the response to any given signal is affected by the other signals that the cell receives simultaneously. The multitude of different receptors, signals and the combinations thereof are the means by which cells are able to respond to an enormous variety of different circumstances.


Enzyme-Linked Receptor

Enzyme-linked receptors have only one transmembrane domain per protein subunit, with an enzymatic catalytic site on the cytoplasmic side of the receptor (see Figure 1-1, C ). For many of these receptors, dimerization activates the receptor to provide the conformational change required for expression of enzymatic activity. The most important cytoplasmic sites have one of the following functions: (1) tyrosine kinase activity, (2) tyrosine phosphatase activity, (3) serine or threonine kinase activity, or (4) guanylyl cyclase activity. For types 1 and 3, autophosphorylation of the receptor also occurs at tyrosine sites and at serine/threonine sites, respectively. Figure 1-2 shows how some of these receptors dimerize after a drug agonist binds.

Many forms of cancer seem to involve mutant variants of enzyme-linked receptors in which the catalytic site or associated nonreceptor protein kinase is continuously activated. Approximately half of all oncogenes discovered to date encode for continuously activated protein kinases.


Abstract

During progression from tumour growth to metastasis, specific integrin signals enable cancer cells to detach from neighbouring cells, re-orientate their polarity during migration, and survive and proliferate in foreign microenvironments. There is increasing evidence that certain integrins associate with receptor tyrosine kinases (RTKs) to activate signalling pathways that are necessary for tumour invasion and metastasis. The effect of these integrins might be especially important in cancer cells that have activating mutations, or amplifications, of the genes that encode these RTKs.


2. Biochemical mechanism of action of tyrosine kinase

Tyrosine kinases are enzymes that selectively phosphorylates tyrosine residue in different substrates. Receptor tyrosine kinases are activated by ligand binding to their extracellular domain. Ligands are extracellular signal molecules (e.g. EGF, PDGF etc) that induce receptor dimerization (except Insulin receptor). Different ligands employ different strategies by which they achieve the stable dimeric conformation. One ligand may bind with two receptor molecules to form 1:2 ligand: receptor complex e.g. growth hormone and growth hormone receptor, while in other cases two ligands binds simultaneously to two receptors 2:2 ligand receptor complex and provides the simplest mechanism of receptor dimerization e.g. VEGF and VEGFR. The receptor dimerization is also stabilized by receptor–receptor interactions. Some ligand receptor is not sufficient for some complex and is stabilized by accessory molecules e.g. FGFs are unable to activate FGFR complex and is stabilized by heparin sulfate proteoglycans (HSPG). Ligand binding to the extracellular domain stabilizes the formation of active dimmers and consequently protein tyrosine kinase activation.

Structural studies of the catalytic core of several RTKs, supported by biochemical and kinetic studies of receptor phosphorylation have provided proof that receptor oligomerization increases the local concentration of the RTKs, leading to efficient transphosphorylation of tyrosine residues in the activation loop of the catalytic domain. Upon tyrosine phosphorylation the activation loop adopts an open conformation that gives access to ATP and substrates and makes ATP transfer from Mg-ATP to tyrosine residue on the receptor itself and on cellular proteins involved in signal transduction.

The ATP binding intracellular catalytic domain that catalyzes receptor autophosphorylation displays the highest level of conservation between the RTKs. The ATP binding site serves as a docking site for specific binding of cytoplasmic signaling proteins containing Src homology-2 (SH2) and protein tyrosine binding (PTB) domains. These proteins in turn recruit additional effector molecules having SH2, SH3, PTB and Pleckstrin homology (PH) domain. This results in the assembly of signaling complexes to the activated receptor and the membrane and subsequent activation of a cascade of intracellular biochemical signals, which leads to the activation or repression of various subsets of genes and thus defines the biological response to signals. During these processes, receptors migrate within the plasma membrane and are internalized through clathrin-coated invagination, which eventually seal off and forms an endocytic vesicle. The endocytic vesicles fuse with the lysosomes and in the process the receptor and ligand may be degraded by the lysosomal enzymes. The receptors are also recycled in some cases. During the whole process of receptor internalization the ligand receptor complex is dissociated and this results in the termination of the signaling reaction.


Results

Evolutionary Relationships among Vertebrate Tyrosine Kinases

We first investigated the evolutionary relationships among the known 58 RTKs and 32 CTKs in human (Robinson et al. 2000 Lemmon and Schlessinger 2010) through phylogenetic analysis of the TK domain ( fig. 1 ). Neither the RTKs nor the CTKs formed clearly distinct monophyletic groups, and many links between RTK and CTK subfamilies were only supported by low bootstrap values. Hence, the phylogeny obtained was not robust enough to establish with certainty a scenario explaining the evolutionary switch between RTKs and CTKs through the gain versus loss of extracellular and transmembrane domains. Neither synteny analysis nor the study of intron phase and position along the TK domain revealed significant additional information to assess relationships between RTKs and CTKs ( fig. 1 , supplementary figs. S1 and S2, Supplementary Material online).

Phylogenetic analysis by Maximum-Likelihood of all trans-membrane receptor tyrosine kinases (in red) and cytoplasmic non-receptor tyrosine kinases (in green) described in human. Alignment is based on the common tyrosine kinase domain and these proteins are rooted by PRKCD and MELK kinases. Bootstrap replicates: 1,000. Name of gene families are given on the right column of the tree. Genes that are in close vicinity in the human genome are also indicated on the right using the same color code other interspaced genes are in grey.

Within RTKs, some of the 20 subfamilies are grouped together in the TK domain molecular phylogeny with significant bootstrap values ( fig. 1 ). Phylogenetic clustering was found for the Tie/Fgfr/Ret/Vegfr/Pdgfr, Met/Ryk/Tam, Alk/Ros1/Insr, and Ddr/Ror/Musk/Trk subfamilies, as well as between Lmr and Styk1 but with a lower bootstrap value. The Ephrin receptors (Eph, subdivided in EphA and EphB) and the Erbb subfamily formed distinct subgroups. Ptk7 was not particularly related to any other RTK.

The RTK subfamily clustering obtained using the TK domain sequence was confirmed by a phylogenetic analysis based on the intron characteristics (supplementary figs. S1 and S2, Supplementary Material online). In addition, 13 out of the 20 RTK subfamilies, including the Tie/Fgfr/Ret/Vegfr/Pdgfr, Met/Ryk/Tam, and Alk/Ros1/Insr groups as well as Lmr and Styk1 are all joined by one phase-2 intron, suggesting a common origin of all these genes. Styk1 is more particularly associated with the Vegfr/Pdgfr/Ret/Fgfr/Tie subfamilies and shares with them three introns. In this group of genes, all but the Tie genes are characterized by a TK domain subdivided in two portions (Lemmon and Schlessinger 2010). The Lmr gene shares common introns with Alk/Ros1/Insr. With their tiny extracellular domain compared with the long and distinct ones characterizing all other RTKs, Lmr, and Styk1 may not be considered as bona fide RTKs, and their phylogenetic position in the TK domain tree suggests that they are indeed divergent ( fig. 1 the human protein kinome database, www.kinase.com/human/kinome Manning et al. 2002). However, they have a transmembrane domain and their kinase domain shares strong sequence similarity with other RTKs. Intron-based homologies suggest that Lmr and Styk1 derive from RTKs the shortness of their extracellular region probably reflects secondary reductions or losses of the extracellular receptor domain.

Within the Eph subfamily, EphA and EphB, which were not clearly separated by the molecular phylogeny of the TK domain sequence, could be distinguished by one intron (EphA has an additional phase-1 intron compared with the EphB).

The RTK Repertoire of Jawed Vertebrates Has Been Extensively Shaped by Ancestral WGDs

We analyzed 7,376 RTK genes (supplementary table S1, Supplementary Material online) that were retrieved from 143 species covering all major clades among jawed vertebrates. Since only few RTKs were described in some species, we concentrated on the 47 species with the largest set of RTKs ( fig. 2 ), but added information from the other species for confirmation (supplementary table S1, Supplementary Material online). Particularly, related species were analyzed when available to differentiate true lineage-specific absence of a gene from incomplete genome assemblies or gene annotations. Evolutionary relationships between genes were assessed by protein sequence phylogeny (supplementary fig. S3, Supplementary Material online) and synteny analysis (supplementary fig. S4, Supplementary Material online). A total of 63 RTK genes representing 20 subfamilies were found in non-teleost-jawed vertebrate species (including spotted gar and elephant shark).

Schematic representation of the occurrence of receptor tyrosine kinases in 47 representative vertebrates species. A white box is used when no sequence, even partial, was found in a species, which is named on the left. Yellow boxes refer to lack of a gene in a taxonomic group of species, which are shown on the right. Other plain colored boxes are used when a sequence, even partial was found. Duplicated genes from the teleost specific whole genome duplication are shown by double-squares. The top phylogeny refers to a Maximum-Likelihood phylogenetic analysis of the RTKs found in human and in other vertebrate species for those that were lost in mammals (ephA4-like, axl-like, ddr2-like, kdr-like, and styk1-like, see details in supplementary fig. S3, Supplementary Material online). All collapsed subfamilies are rooted by non-vertebrate deuterostomes and have a significant bootstrap value. Linkage of the genes tandemly duplicated in the vegfr and pdgfr subfamilies are represented.

To assess the vertebrate-specific evolution of the RTK repertoire, we screened the available non-vertebrate deuterostome genomes, that is, amphioxus, sea squirt, acorn worm, and sea urchin species, using human RTK genes as queries. With the exception of Eph (several genes in non-vertebrates) and Pdgfr (no clear orthologous sequence in non-vertebrates, but see below), 18 out of 20 RTK subfamilies generally had a single representative gene among non-vertebrate deuterostomes ( fig. 3 and supplementary fig. S5, Supplementary Material online). For Met, Tie and Eph, lineage-specific duplications were found in the sea squirt and/or the amphioxus.

Phylogenetic analysis by Maximum-Likelihood of all trans-membrane receptor tyrosine kinase genes observed in human (in red) and those lost in human but found in other species (in blue) (ephA4-like, axl-like, ddr2-like, kdr-like, and styk1-like in red). These genes have been aligned with RTK proteins found in non-vertebrate deuterostomes (in black), which root all RTK subfamilies except the pdgfr subfamily. Bootstrap replicates: 1,000. Only major bootstrap values at key phylogenetic nodes were kept.

Vertebrate-specific expansion was observed for most RTK families ( fig. 3 ). Only five RTK subfamilies out of 20 contain one single gene: Ryk, Ros1, Musk, Ptk7, and Ret. Another five subfamilies are constituted of two genes: Met (Met/Mst1r), Alk (Alk/Ltk), Ror (Ror1/Ror2), Tie (Tie1/Tek), and Styk1 (Styk1/Styk1-like). Four subfamilies are formed by three genes: Insr (Insr/Igfr/Insrr), Ddr (Ddr1/Ddr2/Ddr2-like), Trk (Ntrk1/Ntrk2/Ntrk3), and Lmr (Aatk/Lmtk2/Lmtk3). Four subfamilies have retained the full set of four genes: Erbb (Egfr/Erbb2/Erbb3/Erbb4), Tam (Tyro3/Axl/Axl-like/Mertk), Vegfr (Flt1/Kdr/Kdr-like/Flt4), and Fgfr (Fgfr1/Fgfr2/Fgfr3/Fgfr4). The Pdgfr subfamily is composed of five genes and is subdivided in two monophyletic groups made of Csf1r/Kit/Flt3 and PdgfrA/PdgfrB ( fig. 3 ). Finally, the Eph subfamily contains as many as 15 genes (see below). The observed expansion of the RTK repertoire at the base of the vertebrates is consistent with the involvement of the 1R/2R-WGDs. Up to four copies have been maintained in about 75% of the RTK subfamilies, depending on the rate of gene retention after rediploidization.

