Approximately 90 TK receptors have been identified, 58 of which are transmembrane receptor type (eg: EGFR, Epidermal Growth Factor Receptor) and 32 the cytoplasmic “non receptor” type.

Introduction

TYROSINE KINASE RECEPTORS (RTKs)

Protein tyrosine kinases represent a wide family of homologous enzymes, both transmembranous and cytoplasmic, that catalyze the transfer of the γ-phosphate group from ATP to a hydroxy group of selected tyrosine residues in target protein substrates.

Under physiological conditions, tyrosine phosphorylation represent a fundamental signal transduction mechanism that proceeds hierarchically in a ordered sequence of protein interactions, ensuring cross-talk between cells and regulating key aspects of cell life such as proliferation, differentiation, metabolism, and apoptosis. ¹

Approximately 90 TK receptors have been identified, 58 of which are transmembrane receptor type (eg: EGFR, Epidermal Growth Factor Receptor) and 32 the cytoplasmic “non receptor” type.

Transmembrane RTKs are floating protein in the double layer phospholipid membrane, all of which share a similar structure characterized by three regions: a cytosolic, an extracellular, an intramembrane portion. For their different structural features of domains approximately 20 RTK classes have been identified (Figure 1). The best characterized are ² :

RTK class I (Epidermal Growth Factor (EGF) receptor family) RTK class II (Insulin receptor family)

RTK class III (Platelet-derived Growth factor (PDGF) receptor family) RTK class IV (Fibroblast Growth Factor (FGF) receptor family)

RTK class V (Vascular Endothelial Growth Factor (VEGF) receptors family) RTK class VI (Hepatocyte Growth Factor (HGF) receptor family)

RTK class VII (Tropomyosin-receptor-kinase (Trk) receptor family) RTK class XI (Angiopoeitin (TIE) receptor family)

RTK class XVII (Muscle Specific Kinase (MuSK) receptor family)

Introduction

Figure 1: Human Receptors Tyrosine Kinase

The extracellular region exposes N-terminal domain to the interstitial fluid and presides over the recognition and interaction with the ligand.

Hydrophilic intracellular portion, C-terminal, is equipped with sites that govern signal transduction after ligand-receptor interaction. This portion is characterized by tyrosine residues in specific positions for any receptor and after interaction with the ligand undergo autophosphorylation. It’s the specific arrangement that influenced the interaction with the cytoplasmic proteins and its phosphorylation because those exposes on their surface domains able to recognize specific phosphorylated aminoacid sequences of the catalytic domain of intracellular portion of the receptor. ³

Instead, cytoplasmic receptors are devoid of the extracellular portion and generally allow the signal transduction within the cell only after the activation of transmembrane receptors.

Normally, TK receptors are encoded by protoncogenes, and when undergo mutations or are overexpressed, they become oncogenes and activate enzymes prematurely, leading to the appearance of tumors.

The basis of many cancers are anomalies involving two important classes of genes:

Introduction

• protoncogenes, that stimulate the cell to advance its cell cycle, are affected by dominant mutations with gain of function, which leads to increased or uncontrolled activity of the product, or oncogenes.

So inactivated tumour suppressor genes lead to a lack of controller proteins preventing the excessive cell proliferation, while protoncogenes mutation leads to production of excessive amounts or of an excessively active form of the protein growth promoters.

In both cases the result is the same: an uncontrolled cell growth that degenerates into a tumour. The difference between oncogenic and normal genes may also be related to the replacement of a single amino acid, which often results in a radical change in protein function.

While normal cytoplasmic receptors encoded by protoncogenes are activated by transmembrane receptors, in turn activated by their ligand, those resulting from the respective oncogenes are constitutively endowed with enzymatic activity.

Under physiological conditions the kinase activity is very low while it’s increased in tumor cells: in fact, the increase in intracellular phosphotyrosine, associated with increased cell proliferation, represents a real tumor marker status.

In the last years some RTK have become targets of anti-tumor drugs, which,

inhibiting the TK activity, could block the proliferation of cancer resulting from signal

transduction. ³

Introduction

THE EPIDERMAL GROWTH FACTOR RECEPTOR EGFR

The Epidermal Growth Factor Receptor (EGFR) is one of the most well-studied tyrosine kinases receptor: it’s coded by c-erbB-1 protoncogenes, located on chromosome 7, and is constitutively expressed in some tissues of epithelial, mesenchymal and neural origin. The receptor acts as a result of binding with the specific growth factor EGF (Epidermal Growth Factor) or less specific ligands. ¹

There are four known EGFR family members called ErbB or HER (Human Epidermal Receptor), including: ErbB-1 (EGFR, HER1), ErbB-2 (HER-2), ErbB-3 (HER-3) and ErbB-4 ( HER4) (Table 1) . ⁴

Receptors Synonyms HER1 erbB-1, EGFR HER2 erbB-2 HER3 erbB-3 HER4 erbB-4

Table 1. Synonyms of HER family members

The EGFR is a transmembrane glycoprotein of 170 kDa formed by a single

polypeptide chain containing 1186 amminoacids, and is characterized by three regions

(Figure 2): ⁴

Introduction

• A part of amino-terminal extracellular domain (or ectodomain) which consists of 621 amino acids and comprises four sub-domains. Early studies have shown that the major binding site of the receptor for growth factors is located in a fragment of domain III (or L2), between amino acids 295 and 543. Subsequent studies, instead, have revealed that a portion of domain I (or L1) plays a role, albeit minor, in the binding of growth factors. Domains L1 and L2 are rich in cysteine (Figure 3). Domains II (or CR1) and IV (or CR2) are characterized by several modules linked together through one or two disulfide bridges. Behind the CR1 domain a loop protrudes that binds to another receptor of CR1 during dimerization. The extracellular portion is very variable within the HER family, being characterized by a homology of 30-50% (Figure 4). ^5,6

Figure 3. Extracellular domain to bind EGF

Introduction

Figure 4 Homology of HER family

• The transmembrane region consists of 23 amino acids, from 622 to 644, with hydrophobic characteristics, folded into a single α-helix. This latter extends up to amino acid 647, thus suggesting its continuation even in the internal domain close to the membrane.