Taken together, without considering the Eph gene subfamily, which has a rather complex evolutionary history, and Insrr, which might be the result of a segmental duplication (see below), and if we assume that the Vegfr/Pdgfr subfamilies have been formed from three pre-WGD genes (see below), the retention rate of RTK genes after 1R/2R is 58.75% [47/(20*2*2)]. As a comparison, 31 CTK genes have been kept after 1R/2R duplications of 11 (or 12) subfamily progenitors, with a retention rate of 70.45% (or 64.5%).

After 1R/2R-WGDs, lineage-specific gene losses contributing to differences in RTK repertoire occurred in different groups of vertebrates. Axll was lost in tetrapods, EphA4l and Styk1l in amniotes (sauropsids and mammals), and Ddr2l in mammals. The fourth member of the Vegfr family, that we named Kdrl (Kdr-like) according to the synteny data and the phylogeny of the Vegfr subfamily, was not maintained in eutherians. More restricted gene losses were also detected (for example, the Ltk gene in Carnivora).

Two RTK Subfamilies Have Also Evolved by Ancestral Local Duplications

The Pdgfr (Flt3/Kit/Csf1r, PdgfrA/PdgfrB) and Vegfr (Flt1/Kdr/Flt4) subfamilies are phylogenetically related and very similar in structure, with five and seven immunoglobulin (Ig) domains characterizing the extracellular part of the proteins (Robinson et al. 2000 Lemmon and Schlessinger 2010). Strikingly, genes from the Pdgfr subfamily are clustered with genes from the Vegfr subfamily in human and other vertebrates. Csf1r/PdgfrB are organized in tandem, and Kdr/Kit/PdgfrA are clustered without any other intervening gene ( fig. 4 ). Flt1 and Flt3 are neighbors and only separated by one (non-PTK) gene (Pan3). Flt4 is on the same chromosome as the Csf1rPdgfrB tandem. There is only one gene found in non-vertebrate deuterostomes that roots the Vegfr gene subfamily, and none was identified for the Pdgfr subfamily ( fig. 3 and supplementary fig. S5, Supplementary Material online). Pdgfr genes have the same intron phase and position structure (111210012), which is different by only one change of intron phase and position from the Vegfr genes (111010012) (supplementary fig. S1, Supplementary Material online).

Possible scenarios proposed for the evolution of the vegfr and pdgfr subfamilies. SSD in tandem initiated the amplification of this subfamily. The sequences of their intron phases show the SSD events. Scenario 1 supports partially the phylogeny of vegfr, the most ancient genes. Members of other gene families, Cdx and Chic, in synteny with vegfr and pdgfr subfamilies endorse this scenario. The phylogeny of Kit/Csf1r/Flt3 favors scenario 2. Since the Vegfr are the ancestral genes, and because both syntenic data and phylogeny support the Scenario 1, the gene lost in eutherians should be the named Kdrl.

Putting together, these observations suggest that the ancestor of the Pdgfr genes was duplicated head-to-head in tandem from the ancestor of the Vegfr genes, very early at the base of vertebrates, before the two ancestral WGDs ( fig. 4 ). PdgfrA and PdgfrB genes are more related to each other than to the three other members of the Pdgfr subfamily ( figs. 1 and 2 ). This suggests that the ancestral PdgfrA/B gene was generated from another tandem duplication, tail-to-head this time, from the previously duplicated copy. Thus, the Vegfr and Pdgfr subfamilies probably originated from two tandem duplications of a single ancestor ( fig. 4 ).

The Eph (EphA/B) subfamily presents a more complex picture and the evolutionary relationships between member genes are more difficult to disentangle. Up to 15 genes have been detected in vertebrates. In addition, Eph’s are also duplicated in other chordate lineages, with at least six copies in the sea squirt Ciona and two in amphioxus, and only one Eph gene is present in the more distantly related Ambulacraria superphylum represented by the sea urchin and the acorn worm ( figs. 2 and 3 and supplementary fig. S5, Supplementary Material online). This suggests either independent lineage-specific expansion of the Eph gene family in different groups of chordates or the existence of duplications in chordate ancestors prior to the 1R/2R-WGDs. No obvious case of tandem duplication was detected in this subfamily ( fig. 1 ). Within EphB genes, which can be clearly distinguished from EphA genes by one phase-1 intron, EphB1/EphB2/EphB3/EphB4 form a strongly supported monophyletic group, in which EphB6 is not included ( fig. 3 ). Interestingly, the EphB1/EphB2/EphB3/EphB4 genes are more related to Ciona sequences than to vertebrate EphB6. This suggests an event of gene duplication before the urochordate/vertebrate split, with subsequent 1R/2R-mediated duplications of the EphB1/EphB2/EphB3/EphB4 progenitor in vertebrates.

Other genes might also have been generated by local gene duplications. For example, the EphA1 and Insrr genes are found in sarcopterygian species including the coelacanth, but neither in actinopterygians nor in the elephant shark. A simple explanation for this distribution implies local duplication events at the base of sarcopterygians, even if loss of the genes in cartilaginous and ray-finned fishes but maintenance in coelacanth/tetrapods after 1R/2R-WGDs cannot be excluded.

The RTK Repertoire of Teleost Fish Has Been Shaped by the 3R WGD

Teleost fish genomes have been shaped by a third WGD called the 3R. The fish investigated here are two Ostariophysi (the cave fish, a characiform and the zebrafish, a cypriniform) and nine Percomorpha species, which together with the cod belong to the Neoteleostei. The genome of the gar Lepisosteus oculeatus was also analyzed. This holostean fish diverged from the fish lineage that includes the teleosts before the 3R WGD, and is expected to define the set of genes that were present before the teleost WGD (Amores et al. 2011).

According to the RTK gene set found in the gar, the coelacanth, and the elephant shark, 61 RTK genes probably existed before the 3R event in the lineage leading to teleosts. Among the 14 Eph genes, eight duplicates were retained in all fish from the 3R-WGD up to the split between Neoteleostei and Ostariophysi ( fig. 2 ). One duplicate of EphA4l was then lost in the Neoteleostei, and one duplicate was not maintained in the two representatives of the Ostariophysi for EphA6b and EphB1b. Among the 47 remaining RTK genes, 15 were duplicated in teleosts. Ros1 and Tek were subsequently lost in the Neoteleostei, and one PdgfrB duplicate is lacking in the Ostariophysi.

Taken together, 23 out of 61 RTK genes duplicated by the 3R WGD were kept in a least one major group of teleosts. This makes 84 RTK genes in the representatives of the teleosts used in this analysis, with a retention rate of 68.8% [84/(61*2)]. These values are as high as those obtained for the two ancestral vertebrate 1R/2R-WGDs. Hence, teleosts have a considerably larger repertoire of RTKs than tetrapods.


Part I Introduction 1

1.1 Receptors and Signaling 3

1.1.1 General Aspects of Signaling 3

1.1.2 Verbal and Physiological Signals 3

1.1.3 Criteria for Recognizing Transmitters and Receptors 4

1.1.6 Receptor&ndashEnzyme Similarities 4

1.2 Types of Receptors and Hormones 5

1.2.1 Receptor Superfamilies 5

1.3 Receptors Are the Chemical Expression of Reality 6

2 The Origins of Chemical Thinking 9

2.1 Overview of Early Pharmacological History 9

2.1.1 The Development of a Chemical Hypothesis 9

2.1.2 Chemical Structure and Drug Action 10

2.1.3 The Site of Drug Action 10

2.2 Modern Pharmacology 10

2.2.1 Langley and Ehrlich: the Origins of the Receptor Concept 10

2.2.2 Maturation of the Receptor Concept 13

2.3 Phylogenetics of Signaling 13

2.3.1 The First Communicators 13

Part II Fundamentals 15

3 Membranes and Proteins 17

3.1.1 The Cytoplasmic Membrane &ndash the Importance of Cell Membranes 17

3.1.2 History of Membrane Models 17

3.1.2.1 The Roles of Proteins in Membranes 18

3.1.2.2 Challenges to the Danielli&ndashDavson Model 19

3.1.2.3 A New View of Membrane Proteins 19

3.1.2.4 The Modern Concept of Membranes &ndash the Fluid Mosaic Model 19

3.1.3 Membrane Components 19

3.1.3.2 Asymmetry and Heterogeneity in Membrane Lipids 20

3.1.3.3 Membrane Construction and Insertion of Proteins 20

3.2 The Nature and Function of Proteins 21

3.2.1 Linear andThree-Dimensional Structures 22

3.2.3 Secondary Structure 23

3.2.4 Tertiary Structure 24

4 Hormones as First Messengers 27

4.1 Hormones and Cellular Communication 27

4.1.1 Discovery of Hormones 27

4.2.1 Pheromones for Signaling between Individuals 28

4.2.2 Archaea and Bacteria 28

4.2.3.2 Unikonts &ndash Amoebozoa, Fungi, Animals 29

4.2.3.3 Invertebrate Pheromones 31

4.2.3.4 Vertebrate Pheromones 31

4.3 Vertebrate Hormones and Transmitters 31

4.3.1 Peptide and Non-Peptide Agonists 31

4.3.2 Peptide Hormones of the G-Protein-Coupled Receptors 32

4.3.2.1 Hypothalamic-Pituitary Axis 32

4.3.2.2 The Anterior Pituitary Trophic Hormones 34

4.3.3 Other Neural Peptides 35

4.3.3.2 Non-Opioid Transmitter Peptides 36

4.3.4 Peptides from Non-Neural Sources 36

4.3.4.1 Digestive Tract Hormones 36

4.3.4.2 Hormones from Vascular Tissue 38

4.3.4.3 Hormones from the Blood 38

4.3.4.4 Peptide Hormones from Reproductive Tissues 39

4.3.4.5 Hormones from Other Tissues 39

4.3.5 Non-Peptides Acting on G-Protein-Coupled Receptors 39

4.3.5.1 Transmitters Derived from Amino Acids 39

4.3.5.2 Transmitters Derived from Nucleotides 40

4.3.5.3 Transmitters Derived from Membrane Lipids &ndash Prostaglandins and Cannabinoids 41

4.3.6 Transmitters of the Ion Channels 41

4.3.7 Hormones of the Receptor Kinases &ndash Growth Factor Receptors 43

4.3.7.2 Insulin-Like Growth Factors 43

4.3.7.3 Natriuretic Peptides 43

4.3.7.4 Peptide Signal Molecules Important in Embryogenesis 43

4.3.7.5 Pituitary Gland Hormones &ndash Somatotropin and Prolactin 43

4.3.8 Hormones of the Nuclear Receptors 44

4.3.8.2 Non-Steroid Nuclear Hormones 46

4.4 Analgesics and Venoms as Receptor Ligands 46

5.1 The Materialization of Receptors 47

5.2.1 Binding of Agonist to Receptor 48

5.3.1 Early Approaches to Understanding Receptor Action 49

5.3.1.1 The Occupancy Model 49

5.3.1.2 Processes That Follow Receptor Activation 52

5.3.1.3 Efficacy and Spare Receptors 52

5.3.2 Modern Approaches to Receptor Theory 52

5.3.2.1 The Two-State Model 52

5.3.2.2 The Ternary Complex Model 53

5.3.2.4 Cubic Ternary Complex (CTC) Model 55

5.3.3 Summary of Model States 55

5.4 Visualizing Receptor Structure and Function 55

5.4.1 Determination of Receptor Kd 55

5.4.2 Visualizing Ligand Binding 57

5.4.2.1 Receptor Preparation 58

5.4.2.2 Equilibrium Binding Studies 58

5.4.2.3 Competition Studies 58

5.4.3 X-ray Crystallography of Native and Agonist-Bound Receptors 59

5.4.4 Probe Tagging (Fluorescent and Photoaffinity) 60

5.5 Proteomics Approaches to Receptor Efficacy 60

5.6 Physical Factors Affecting Receptor Binding 61

5.6.2 Relation of Agonist Affinity and Efficacy to Distance Traveled Following Release 61

Part III Receptor Types and Function 63

6 Transduction I: Ion Channels and Transporters 65

6.1.1 Family Relationships 65

6.2 Small Molecule Channels 66

6.2.1 Osmotic and Stretch Detectors 66

6.2.2 Voltage-Gated Cation Channels 66

6.2.2.1 History of Studies on Voltage-Gated Channels 66

6.2.2.2 Structure and Physiology of Ion Channels 68

6.2.3 Potassium Channels 68

6.2.4.1 Bacterial Na+ Channels 70

6.2.4.2 Vertebrate Na+ Channels 70

6.2.6 Non-Voltage-Gated Cation Channels &ndash Transient Receptor Potential (TRP) Channels 72

6.3.1 Pumps and Facilitated Diffusion 73

6.3.2 The Chloride Channel 76

6.4 Major Intrinsic Proteins 76

6.4.2 Glycerol Transporters 77

6.5 Ligand-Gated Ion Channels 77

6.5.1 Four-TM Domains &ndash the Cys-Loop Receptors 77

6.5.1.1 The Four-TM Channels for Cations 78

6.5.1.2 The Four-TM Channels for Anions 80

6.5.2 Three-TM Domains &ndash Ionotropic Glutamate Receptors 82

6.5.2.1 Glutamate-Gated Channels 82

6.5.2.2 N-Methyl-D-aspartate (NMDA) Receptor 82

6.5.2.3 Non-NMDA Receptors 82

6.5.3 Two-TM Domains &ndash ATP-Gated Receptors (P2X) 82

7 Transduction II: G-Protein-Coupled Receptors 85

7.1.2 Sensory Transduction 87

7.1.2.1 Chemoreception in Non-Mammals 87

7.1.2.2 Chemoreception in Mammals 87

7.2 Families of G-Protein-Coupled Receptors 89

7.3 Transduction Mechanisms 89

7.3.1 Discovery of Receptor Control of Metabolism &ndash Cyclic AMP and G Proteins 89

7.3.1.1 Components of the Process of Metabolic Activation 89

7.3.1.2 Discovery of Cyclic AMP 90

7.3.1.3 Discovery of G Proteins 90

7.3.2 Actions of G Proteins 91

7.3.2.2 Roles of the Beta and Gamma Subunits 95

7.3.3 Proteins That Enhance (GEF) or Inhibit (GAP) GTP Binding 96

7.3.4 Signal Amplification 97

7.3.5 Signal Cessation &ndash Several Processes Decrease Receptor Activity 97

7.3.6 Interactions between Receptors and G Proteins 97

7.3.7 Summary of Actions of GPCRs: Agonists, Receptors, G Proteins, and Signaling Cascades 98

7.4 The Major Families of G Protein-Coupled Receptors 99

7.4.1 Family A &ndash Rhodopsin-Like 99

7.4.2 Family B &ndash Secretin-Like 104

7.4.3 Family C &ndash Metabotropic Glutamate and Sweet/Umami Taste Receptors 104

7.4.3.1 Taste 1 Receptors (T1Rs) 105

7.4.3.2 Calcium-Sensing Receptors 106

7.4.4 Family D &ndash Adhesion Receptors 106

7.4.5 Family F &ndash Frizzled-Smoothened Receptors 106

7.4.6 Family E &ndash Cyclic AMP Receptors 106

7.4.7 Other G-Protein-Coupled Receptor Types in Eukaryotes 106

7.4.7.1 Yeast Mating Pheromone Receptors 106

7.4.7.2 Insect Taste Receptors 106

7.4.7.3 Nematode Chemoreceptors 106

8 Transduction III: Receptor Kinases and Immunoglobulins 107

8.2 Receptors for Cell Division and Metabolism 108

8.2.1 Overview of Family Members 108

8.2.2 Overall Functions of RTK 108

8.2.2.1 Extracellular Domains 108

8.2.2.2 Intracellular Domains 109

8.2.3 Receptor Tyrosine Kinase Subfamilies 110

8.2.3.1 EGF Receptor Subfamily 111

8.2.3.2 Insulin Receptor Subfamily 111

8.2.3.3 FGF and PDGF Receptor Subfamilies 111

8.2.3.4 NGF Receptor Subfamily 111

8.3 Receptor Serine/Threonine Kinases 112

8.3.1 Transforming Growth Factor-Beta (TGF-&beta) Receptor 112

8.4 The Guanylyl Cyclase Receptor Subfamily &ndash Natriuretic Peptide Receptors 112

8.5 Non-Kinase Molecules &ndash LDL Receptors 113

8.5.1 Cholesterol Transport 113

8.5.2 The Low-Density Lipoprotein (LDL) Receptor 114

8.5.2.1 Clathrin-Coated Pits 114

8.6 Cell&ndashCell Contact Signaling 115

8.6.1 Notch&ndashDelta Signaling 115

8.7 Immune System Receptors, Antibodies, and Cytokines 115

8.7.1 The Innate Immune Responses 115

8.7.2 The Cells and Molecules of the Adaptive Immune System 116

8.7.3 T-Cell Receptors and Immunoglobulins 116

8.7.4 Cell-Surface Molecules 117

8.7.4.1 The MHC Proteins 117

8.7.4.2 Receptors of the B and T Cells 118

9 Transduction IV: Nuclear Receptors 121

9.2 Genomic Actions of Nuclear Receptors 122

9.2.1 Families of Nuclear Receptors 122

9.2.2 Transcription Control 122

9.2.3 Constitutively Active Nuclear Receptors 122

9.2.4 Liganded Receptors 122

9.2.5 History of Steroid Receptor Studies 123

9.2.6 Receptor Structure 123

9.2.7 The Ligand-Binding Module 124

9.2.8 The DNA-BindingModule 125

9.2.9 Specific Nuclear Actions 125

9.2.9.1 Family 1 &ndashThyroid Hormone and Vitamins A and D Receptors 125

9.2.9.2 Family 2 &ndash Fatty Acid (HNF4) and Retinoic X Receptors (RXR) 127

9.2.9.3 Family 3 &ndash Steroid Receptors for Estrogens, Androgens, Progestogens, Mineralocorticoids, and Glucocorticoids 128

9.3 Actions of Receptor Antagonists 129

9.4 Non-Traditional Actions of Steroid-Like Hormones andTheir Receptors 130

9.4.1 Cell-Membrane Progesterone Receptors 131

9.4.2 Cell-Membrane Mineralocorticoid and Glucocorticoid Receptors 131

9.4.3 Cell-MembraneThyroid Hormone and Vitamin A/D Receptors 131

9.4.4 Ligand-Independent Activation of Transcription 131

Part IV Applications 133

10 Signaling Complexity 135

10.2 Experimental Determination of Signaling Cascades 135

10.2.2 MAPK: a Phosphorylation Cascade 136

10.3 Transduction across theMembrane 138

10.3.2 G-Protein-Coupled Receptors 138

10.3.2.1 Other G-Protein-Like Transducers &ndash Ras 139

10.3.2.2 Other G-Protein-Like Transducers &ndash Ran 139

10.3.3 Cell Aggregation and Development 140

10.3.3.1 Coaggregation in Bacteria 140

10.3.3.2 Aggregation in Eukaryotes 140

10.3.3.3 The Molecules of Cell Adhesion 141

10.4 Complexity in Cross Talk &ndash Roles of PIP3, Akt, and PDK1 141

10.4.1 Signaling Cascades Using PIP3 142

10.4.3 Receptor Tyrosine Kinases 144

10.4.4 Cytokine Receptors and the JAK/STAT Proteins 144

10.4.5 Combined Cellular Signaling &ndash GPCR and RTK Actions 144

10.5.1 Constitutive versus Inducible Activation 144

10.6 Signaling Mediated by Gas Molecules 146

10.6.3 Hydrogen Sulfide 148

11 Cellular Interactions in Development 149

11.2 The Origins of Multicellularity 150

11.2.1 Multicellular Lineages in Prokaryotes 150

11.2.2 Multicellular Lineages in Eukaryotes 150

11.2.2.1 Chromalveolates &ndash Generally Unicellular but with One Multicellular Clade 151

11.2.2.2 Archaeplastida &ndash Algae and Plants 151

11.2.2.3 Amoebozoans, Fungi, Choanoflagellates, and Animals 151

11.3 The Origin of Symmetry and Axes 152

11.3.1 The Multicellular Body Plan 152

11.3.2 The Porifera &ndash Asymmetric with a Single Cell Layer 152

11.3.3 Cnidaria &ndash Radial Symmetry, Two Cell Layers, Tissues 153

11.4 Fertilization and Organization of the Multicellular Body Plan 154

11.4.1 Sperm&ndashEgg Recognition 154

11.4.1.1 Sea Urchin Fertilization 154

11.4.1.2 Mammalian Fertilization 157

11.5 Differentiation of Triploblastic Embryos &ndash Organogenesis 158

11.5.2 The Origin of Triploblastic Animals 158

11.5.3 Development in Protostomes 159

11.5.3.1 Segmentation and Organ Formation in Drosophila 159

11.5.3.2 Cellular Interactions in Later Drosophila Development 161

11.5.4 Development in Deuterostomes 162

11.5.4.1 Early Frog Development 162

11.6 Programmed Cell Death (Apoptosis) 165

11.6.1 Apoptosis During Development 166

11.6.2 Apoptosis During Adult Life 166

12 Receptor Mechanisms in Disease Processes 169

12.1 Genetic Basis for Receptor Function 169

12.1.1 Genotype and Phenotype 169

12.1.2 Classical Dominance Mechanisms 169

12.1.3 Other Levels of Gene Expression 170

12.1.4 Pre-receptor Mutations 170

12.1.5 Receptor Mutations 171

12.1.6 Post-receptor Mutations 171

12.2 Receptor Pathologies 171

12.2.1 Ion Channel Superfamily 171

12.2.1.1 Calcium Channels 172

12.2.1.2 Transient Receptor Protein (TRP) Channels 172

12.2.1.3 Voltage-Gated Na+ Channels 172

12.2.1.4 Ligand-Gated Na+ Channels 172

12.2.1.5 Chloride Transporter &ndash Cystic Fibrosis 172

12.2.2 G-Protein-Coupled Receptor Superfamily 172

12.2.2.2 Thyroid Diseases 173

12.2.2.3 Cardiovascular Disease 173

12.2.3 Immunoglobulin Superfamily 176

12.2.3.1 Diabetes Mellitus 176

12.2.3.2 Atherosclerosis 176

12.2.4 Nuclear Receptor Superfamily &ndash Steroid Receptors 176

12.2.4.1 Alterations in Transcription 176

12.2.4.2 Additional Effects 177

12.3 Signaling Mutations Leading to Cancer 177

12.3.1 Pathogenesis of Cancer 177

12.3.2 Cancer as a Disease of Signaling Molecules 178

12.3.2.1 Oncogenes that Encode Mutated Transmitters 178

12.3.2.2 Oncogenes that Encode Mutated RTKs 178

12.3.2.3 Oncogenes that Encode Mutated G Proteins 179

12.3.2.4 Oncogenes that Encode Mutated Transcription Factors &ndash Steroid Receptors 180

13 Receptors and the Mind 181

13.1 Origins of Behavior 181

13.1.1 Bacterial Short-Term Memory 181

13.1.2 AnimalsWithout True Neural Organization:The Porifera 182

13.1.3 Animals with Neural Networks: The Cnidaria 182

13.1.4 Bilaterally Symmetrical Animals: The Acoela 183

13.2.3.1 Synthesis and Release of Brain Transmitters 185

13.2.3.2 Converting Short-Term Memory to Long Term 186

13.3 Animal Memory: Invertebrates 186

13.3.1 Discovery of the Signaling Contribution to Memory 186

13.3.2 Receptor Mechanisms of Nerve Cell Interactions 186

13.3.2.1 The GillWithdrawal Reflex of Aplysia 186

13.3.2.2 Mechanisms Underlying Sensitization and Short-Term Memory 187

13.3.2.3 Ion Flows in Nerve Action Potentials 187

13.3.2.4 Consolidation into Long-Term Memory (LTP) 188

13.4 Animal Memory: Vertebrates 188

13.4.1 Intracellular Mechanisms of Potentiation 188

13.5 Receptors and Behavior: Addiction, Tolerance, and Dependence 190

13.5.1 Opioid Receptors 190

13.5.1.1 Opioid Neuron Pathways in the Brain 191

13.5.1.2 The Opioid Peptides and Receptors 192

13.5.1.3 Mechanisms of Transduction 192

13.5.1.4 Characteristics of Responses to Continued Drug Presence 192

13.5.2 Individual and Cultural Distributions of Depression 193

13.5.2.2 Polymorphisms in Neurotransmitter Transporters 194

13.5.2.3 Polymorphisms in Opioid Receptor Subtypes 194

13.5.2.4 Polymorphisms in Enzymes for Transmitter Disposition 194

13.5.2.5 Society-Level Actions 194

13.5.2.6 Possible Mechanisms 195

14 Evolution of Receptors, Transmitters, and Hormones 197

14.1.1 Phylogeny of Communication: General Ideas 197

14.2 Origins of Transmitters and Receptors 197

14.2.1 Evolution of Signaling Processes 197

14.2.2 Homologous Sequences 198

14.2.2.1 Orthologous and Paralogous Sequences 198

14.2.3 Phylogenetic Inference 199

14.2.4 Phylogenetic Illustration of Protein Relationships 199

14.2.5 Whole-Genome Duplication (WGD) 200

14.2.6 Origins of Novel Domains 201

14.2.7 Adaptation of Receptor Systems 201

14.2.8 Coevolution of Components of Signaling Pathways 202

14.2.9 Peptide Hormones and Their Receptors 202

14.2.10 Receptors and Their Non-Peptide Hormones 202

14.3 Evolution of Hormones 202

14.3.1 Peptide Hormones for G Protein-Coupled Receptors 202

14.3.1.1 The Yeast Mating Pheromones 203

14.3.1.2 The Anterior Pituitary Trophic Hormones 203

14.3.1.3 The Hypothalamic Releasing Hormones 203

14.3.1.4 The Posterior Pituitary Hormones 203

14.3.1.5 Miscellaneous Peptide Hormones 204

14.3.2 Hormones of the Receptor Tyrosine Kinases 204

14.3.2.1 The Insulin Family 204

14.3.2.2 The Neurotrophins 204

14.3.2.3 The Growth Hormone Family 204

14.4 Evolution of Receptor Superfamilies 205

14.4.1.1 Voltage-Gated Channels 205

14.4.1.2 Ligand-Gated Channels 205

14.4.2 G Protein-Coupled Receptors 206

14.4.2.1 G-Protein-Coupled Receptor Types 206

14.4.2.2 Family A Receptors &ndash Rhodopsin Family 206

14.4.2.3 Family B &ndash Secretin and Adhesion Receptors 207

14.4.2.4 Family F &ndash Frizzled and Smoothened Receptors 208

14.4.2.5 Elements of the GPCR Transduction Pathway 208

14.4.3 The Immunoglobulin Superfamily 210

14.4.3.1 The Receptor Tyrosine Kinases 210

14.4.3.2 Molecules of the Adaptive Immune System 211

14.4.4 Steroid, Vitamin A/D, andThyroid Hormone Receptors 211

14.4.4.1 Origin of Nuclear Receptors: The Role of Ligands 211

14.4.4.2 The Nuclear Receptor Families 211

14.4.4.3 Later Evolution of Nuclear Receptors &ndash Ligand Exploitation 212


8.5: Receptor Tyrosine Kinases (RTKs) - Biology

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Lesson 1: Cell Signaling

Overview/Objectives

After completing this lesson, you should be able to:

  1. Define the term &ldquocell signaling.&rdquo
  2. Outline the general principle behind signal transduction.
  3. List the general types of signals.

Readings and Activities

  1. Read &ldquoCell Signaling&rdquo (pages 196&ndash198 of the textbook).
  2. You can also watch two video lectures on Signaling Mechanisms for all of the lessons in this unit:

    (A link to this is also provided on page 196 of the textbook.)

    (Note that the current version of textbook does not provide this URL.)

    Commentary

    All organisms, from unicellular to complex multicellular ones, are able to respond to their environment and coordinate their many cellular activities through signal transduction mechanisms. Signal transduction follows a simple general principle:

    Signal binds receptor &rarr Pathway of intermediates &rarr Target cell response

    This general principle is illustrated on page 197 of the textbook. An overview diagram is below:

    Adapted from: University of Tokyo, A Comprehensive Guide to Life Science, Fig. 14𔂫B, https://csls-text3.c.u-tokyo.ac.jp/active/14_01.html

    There are many types of receptors on the membrane surface of a cell, which transduce signals from the environment. The molecules that bind specifically to those receptors are called ligands or signal molecules. Signal molecules that mediate signal transduction between cells are called first messengers, and those that are produced and mobilized inside of cells in the pathway are referred to as second messengers.

    Binding of a signal molecule from outside a cell to a receptor causes a receptor to transform and stimulate other molecules, referred to as activation. These activated receptors then activate other molecules in the cell. This pathway transmits information from the environment and amplifies the cell response, ensuring that the cell has an appropriate response to the environmental signal. This information may either be passed to the nucleus, where it will influence gene expression, or passed to intracellular proteins or organelles involved in cellular functions.

    Now that you have an understanding of signal transduction, it is important to see how these general concepts fit into the larger, more detailed picture of this important cellular process. A detailed figure of the different signal transduction pathways is below:

    File:Signal transduction pathways.svg. By cybertory, 2010. GFDL or CC BY-SA 3.0 via Wikimedia Commons, https://commons.wikimedia.org/wiki/File:Signal_transduction_pathways.svg.

    This detailed figure shows the complexity of some signal transduction pathways in the cell. As this figure demonstrates, there are a number of pathways in cells, with many steps and second messengers culminating in a variety of cellular responses, which are dependent on the initial signal received. (You are responsible only for the material highlighted in the textbook and in the study guide commentary you are not expected to know all the steps shown for every signal transduction pathway.)

    There are many excellent figures online for signal transduction, which you may wish to consult as well. Even in its complexity, this figure does not outline all of the possible cell responses and second messengers. As you proceed through the other lessons in this unit, and learn about each type of receptor and resulting transduction pathway, you may wish to come back to this figure or others that you find to see how they all fit together.

    Study Questions

    1. On a cell surface, why are there so many different types of receptors with binding specificities to different molecules?
    2. What are the three general steps in signal transduction?
    3. List two general ways that signal transduction causes a change or response in a cell.

    If you wish to discuss any of these questions or need assistance with the material, please contact your Academic Expert (AE) by emailing the Student Success Centre at [email protected]


    Background

    Breast cancer is a major cause of morbidity and mortality among women population worldwide. The incidence of breast cancer differs considerably worldwide. It is expected to affect 0.2 million and would result in an estimated 41,070 deaths in 2017 in USA [1]. Breast cancer emerges as a consequence of dysregulation of different signaling pathways in mammary epithelial cells. Growth factors and chemokines activate various signaling cascades which cross-talk in tumor microenvironment leading to cancer progression. They bind to different families of receptors. Receptor Tyrosine Kinases (RTKs) comprise one such family. RTKs are single-pass transmembrane proteins, expressed on various cell types including the ones in the tumor microenvironment. Overexpression of various types of RTKs such as epidermal growth factor receptors (EGFRs), vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor receptors (PDGFRs), insulin-like growth factor receptors (IGFRs), and fibroblast growth factor receptors (FGFRs) is found in different types of cancer including breast [2,3,4]. Elevated levels of RTKs are associated with increased breast cancer aggressiveness and decreased overall and disease-free survival [5]. Ligand binding leads to conformational changes in RTKs that result in activation of downstream signaling molecules. The important pathways that are known to be activated by RTKs include mitogen-activated protein kinase (MAPK), Janus kinase (JAK)/ signal transducer and activator of transcription (STAT) and phosphoinositide 3-kinase (PI3K)/Akt [6,7,8,9,10]. RTK-regulated pathways play key roles in various facets of cancer progression. RTK-activated signaling also induces cancer stem cell (CSC) phenotype that exhibit resistance to therapeutic regimens [6, 9]. Cancer progression is not only regulated by autonomous signaling networks but also context-dependent molecular signals received from tumor stroma. Tumor stroma consists of various types of non-cancerous cells such as fibroblasts, endothelial cells, macrophages and other immune cells [11]. RTK signaling-regulated interplay between the tumor and stromal cells contributes to tissue remodeling, stromal cell recruitment and activation. Survival of disseminated cancer cells in metastatic sites requires formation of the pre-metastatic niche by stromal cells. Stromal cells expressing RTKs are known to be recruited to metastatic sites and have been found to form pre-metastatic niche through the RTK-regulated signaling [8]. RTKs also regulate trans-differentiation of cancer cells to endothelial cells to form new blood vessels in a process known as vasculogenic mimicry [12, 13]. Since RTKs play important roles in different aspects of breast cancer progression, targeting RTKs might be useful in cancer treatment. Over the years, several RTK inhibitors have been screened and tested in clinical trials. Some of them such as lapatinib, trastuzumab and bevacizumab have been approved by Food and Drug Administration (FDA), USA for clinical management of breast cancer. Interestingly, RTK inhibitors revert conventional therapy-induced multidrug resistance and improve the disease-free survival in metastatic breast cancer patients [14]. Even though anti-RTK therapy shows clinical benefits in breast cancer patients, unfortunately, cancer cells develop de novo or acquired resistance that limits the success of RTK-targeted therapy [15]. In this review, we deal with EGFR, VEGFR, PDGFR and FGFR signaling in breast cancer progression, maintenance of cancer stem cell phenotype, tumor-stroma interaction and drug resistance. Moreover, this review also discusses the major challenges in targeting RTKs for the successful treatment of breast cancer.

    Structure and classification of RTKs

    Fifty eight different RTKs have been characterized in humans and they have been classified into 20 different subfamilies on the basis of structural features. Each RTK subfamily exhibits a prototype structural organization along with class-specific characteristics. A prototype RTK has an extracellular ligand-binding domain and intracellular tyrosine kinase domain separated by a transmembrane domain. The subfamilies of RTKs are (1) EGFR, (2) InsR, (3) PDGFR, (4) VEGFR, (5) FGFR, (6) PTK7/CCK4, (7) Trk, (8) Ror, (9) MuSK, (10) Met, (11) Axl, (12) Tie, (13) EphA/B, (14) Ret, (15) Ryk, (16) DDR1/2, (17) Ros, (18) LMR, (19) ALK and (20) SuRTK106/STYK1. The intracellular domain of RTKs has tyrosine kinase activity (tyrosine kinase domain TKD). This tyrosine kinase domain can phosphorylate tyrosine residues in cis (within the same molecule) or in trans (residing on a different molecule) (Fig. 1). This consensus design of RTKs has been found to be conserved across evolution. Mutations in RTKs that result in structural abnormalities have been found to lead various disorders.

    Structure of prototype of receptor tyrosine kinase and mechanism of activation. Receptor tyrosine kinases (RTKs) have the following structural segments from N- to C-terminal: immunoglobulin folds, transmembrane region, juxtamembrane region, N-lobe, activation loop, C-lobe and cytoplasmic tail. RTKs reside at the plasma membrane as a monomer. Ligand binding crosslinks receptor molecules and induces conformational changes that lead to receptor autophosphorylation and activation. Phosphorylated RTK either serves as a docking site for adaptor proteins (B) or may directly phosphorylate signaling molecules (A). Adaptor proteins or signaling molecules bind to phosphorylated receptor through Src homology 2 (SH2) or phosphotyrosine-binding (PTB) domain. Docked adaptor proteins further transduce signal by phosphorylating other downstream molecules (C, D)

    RTKs are activated by binding of soluble ligands. Some of the RTKs (DDR1, DDR2) are activated not by soluble ligands but by collagen fibers of the extracellular matrix [16]. Two compulsory events in RTK activation are ligand binding and receptor dimerization. Although the earlier idea was that cognate ligand binding ultimately results in the receptor dimerization, it has been found that few RTKs are oligomeric even in the absence of ligands [17]. EGFR is mostly present as a monomer whereas insulin receptor is present as a dimer on the cell membrane [18]. Nonetheless, receptor activation requires binding of ligand and consequent dimerization or oligomerization of the former in an active state. Different mechanisms for ligand binding-induced receptor dimerization have been explained for different classes of RTKs by different research groups. The mechanisms include two extremes where the dimer interface is formed entirely either by the ligand or the receptor molecules. The two other mechanisms include the participation of both ligand and receptor for the formation of the dimer interface and in another case participation of an accessory molecule. An example of the first mechanism is activation of nerve growth factor (NGF) receptor, TrkA where only two NGF molecules form the dimer interface and none of receptor extracellular domains make physical contact to the neighboring molecule [19, 20]. The ligands that activate members of the EGFR family do not themselves form dimers rather they bind two different domains of the same molecule and induce favorable conformational changes that lead to the formation of dimer interface by the receptor molecules [21]. Stem cell factor (SCF) binds to its receptor, KIT and induces receptor dimerization where the dimer interface is formed by both the ligand and receptor molecules [22]. In case of FGFR, heparin molecule stabilizes FGFR dimer configuration following ligand (fibroblast Growth factor (FGF)) binding [23].

    In the absence of cognate ligands, the RTKs are held in an inactive state by autoinhibitory mechanisms. Two different autoinhibitory mechanisms have been described for different families of RTKs. The TKD of the RTKs contains three essential elements, N lobe, C lobe and activation loop [24]. In the activation loop-mediated autoinhibitory mechanism, the activation loop makes physical contact with the active site of TKD. A critical tyrosine residue in the activation loop is phosphorylated and the tyrosine kinase activity is autoinhibited in cis [25]. In the other mechanism, juxtamembrane sequences make extensive contact with the active site of the TKD and the latter is arrested in an autoinhibited inactive conformation [26,27,28]. Ligand binding induces favorable conformational changes that get rid of autoinhibitions following receptor dimerization. Activated RTKs can recruit many downstream effector molecules. These molecules contain SH2 or PTB domains which bind phosphotyrosine residues on RTKs [29]. These proteins can either interact directly with the activated RTKs or they may interact with other docking proteins which are tyrosine phosphorylated by RTKs. Some of the well-known docking proteins which orchestrate the formation of large protein complexes downstream of RTK activation are FGF receptor substrate 2 (FRS2), insulin receptor substrate 1 (IRS1) and Grb2-associated binder 1 (Gab1). Some of the docking proteins have specificity in terms of which classes of RTKs they bind whereas other docking proteins bind RTK members across different families. A single RTK can bind different ligands. EGFR binds seven different ligands [30]. The strength of interaction with RTK varies for these different ligand molecules. The attributes of the active conformation of dimerized receptor differ greatly for different ligands. Different active dimer conformations of RTK activate different downstream signaling cascades [31]. Gene rearrangements and mutations confer certain structural features to RTKs that result in ligand-independent receptor dimerization and activation. Aberrant activation of RTKs by such means can lead to different pathophysiology. Gene rearrangements can lead to an abnormal coiled coil and leucine zipper conformations of the extracellular domain that induce ligand-independent association of RTKs. Mutations resulting in cysteine residues in the extracellular domain also can induce permanent association of two RTK monomers [32]. Transmembrane domain mutations also can result in constitutive dimerization of RTKs leading to certain pathophysiologies [33]. Apart from the classification outlined above, RTKs have also been categorized based on the commonality of downstream signaling and expression pattern across tissues. Three such classes are (1) EGFR/FGFR1/c-Met, (2) IGF-1R/NTRK2 and (3) PDGFRβ [34].

    Breast cancer stem cells and drug resistance

    Despite the advent of new therapeutic avenues, tumor relapse remains to be a greater challenge in breast cancer management. There are various reasons for tumor recurrence including breast cancer stem-like cells (BCSCs) residing at primary tumor as well as at metastatic sites. CSCs are subpopulation of tumor cells which have the potential to self-renew and drive tumorigenesis. BCSCs are characterized by the expression of specific cell surface markers including EpCAM + /CD24 - /CD44 + [35]. Moreover, it has been reported that CSCs also express high level of aldehyde dehydrogenase (ALDH) and it is associated with poor clinical outcome [36]. However, a recent study suggests that EpCAM + /CD24 - /CD44 + CSCs are anatomically distinct from ALDH+ve CSCs. Molecular profiling of EpCAM + /CD24 - / CD44 + and ALDH+ve CSCs revealed that the former sub-populations exhibit quiescent, epithelial to mesenchymal transition (EMT) phenotype whereas ALDH+ve CSCs show epithelial phenotype with self-renewal capacity [37]. Tumor microenvironment consists of cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), mesenchymal stem cells (MSCs) and other immune and vascular cells and involved in the maintenance of CSCs in breast cancer [11, 38]. RTK signaling in tumor and stromal cells plays a critical role in the regulation of both CD24 - and CD44 + and ALDH+ve CSC phenotypes. CSCs exhibit major impact on cancer therapy as they show resistance to conventional chemo therapies by expressing multi-drug resistance (MDR) genes. The CD44 + /CD24 - tumor cell fraction is increased in breast cancer patients upon administration of neoadjuvant chemotherapy [39]. Moreover, paclitaxel and epirubicin-based chemotherapy is associated with enrichment of ALDH+ve cells in breast tumors [40]. Altered expression/dysregulation of RTKs is associated with BCSC phenotype and drug resistance. Several reports suggest the treatment of breast cancer with RTK-based therapies reverses the multidrug resistance [41,42,43]. The role of RTK signaling in regulation of CSC phenotype and drug resistance has been discussed further.

    Role of receptor tyrosine kinase (RTK) signaling in breast cancer progression

    EGFR: A key regulator of cancer stem cell phenotype and metastasis in inflammatory breast cancer

    EGFR is overexpressed in breast cancer tissues and is associated with higher aggressiveness and poor clinical outcomes [44, 45]. EGFR is a classic RTK and it undergoes homo or heterodimerization and trans-autophosphorylation upon ligand binding. EGFRs possess seven different cognate ligands including EGF, TGFα, betacellulin (BTC), heparin-binding EGF, amphiregulin (AREG), epiregulin, and epigen. The EGFR family consists of EGFR1 (EGFR, HER1, c-erbB1), HER2 (EGFR2, c-erbB2), EGFR3 (c-erbB3, HER3) and EGFR4 (c-erbB4, HER4) [46, 47]. Witton et al. have examined the expression of EGFR1, HER2, EGFR3 and EGFR4 using immunohistochemistry in 220 breast cancer patients and found overexpression of EGFR1 in 16.4%, HER2 in 22.8%, EGFR3 in 17.5%, and EGFR4 in 11.9% of breast cancer tissues. Increased expressions of EGFR1, HER2 or EGFR3 were associated with reduced survival whereas elevated level of EGFR4 was connected with better survival of breast cancer patients. It has been also reported that increased expressions of EGFR1, HER2 and EGFR3 were coupled with reduced expression of estrogen receptor (ER) [48]. Upon binding to the ligand, EGFR activates various downstream signaling molecules including Ras, PI3K, phospholipase C-γ (PLC-γ), and JAK leading to cell survival, cell growth, and tumor progression (Fig. 2) [6, 49, 50]. Various studies found that ER expression is inversely correlated with EGFR or cancer stem cell phenotype and that is well supported by the data that indicate higher expression of EGFR and presence of stem cell population in TNBCs which lack ER expression [51]. To investigate whether EGFR regulates stemness in breast cancer, Wise et al. have studied the enrichment of cancer stem cells under EGFR activation. They found that metalloproteinase-dependent activation of EGFR enriches CD44 + /CD24 - stem cells in TNBC through the MAPK/ERK pathway (Fig. 2) [6]. Inflammatory breast cancer (IBC) (especially inflammatory TNBC) is a more lethal and aggressive form of breast cancer characterized by enrichment of chemo- and radio-resistant CSCs [52, 53]. Various reports suggest that EGFR signaling is important for IBC pathogenesis and progression [54, 55]. Activation of NF-κB in IBC leads to ER downregulation and EGFR and/or ErbB2 overexpression and MAPK hyper-activation. MAPK signature distinguishes IBC from non-IBC tumors better than ER-based stratification (54). Wang et al. have identified that EGFR/cyclooxygenase-2 (COX-2) axis-regulated nodal signaling promotes CSC phenotype and increases invasiveness of IBC cells through induction of EMT (Fig. 2) [55]. TGF-β-elicited EMT program augments expression of RTKs such as EGFR and IGF-1R which form cytoplasmic complexes with ER-α and Src leading to anti-estrogen resistance in breast cancer [56]. Syndecan-1 (CD138) is overexpressed and associated with cell proliferation and invasion, and emerged as an important drug target in IBC. Ibrahim et al. have established the relation between Syndecan-1 and EGFR in the regulation of cancer stem cell phenotype in inflammatory TNBC. Their studies revealed that Syndecan-1 regulates EGFR expression through activation of Notch signaling. Syndecan-1/Notch/EGFR crosstalk modulates interleukin-6 (IL-6), gp130 and other inflammatory cytokine expressions thereby promotes colony formation and stem cell marker expression through Akt-mediated NFκB activation (Fig. 2) [9].

    RTK-regulated signaling in breast cancer progression. VEGFR activates JAK/STAT signaling pathway to induce cancer stem cell phenotype through Myc and Sox2 expression. Mutant p53 induces the expression of VEGFR through the interaction with SWI/SNF complex. EGFR-regulated signaling also plays pivotal role in angiogenesis and metastasis. EGFR regulates the activation of JAK/STAT and MAPK signaling pathway to induce expression of Sox2 and other stem cell markers leading to enrichment of cancer stem cells. EGFR induces Akt phosphorylation to promote inflammation. PDGFR is expressed on stromal cells such as fibroblasts and is a marker of fibroblast activation. PDGFR-regulated STAT activation is involved in regulation of miR-9-mediated differentiation of cancer cells to endothelial cells leading to angiogenesis. FGFR-activated MAPK pathway induces EMT and CSC phenotype. Cooperation between the FGFR and HER2 regulates nuclear translocation of Cyclin D1 leading to enhanced cancer cell proliferation

    Autophagy exhibits double-edged role in tumor progression depending on the context of a tumor. A recent study has revealed that autophagy regulates enrichment of ALDH+ve cancer stem-like cells via EGFR/Stat3 signaling in PyMT murine mammary cancer (Fig. 2) [57]. Tumor stroma also induces cancer stem cell phenotype by interacting with EGFR that is present on cancer cells through different downstream molecular players [58]. In the similar line of evidence, Yang et al. have reported that activation of EGFRs in cancer cells by TAMs leads to the Stat3-mediated Sox2 expression that resulted in increased cancer stem cell population and metastasis in murine breast cancer models (Fig. 2) [59].

    VEGFRs: Master nodes in VEGF-regulated metastasis, tumor angiogenesis and lymphangiogenesis

    Various studies established that angiogenesis is indispensable for breast tumor progression. VEGFs are potent proangiogenic factors that bind to three different types of VEGFRs, VEGFR1 (Flt1), VEGFR2 (KDR or murine homolog, Flk1). VEGFRs are expressed on cancer, endothelial and other stromal cells. VEGFRs are typical RTKs contain an extracellular domain for ligand binding, a transmembrane domain, and a cytoplasmic domain which includes a tyrosine kinase domain (TKD) [38]. VEGF-A binds to both VEGFR1 and VEGFR2 to induce tumor angiogenesis whereas VEGF-C and D interact with VEGFR3 to promote lymphangiogenesis in different types of cancer [38, 60]. However, Laakkonen et al. have reported that VEGF-C and VEGF-D-regulated VEGFR3 signaling induces tumor angiogenesis [61]. Chakraborty et al. have shown that osteopontin (OPN) augments VEGF-A expression in breast cancer cells and induces tumor growth and angiogenesis by regulating autocrine, paracrine and juxtacrine VEGF/VEGFR signaling in cancer and endothelial cells [62]. Srabovic et al. have reported that expression of VEGFR1 is significantly increased in breast tumor tissues as compared to benign tumors or healthy surrounding tissues, irrespective of the status of lymph node metastasis [63]. Kosaka et al. have identified elevated levels of VEGFR1 mRNA in peripheral blood of breast cancer patients and that is associated with cancer metastasis and recurrence and might be used for prognosis of breast cancer with basal-like and luminal type diseases [64]. In a recent study, Kapahi et al. have revealed that VEGFR1−710C/T polymorphism is associated with higher risk of breast cancer in North Indian population [65]. Ning et al. have revealed that VEGFR1 activation induces EMT of cancer cells thus promoting invasion and metastasis in breast cancer models [66]. Accumulated evidence suggests that infiltrated macrophages in tumor microenvironment promote malignant progression and enhance metastasis [11, 67]. A recent report has suggested that VEGFR1 signaling regulates obesity-induced tumorigenesis. Ablation of VEGF1 in obese animals reduced breast cancer growth and lung metastasis by decreasing M2 macrophage polarization and affecting glucose metabolism (Fig. 2) [67]. A recent evidence suggests that Flt1+ve metastasis-associated macrophages (MAMs), a subset of TAMs are enriched in metastatic breast cancer as compared to primary tumors. Flt1 signaling in MAMs regulates a set of inflammatory genes imperative for cancer cell survival after metastatic seeding. In addition, circulating VEGFR1+ve myeloid cells are involved in pre-metastatic niche formation [8, 68]. CYP4A polarized TAMs stimulate pre-metastatic niche formation and metastasis in lungs by mobilizing and recruiting VEGFR1+ve myeloid cells (Fig. 2) [68]. VEGR-2 is a key regulator of angiogenesis and overexpressed in breast cancer tissues [69]. Pfister et al. have studied the activation of VEGFR2 gene expression by mutant p53 in triple-negative breast cancer. In this study, they have shown that mutant p53 interacts with SWI/SNF and recruits to the promoter of VEGFR2 where this complex remodels the VEGFR2 promoter and induces the transcription leading to VEGFR-mediated breast tumor progression. These results indicate that mutant p53 gain of function is mediated by activation of VEGFR2 expression (Fig. 2) [70]. Collective evidences suggest that VEGFR2 exhibits prominent role in metastasis of breast cancer. However, the role of VEGFR2 in cancer cell invasion and migration is context-dependent. In breast tumor microenvironment, hypoxia induces c-Met/β1 integrin complex formation that results in higher invasion and migration potential of cancer cells. However, VEGF-activated VEGFR2 binds directly with c-Met and β1 integrin to prevent complex formation thus leading to sequestration of c-Met and β1 integrin [71]. Zhao et al. have found that VEGF drives VEGFR2 expression and subsequently activates JAK2/STAT3 signaling-mediated Myc and Sox2 expression. VEGF/VEGFR2 axis-established autocrine loop consisting of STAT3, Myc and Sox2 which implicated in enhancement of cancer stem-like cell phenotype in TNBC (Fig. 2) [10]. Nonetheless, CSCs are responsible for cancer cell metastasis, drug resistance, and tumor relapse, perturbing VEGFR2/STAT3/Myc/Sox2 axis might be useful in overcoming the chemo-resistance in triple-negative breast cancer.

    Lymphangiogenesis, formation of new lymphatic vessel plays a major role in cancer cell dissemination and distant metastasis. Hence, lymphangiogenesis is proved to be a promising target for the treatment of breast cancer. However, unavailability of specific markers for studying lymphatic vessels and lymphogenic metastasis delays the development of anti-lymphangiogenic therapy for management of different types of cancer [72]. VEGFR3 is a RTK expressed on lymphatic endothelial cells (LECs) and it plays a key role in lymphangiogenesis [20]. A recent study suggested that CCL21/CCR7 chemokine axis expressed on breast cancer cells interacts with VEGFR3 present on LECs to induce tumor-dependant lymphatic vascular recruitment and thereby lymphangiogenesis in breast cancer [73]. Lymphangiogenesis is also imperative for metastasis in postpartum breast cancer. Recent reports suggest that COX-2 induces VEGFR3 expression and lymphangiogenesis via VEGF-C/VEGFR3 axis to promote nodal metastasis of postpartum breast cancer [74, 75]. VEGFR3 is indispensable for galectin-8-mediated-crosstalk involving the VEGF-C, podoplanin and integrin pathways leading to lymphangiogenesis in breast cancer [76]. Based on above findings, targeting lymphangiogenesis using anti-VEGFR3 therapy might be useful in preventing tumor cell metastasis and increasing survival of breast cancer patients.

    PDGFR: promising role in tumor-stroma interaction in breast carcinoma

    PDGFRs are type III RTKs that are highly expressed in breast tumor and stromal cells. The PDGFR family consists of PDGFR-α and β and both show similar kind of functions. PDGFR-α and β are structurally similar and contain extracellular domain which consists of five immunoglobulin (Ig) - like folds and intracellular domains that exhibit kinase activity and consists of 100 amino acid residues dissimilar to other RTKs. PDGFs mostly bind to Ig-like domains 2 and 3, and induce homo or heterodimerization of the receptors. Moreover, these receptors are further stabilized by direct receptor-receptor interactions through Ig-like domain 4 after dimerization [77]. Aberrant activity of PDGFRs in different types of cancer including breast drives tumorigenesis. Various studies reported that PDGFR expression is associated with poor prognosis of breast cancer patients and it has prognostic and predictive potentials [78,79,80]. PDGFR is known to regulate various downstream signaling networks including Stat3 to support breast tumor initiation and progression [72]. Park et al. have reported that AF1q-induced STAT3 activation enhances breast cancer cell proliferation, angiogenesis and metastasis through PDGFR/Src signaling cascade [7]. Apart from directly regulating cancer cells, PDGFRs are also found to be expressed in reactive desmoplastic stroma that shows its possible role in tumor-stroma interaction. Bhardwaj et al. have found that PDGFR is expressed by α-SMA-positive myofibroblasts (cancer associated fibroblasts, CAFs) and endothelial cells in the periepithelial stroma of breast cancer tissues (Fig. 2) [79]. Paulsson et al. have examined the prognostic role of stromal PDGFR-β expression using tissue microarrays (TMAs) of breast cancer. Their findings suggested that stromal PDGFR-β exhibits most prominent prognostic significance in the subset of breast tumors. They also found that enhanced PDGFR expression is associated with reduced ER and PR and higher HER2 expression as well as inceased proliferation rate and tumor size [80]. In a similar line of evidence, Pinto et al. have shown that malignant stroma induces luminal breast cancer cell proliferation and angiogenesis in estrogen free-conditions through the PDGFR signaling cascade [81]. These results indicate the major role of PDGFR in breast cancer progression in absence of ER signaling. This notion is further supported by the fact that PDGFR induces endothelial differentiation of TNBC cells using in vitro tube formation and in vivo xenograft models. Moreover, D'Ippolito et al. have delineated the molecular mechanism by which PDGFR-regulates endothelial differentiation of tumor cells in TNBC. PDGFR induced miR-9 expression promotes vasculogenic properties by targeting STARD13 and downregulating miR-200 in TNBC (Fig. 2) [13]. These results indicate that targeting PDGF/PDGFR in tumor microenvironment might be the promising therapeutic approaches for the treatment of TNBC.

    FGFR: aberrantly expressed in breast cancer and implications in targeted therapy

    The FGFR family members (FGFR1, FGFR2, FGFR3 and FGFR4) are comprised of an extracellular ligand-binding domain, a transmembrane domain and an intracellular tyrosine kinase (TK) domain. The extracellular domain has three Ig-like domains (IgI-III). The FGFs binding to FGFR leads to dimerization and subsequent activation of the intracellular kinase domain resulting in cross-phosphorylation of tyrosine residues present on the cytoplasmic tail of the receptor [82]. Ras/MAPK and PI3K/Akt pathways are activated downstream to these receptors upon ligand stimulation. These pathways are known to be aberrantly activated in breast cancer and are involved in cell survival, proliferation, apoptosis and migration [83, 84]. The FGFRs harbour genetic aberrations such as amplifications of FGFR1, FGFR2 and FGFR4 and mutations in FGFR2 and FGFR4 genes in breast cancer [84,85,86,87]. Metastatic lobular breast carcinoma which shows poor response to chemotherapy demonstrates amplification of FGFR1 gene with implications in targeted therapy [86]. Formisano et al. have demonstrated that ER+ breast cancer shows amplification of FGFR1. They found that FGFR associates with ERα in nuclei of breast cancer cells and regulates ER-dependent genes in the presence of estrogen deprivation. In addition to ER+ breast cancer, amplification of FGFR1 gene correlated with poor prognosis in HER2- breast cancer [88]. Moreover, elevation of FGFR regulates tumor stroma remodelling and tumor recurrence in FGFR1-driven breast cancer [2]. Hence, studies with combinational therapies, targeting FGFR1 and other RTKs showed better results in cancer treatment as compared to targeting a single RTK. Single nucleotide polymorphisms (SNPs) in FGFR2 have been associated with an increased risk of ER+ and PR+ breast cancer [89]. Cerliani et al. have observed the interaction of FGFR2 with progesterone and STAT5 in breast tumor resulted in increased transcription of PR/STAT5-regulated genes [90]. Association of FGFR2 and FGFR3 expression with ER+ breast cancer progression was observed [91]. Even though, role of FGFR3 in breast cancer progression has not been studied well, splice variants of FGFR3 are known to localize to nucleus of breast epithelial cancer cells [92]. Koziczak et al. have shown that FGFR4 and ErbB2 co-operately regulate cyclin D1 expression to promote cell proliferation in breast cancer [93]. FGFR signaling-regulated ERK1/2- mediated Twist1 positive feedback loop stabilizes a CD44 high drug-resistant phenotype following ErbB inhibition (Fig. 2) [94]. Based on above findings, it is clear that FGFRs are mechanistically linked to the functions of other RTKs and drug resistance and may be a potential targets for treatment of breast cancer.

    Role of miRNAs and lncRNAs in regulation of RTK signaling

    In recent years, several studies have reported the role of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) in regulating the expression of components of different RTK signaling pathways. Tan et al. have shown that the level of ErbB2 in tamoxifen-resistant ER + breast cancer is tightly regulated by interplay between miR-26a/b and human antigen R (HuR) (Fig. 2) [95]. miR-34a and miR-155 also regulate expression of ErbB2 at the post-transcriptional level (Fig. 2) [96, 97]. miR-24 targets two regulators (tyrosine-protein phosphatase non-receptor type 9 (PTPN9) and receptor type tyrosine protein phosphatase F (PTPRF)) of EGFR activation, thereby promoting metastasis of breast cancer [98]. EGFR is a direct target of miR-206 in breast cancer and the latter is induced in nuclear factor (erythroid-derived 2)-like 2 (NRF2)-deficient breast cancer [99]. In human breast cancer, H19 lncRNA-derived miR675 targets c-Cbl and Cbl-b, E3 ubiquitin ligases which are known to degrade EGFR and c-MET thereby increases the stability of latter [100]. lncRNA CYTOR regulates the breast cancer progression through EGFR dependent pathway [101]. Another lncRNA, BCAR4 enhances the activity of ErbB2/3 receptors [102]. Role of different miRNAs and lnRNAs in the regulation of RTK signaling components are listed in Table 1.

    Role of RTK signaling in drug resistance

    Endocrine therapy is the treatment that specifically blocks the function of ER signaling using antagonists (tamoxifen, fulvestrant) or estrogen deprivation [103]. Almost 20% of the patients acquire resistance to ER-targeted therapy via activation of escape signaling pathways to overcome estrogen dependency [104]. Overexpression or activation of RTKs such as EGFR, HER2 and IGF1R leads to downregulation of ER and resistance to tamoxifen through activation of PI3K/Akt and MAPK pathways (Fig. 3) [105, 106]. EGFR/MAPK axis promotes phosphorylation of AF-1 domain of ER to enhance the ligand-independent activation of ER signaling [106, 107]. Activation of EGFR/ErbB2 signaling in tamoxifen-resistant ER+ breast cancer cells induces highly aggressive stem cell phenotype in these cells [108,109,110]. Inhibition of EGFR signaling using erlotinib considerably reduces the cancer stemness and reverses the endocrine resistance by inducing the expression of ER [111]. Moreover, HER2 amplification in ER-resistant breast cancer correlates with the ALDH+ stem cell population [108]. CSC population expresses a very high level of HER2 mRNA and protein as compared to the non-CSC population in endocrine-resistant patients. Higher activation of EGFR/HER2 might be the driving force in enriching CSC population in tamoxifen-resistant breast cancer [36, 108]. Association of HER2 expression with ER resistance has been explained in several reports. Whole exome sequencing studies revealed 13 mutations in different domains of HER2 in ER+ endocrine-resistant metastatic breast cancer patients [112]. These mutations produce different level of resistance to tamoxifen and fulvestrant in ER+ breast cancer cell lines. Moreover, ER cofactors, HOXB3 and HOXB7 are found to be overexpressed in tamoxifen-resistant breast cancer cells and enhance CSC phenotype. Myc-mediated transcriptional repression of miR-375 and miR-196a enhances the expression of HOXB3 and HOXB7 respectively [113, 114]. Retinoblastoma binding protein 2 (RBP2), an ER co-regulator is overexpressed in tamoxifen-resistant breast cancer patients and increases the stability of RTKs such as EGFR and HER2. Moreover, RBP2-ER-NRIP1-HDAC1 complex activates IGF1R through transcriptional repression of IGFBP4 and 5 [115]. Another ER transcriptional coactivator, mediator subunit 1 (MED1) is overexpressed in circulating tumor cells and primary breast tumor tissues following tamoxifen treatment leading to HER2-mediated ER resistance. HER2-mediated phosphorylation of MED1 recruits the transcriptional corepressors such as HDAC1, N-CoR and SMART to the promoter of the ER-regulated genes in HER+ tamoxifen-resistant cells [116, 117].

    RTK signaling in drug resistance. a Conventional chemotherapeutic agents reduce the cancer progression through the inhibition of MAPK/PI3K/Akt signaling axis. Amplification and overexpression of RTKs including EGFR, HER2 and PDGFR reinforce the activation of PI3K/Akt/YB-1/RTK axis to maintain drug resistance increases the kinase activity and thereby leading to cancer progression, drug efflux and cancer stemness. b Cancer cells exhibit resistance to RTK therapy due to disruption of interaction between drug and receptor or activation of alternate RTK signaling

    Apart from the endocrine therapy, other types of treatment such as surgery, radiation therapy and cytotoxic drugs are also available for breast cancer. Mainly, anthracyclines (DNA damaging agents) and taxanes (microtubule-stabilizing agents) are widely used for breast cancer as adjuvant or neoadjuvant therapies [118]. However, the resistance to cytotoxic cancer drugs is the major drawback in cancer treatment. Multidrug resistance is mainly associated with cancer stemness and drug efflux driven by various survival signals [119]. Importantly, RTKs are key regulators of cancer stemness and associated with drug resistance in breast cancer cells. In general, various RTKs activate PI3K/Akt signaling to induce the expression of cancer stemness factors, multidrug resistance associated proteins and membrane transporters in cancer cells. Accumulating evidence clearly suggest that upregulation of RTKs including EGFR, HER2, VEGFR and IGF-1R in course of chemotherapy is associated with overexpression/activation of drug efflux transporters [41, 42]. Jin et al. have shown the strong positive correlation between p-glycoprotein expression and EGFR with overall and disease-free survival [43]. Moreover, higher expressions of EGFR and HER2 are detected in doxorubicin-resistant MCF7 cells as compared to the doxorubicin-sensitive MCF7 cells. Overexpression of HER2 also induces resistance to various chemotherapeutic agents such as taxane, cyclophosphamide, methotrexate, epirubicin in breast cancer [120]. Moreover, HER2 expressing circulating tumor cells (CTCs) shows less sensitivity to the various chemotherapeutic agents including doxorubicin, docetaxel and 5-fluorouracil as compared to HER-negative CTCs [121]. Overexpression of RTKs is correlated with expression of transcription factors linked to drug resistance in breast cancer. YB-1 is a transcriptional/translational regulator and overexpressed in cancer stem cells. Nuclear localization of YB-1 is reported in cancer relapse and drug-resistant patients irrespective of ER and HER2 status. RTK-regulated PI3K/Akt phosphorylates YB-1 at Ser-102 to facilitate the nuclear localization. Furthermore, nuclear YB-1 binds to the specific promoter region and transcriptionally activates the expression of RTKs including EGFR, HER2 and VEGFR. Disturbance in YB-1/RTKs self-reinforcing loop significantly reduces the cancer stemness and drug efflux in breast cancer cells [122]. Moreover, YB-1 transcriptionally increases the expression of p-glycoproteins (MDR-1 and MDR-3) provokes the multidrug resistance in breast cancer (Fig. 3) [123, 124]. TAMs are known to influence the maintenance of suitable microenvironment for cancer stem cells and sustained drug resistance in breast cancer. TAMs produce the higher level of cytokines, TGFα, EGF, FGF and VEGF in the tumor microenvironment. Higher levels of these ligands activate RTK signaling in breast cancer as well as macrophages [125]. A strong correlation between EGFR expression and CD163+ macrophages were found in tamoxifen-resistant breast cancer patients [126]. Moreover, TAMs upregulate the cancer stemness associated genes along with increased drug efflux and chemoresistance in preclinical breast cancer model [127].

    Receptor tyrosine kinase (RTK)-targeted cancer therapeutics

    Breast cancer is a heterogeneous disease which has been characterized molecularly into five subtypes depending on expression of ER, PR and HER2. These subtypes consist of Luminal A (low grade, ER+/PR+, HER2-, low Ki67), Luminal B (ER+/PR+, HER2+ or HER2-, high Ki67), TNBC or basal-like (ER-/PR- and HER2-), HER2-enriched and normal-like breast cancer [128]. For hormone receptor-positive breast cancer (luminal A and B), hormone therapy consists of selective estrogen receptor modulators (tamoxifen and raloxifene) is routinely used as adjuvant therapy [129]. Since TNBC or basal-like and HER-enriched breast cancer do not express hormone receptors so that hormone therapy is not effective in these subtypes. However, due to the prominent expression of RTKs in TNBC and HER2-enriched subtypes, blocking the functions of RTKs is one of the promising approaches for management of TNBC and HER2-enriched breast cancer. So far, various strategies have been adopted for inhibition of RTK-dependent signaling. Mutations or overexpression of EGFR genes leads to tumor progression and drug resistance in various cancer types including breast [127]. Therefore, EGFR holds the potential to be an attractive drug target in breast cancer, and the EGFR inhibitors, including small molecule inhibitors and monoclonal antibodies (mAbs), have been developed and some are currently used in clinics. Overexpression of HER2 is frequently found in breast cancer. Several HER2-targeting drugs were developed and are currently used for the treatment of breast cancer.

    Trastuzumab (Herceptin) is a humanized mAb which targets the extracellular domain of HER2 in HER2+ breast cancer and it has been reported to enhance survival of patients at early and late stages of breast cancer [130]. However, the exact mechanism through which trastuzumab exhibits its therapeutic effect is not well understood. De et al. have reported that trastuzumab inhibits HER2-HER3 heterodimerization which is known to occur in a ligand-independent manner in HER2+ breast cancer. Several reports also suggested that trastuzumab might induce HER2 degradation but the underlying mechanism is unexplored [131]. Although treatment with trastuzumab significantly improves disease outcome, resistance to trastuzumab is a major barrier to treat HER2-positive breast cancer. Approximately 65 % of HER2-positive breast cancer patients do not respond to primary trastuzumab treatment. Moreover, a majority of patients those who originally respond well to trastuzumab therapy show tumor relapse later [132, 133]. In 2013, FDA approved an antibody-drug conjugate T-DM1 or trastuzumab emtansine or ado trastuzumab emtansine (trade name Kadcyla) for the treatment of HER-positive metastatic breast cancer patients who has been previously treated with trastuzumab and a taxane. T-DM1 consists of trastuzumab and cytotoxic agent emtansine (DM1) which kills the cancer cells by binding to tubulin [134]. A random trial on 991patients with HER2-positive advanced breast cancer showed higher median progression-free survival in T-DM1-treated patients compared to lapatinib plus capecitabine-treated ones [135]. However, a recently completed phase III trial using trastuzumab plus taxane, T-DM1 plus placebo, T-DM1, or T-DM1 plus pertuzumab regimens at standard doses in 1095 HER2-positive advanced breast cancer patients. No significant increase in progression-free survival in T-DM1 and T-DM1 plus pertuzumab groups was observed as compared with trastuzumab plus taxane although, T-DM1 containing arms showed better tolerability [136]. Pertuzumab (trade name perjeta) is another monoclonal antibody against HER2 which has been approved for neo-adjuvant or adjuvant therapy of HER2-positive advanced breast cancer in a combination with trastuzumab and docetaxel. Clinical trials have demonstrated that breast cancer patient’s administered with combination of pertuzumab, trastuzumab and docetaxel had enhanced progression-free survival compared to control group [137, 138].

    TNBC or basal-like breast cancer is known to be negative for HER2, shown to express EGFR in 40% of the patients, of those 18% of patients are reported to have amplified EGFR gene. Hence, EGFR is one of the important targets for HER2 negative breast cancer including TNBCs. Lapatinib (Tykerb), a dual tyrosine kinase inhibitor, binds to ATP binding pocket of EGFR and HER2 kinase domain and blocks ATP binding thereby leading to inhibition of EGFR and HER2 kinase activity. The tyrosine kinase inhibitors (TKIs) are known to be used as an alternate therapeutic regimen in HER2+ breast cancer patients with trastuzumab resistance [139, 140]. Moreover, lapatinib has been used in combination with other anticancer drugs, capecitabine or letrozole. These combination therapies showed higher disease-free survival in HER2+ metastatic breast cancer patients [141, 142]. Multiple clinical trials have been conducted to assess the efficacy and toxicity of TKIs either alone or in combination with other drugs in breast cancer. Unfortunately, the outcomes of these trials have been disappointed so far. Few trials and their outcomes are enlisted in Table 2. Phase II clinical trials of gefitinib or erlotinib have shown poor overall response rate (ORR) while clinical trial with gefitinib in combination with epirubicin and cyclophosphamide showed no significant difference in pathologic complete response in ER-negative breast cancer [142,143,144,145,146]. Further, afatinib, a second-generation irreversible EGFR TKI, has shown no objective responses in phase II trial in metastatic TNBC patients [147].

    There have been six clinical trials with anti-EGFR mAbs to explore their efficacy and safety in TNBC patients as given in Table 2. Carey et al. have performed a clinical trial in metastatic advanced recurrent breast cancer to examine the efficacy of cetuximab or cetuximab in combination with carboplatin. Cetuximab in combination with carboplatin demonstrated higher response rate as compared to carboplatin alone. However, 13 out of 18 treated patients showed active EGFR signaling that indicates cetuximab failed to inhibit the EGFR pathway [148]. Higher response rate in cisplatin-cetuximab treated patients (20%) as compared to cisplatin-treated group (10%) has been reported in advanced TNBC. However, the outcomes were not statistically significant [149]. Similarly, a phase II trial of ixabepilone alone and ixabepilone plus cetuximab in patients with advanced/metastatic TNBC was conducted by Tredan et al. This study has shown no improvement in response rate [150]. Meanwhile, irinotecan and cetuximab were shown increased response rate in TNBC patients compared to other subtypes, however, the results were not statistically significant [151]. Modest response was observed when operable TNBC patients were treated with standard FEC (5-fluorouracil, epidoxorubicin, and cyclophosphamide) following preoperative chemotherapy consisting of panitumumab or cetuximab combined with docetaxel [152, 153]. Higher CD8+ tumor infiltrating lymphocytes (TILs) were spotted in the tumor microenvironment in response to EGFR mAb neoadjuvant therapy. Overall, the outcome of clinical trials of EGFR mAbs in TNBC seems to be slightly better than that of EGFR TKIs. Several trials using anti-RTK therapy and their outcomes are enlisted in Table 2 [146, 154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174].

    Challenges in targeting RTKs in breast cancer: emphasis on compensatory elements

    RTK-targeting therapeutic drugs are known to reduce multidrug resistance and CSC phenotype in breast cancer cells. However, cancer cells exhibit the resistance to RTK inhibitors in clinical and preclinical models. For example, HER2-targeted therapies (trastuzumab, pertuzumab, TDM1 and lapatinib) are known to impede primary tumor progression and cancer relapse but still drug resistance is observed in approximately 80% of HER2+ metastatic breast cancer patients [142]. Similarly, many cancer types including breast often acquire resistance to various RTK inhibitors such as VEGFR inhibitors (bevacizumab) [175], EGFR inhibitors (gefitinib) [176], FGFR inhibitors (AZD4547) [177]. Several mechanisms have been derived to describe the occurrence of resistance to RTK inhibitors. Several mutations in RTKs and their downstream targets and the activation of multiple other RTKs are the major compensatory elements instigated the survival pathways and resistance to anti-RTK therapies in breast cancer. IGF1R, EGFR, AXL, VEGFR are other RTK members share common downstream signaling molecules such as PI3K/Akt/mTOR and MAPK with HER2 in breast cancer [178]. Moreover, IGF1R overexpressed in HER2+ breast cancer and forms a heteromeric complex with HER2 and HER3 to activate PI3K signaling pathway. These heteromeric complex formation with HER family proteins have been associated with trastuzumab resistance in HER2+ metastatic breast cancer patients [179]. Combination of anti-HER2 drugs with anti-IGF1R mAbs (metformin and figitumumab) have reported to produce synergetic effects in breast cancer cells. C-Met is the RTK, frequently expressed in HER2+ breast cancer patients and contributes to trastuzumab resistance. Upregulation of c-Met protects the cancer cells from trastuzumab via abrogating p27-induction whereas inhibition of c-Met sensitizes the cancer cells to trastuzumab treatment [180]. c-Src-mediated phosphorylation of EGFR at Tyr845, Tyr992, and Tyr1086 is associated with resistance to anti-EGFR therapy in breast cancer. Activation of c-Met during EGFR treatment facilitates c-Src kinase-associated phosphorylation and cell growth in breast cancer cells. Furthermore, a combination of c-Met targeting small molecule inhibitors along with EGFR inhibitor decreases EGFR phosphorylation and kinase activity via inhibiting c-Src kinase thereby reduces the EGFR resistance [181]. Increased copy number of FGF3/4/19 has been reported in lapatinib and trastuzamab-resistant tumors. Higher expression and phosphorylation of FGFR is correlated with reduced disease-free survival and anti-HER2 therapy resistance in breast cancer patients. Activation of FGFR further stimulates the phosphorylation of non-receptor kinases such as MAPK and PI3K/Akt through the activation of phospholipase Cγ in tamoxifen-resistant breast cancer [182]. Amplifications and mutations in RTK dependent downstream target genes (PI3KCA or Akt) bypass the role of RTKs in their activation so that produce uninterrupted activation of growth signaling in breast cancer cells. Mutation in PI3CA is strongly associated with ErbB2-overexpression and lymph node metastasis [183].

    Bevacizumab is the first anti-VEGFR drug approved by US FDA for the treatment of breast cancer but it is discontinued eventually due to the occurrence of resistance to it. Anti-VEGFR therapy induces hypoxia in the tumor microenvironment and its lead to increase in the aggressiveness of breast cancer. Under hypoxic stimuli, stromal cells secrete very high level of cytokines that activate alternate angiogenic pathways and increase the cancer stemness and autophagy [175]. Ephrin- A1 and B2 are proangiogenic factors, important for the remodeling and maturation of new blood vessels. Hypoxia mediates the upregulation of ephrin and the expression of ephrins is strongly associated with resistance to VEGFR therapy. Several proangiogenic factors such as angiopoietin 2 (ANG-2), EGF, bFGF, keratinocyte growth factor, IGF-1, TGF-β, TNF-α and interleukins (IL-1, IL-8, IL-12 and IL-17) have been implicated in hypoxia-associated tumor refractoriness to anti-VEGFR therapy [184]. Secretion of IL-17, G-CSF, IL-6 and SDF1in tumor microenvironment recruits CD11b+Gr1+ myeloid cells to tumor and conferring Bv8-associated VEGFR-independent angiogenesis leads to resistance to anti-VEGFR therapy. Depletion of CD11b+Gr1+ myeloid cell infiltration by Bv8 neutralizing antibodies sensitizes the cancer cells to VEGFR-targeted therapy [185].

    Impaired interaction between anti-RTK agents and its respective receptor is another reason behind the development of resistance. This might be due to the higher existence of masking proteins in close proximity to the receptors, structural changes in the receptor and lack of expression of targeted domain. Mucin-4 and CD44 are the cell surface proteins overexpressed in trastuzumab resistant breast cancer patients. Expression of these proteins in close proximity to the HER2 epitope masks the interaction between trastuzumab and HER2 and increase the breast cancer growth [186, 187]. On other hand, expression of a truncated version of HER2 overrides trastuzumab sensitivity in breast cancer. p95 HER2 forms heterodimer with HER3 protein and activates downstream signaling in a ligand-independent manner (Fig. 3) [188]. Eliyatkin et al. have shown that 28% of the patients who develop trastuzumab resistance have higher expression of p95 HER2 . However, low level of p95 HER2 expression is found in trastuzumab-sensitive patients as well [189]. Moreover, mutations in HER2 could perturb the antibody recognition or physical interaction between drug and receptor. T798M mutation in HER2 showed increased autocatalytic activity and expression of EGFR ligands lead to 10-fold changes in IC50 of lapatinib in human breast cancer cells. Moreover, EGFR targeting antibody, cetuximab or lapatinib revert the trastuzumab resistance in these T798M specific cells [190]. Hanker et al. have shown that patients with HER2 L869R mutation acquire secondary mutation at HER2 T798I as subsequent response to neratinib treatment. Molecular modeling studies suggested that HER2 T798I has increased isoleucine content in its protein structure and that reduces the binding between neratinib and HER2 [191].


    MiR-219-5p inhibits receptor tyrosine kinase pathway by targeting EGFR in glioblastoma

    Glioblastoma is one of the common types of primary brain tumors with a median survival of 12-15 months. The receptor tyrosine kinase (RTK) pathway is known to be deregulated in 88% of the patients with glioblastoma. 45% of GBM patients show amplifications and activating mutations in EGFR gene leading to the upregulation of the pathway. In the present study, we demonstrate that a brain specific miRNA, miR-219-5p, repressed EGFR by directly binding to its 3'-UTR. The expression of miR-219-5p was downregulated in glioblastoma and the overexpression of miR-219-5p in glioma cell lines inhibited the proliferation, anchorage independent growth and migration. In addition, miR-219-5p inhibited MAPK and PI3K pathways in glioma cell lines in concordance with its ability to target EGFR. The inhibitory effect of miR-219-5p on MAPK and PI3K pathways and glioma cell migration could be rescued by the overexpression of wild type EGFR and vIII mutant of EGFR (both lacking 3'-UTR and thus being insensitive to miR-219-5p) suggesting that the inhibitory effects of miR-219-5p were indeed because of its ability to target EGFR. We also found significant negative correlation between miR-219-5p levels and total as well as phosphorylated forms of EGFR in glioblastoma patient samples. This indicated that the downregulation of miR-219-5p in glioblastoma patients contribute to the increased activity of the RTK pathway by the upregulation of EGFR. Thus, we have identified and characterized miR-219-5p as the RTK regulating novel tumor suppressor miRNA in glioblastoma.

    Conflict of interest statement

    Competing Interests: The authors have declared that no competing interests exist.

    Figures

    Figure 1. miR-219-5p is downregulated in glioblastoma.

    Figure 1. miR-219-5p is downregulated in glioblastoma.

    Expression of miR-219-5p in malignant astrocytoma [anaplastic astrocytoma…

    Figure 2. Overexpression of miR-219-5p in glioma…

    Figure 2. Overexpression of miR-219-5p in glioma cell lines decreased proliferation, anchorage independent growth and…

    Figure 3. miR-219-5p targets EGFR.

    Figure 3. miR-219-5p targets EGFR.

    Figure 4. Overexpression of miR-219-5p reduced the…

    Figure 4. Overexpression of miR-219-5p reduced the activities of MAPK and PI3K pathways.

    Figure 5. Inhibition of MAPK/PI3K pathways and…

    Figure 5. Inhibition of MAPK/PI3K pathways and glioma cell migration by miR-219-5p are mediated by…

    Figure 6. EGFR protein levels negatively correlated…

    Figure 6. EGFR protein levels negatively correlated with miR-219-5p expression levels in glioblastoma samples.


    Watch the video: Lecture 08, concept 19: Signaling - Receptor Tyrosine Kinases RTKs (December 2021).