• The cytoplasmic domain consists in a kinase activity portion extending from amino acid 644 to 955 and is characterized by high homology between members of the HER family (59-82%) and in a carboxyl terminus (between amino acids 956 and 1186), more variable (homology around 30%), containing sites for tyrosine phosphorylation (indicated by Y or Tyr): Y992, Y1068, Y1148 and Y1173. ^4-6

The catalytic domain consists of about 250 amino acids folded to form two distinct lobes. The minor lobe has the function to link ATP, allowing γ-phosphate transfer, the larger lobe recognizes the substrate and properly orients the γ-phosphate. The minor lobe is characterized by β-sheets, while the major is dominated by α-helices with a small β-sheet, near the cavity between the two lobes.

The more conserved structure among HER family members is the ATP binding site,

composed of a lysine (Lys721), a Gly-X-Gly-X-X-Gly sequence and a tyrosine

(Tyr845). The sequence rich in glycine is deputed to the formation of hydrogen bonds

with the γ-phosphate of ATP, the nucleotide is further stabilized by the presence of

Lys721 and Glu738. Adenine is inserted into a hydrophobic pocket and makes hydrogen

Introduction

GROWTH FACTORS AND RECEPTOR ACTIVATION

Growth factors bind to HER family members with different affinities, only HER2 has no specific ligands. Usually they are divided into three groups: the first includes EGF (Epidermal Growth Factor), TGF-α (Trasforming Growth Factor-α) and AR (anfiregulin) that bind only to EGFR, the second includes factors that make a specific link both with EGFR, and with ErbB4, as BTC (betacellulin), HB-EGF (Heparin- binding EGF), EPR (epiregulin) and the third group includes NRG (neuroregulin), which can be divided into two subgroups according to their ability to bind ErbB3 and ErbB4 (NRG-1, NRG-2) or only ErbB4 (NRG-3 and NRG-4) (Table). ^5,6

Table 2. Affinity of ligands

The presence of HER receptors in the basolateral membrane of epithelial cells, and the discovery of many HER ligands such as EGF, TGFα, AR, BTC and NRGs in the extracellular matrix suggest that HER play an important role in mediating the signal between the epithelium and stroma.

Interaction with the ligand in both domain I and III induces a change in the receptor

conformation, allowing to expose an interface that was previously hidden and activating

the process of dimerization. (Figure 5). ⁸

Introduction

Figure 5. Change of receptor conformation after binding with EGF

In the inactive state the receptor exists as a monomer on the surface of the cell membrane. Binding of growth factor stabilizes the dimeric form (active) and causes its autophosphorylation: the catalytic site of each chain phosphorylates tyrosine residues located in the carboxy-terminal portion of the other chain. (Figure 6).

Figure 6 . Dimerization and autophosphorylation of receptor

Introduction

The interaction with the ligand may promote homo-dimerization (between monomers of the same receptor, eg HER1/HER1) or hetero-dimerization between the receptor and other members of the HER family (eg HER1/HER2), increasing its tyrosine kinase activity. Quantitative studies at the cell surface have shown that one of the most frequent association is the complex HER1/HER2. ⁸

In both types of dimerizations the autophosphorylated receptors activate (through phosphorylation), other substrates that recruit the cytoplasmic adapter proteins and trigger a cascade of signals that drive important cellular functions like cell growth, proliferation, cytoskeleton rearrangement, apoptosis, motility, changes of gene expression and angiogenesis.

EGFR SIGNALLING PATHWAYS ^6,9

The most studied signal transduction mechanism include the Ras/Raf/MEK/ERK and PI3K/PDK1/Akt cascade, but also the phospholipase PLC-γ and JAK /STAT can be engaged by activation of EGFR through overlapping or independent mechanism. It is still not clear which are the principal pathways in cancer initiation and progression.

In the Ras dependent EGFR activation the key role is played by GRB-2 (Growth Factor Receptor Bound protein 2) adapter protein, through its ability to bind directly to the receptor in its portions Y1068 and Y1086 or indirectly through phosphorylated Shc protein .

GRB-2 and Shc mediate the activation of transduction of different signals, depending on the type of ligand, the level of expression of the receptor and on the type of EGFR receptor dimerizes with. The study of this cellular process suggested that the association of Shc with EGFR through its domains, which involves tyrosine phosphorylation and recruitment of GRB-2, may be a critical step in the mechanism of activation of EGFR by the employee Ras / MAPK .

The molecules at the top of the cascade of intracellular signal transduction are

characterized by the presence of regions of homology with the src oncogene, the so-

called SH2 domains (src homology 2) and SH3 (src homology 3). These domains are

essential for the interaction between molecules in the chain of transmission. SH2

Introduction

domains consist of amino acid sequences that bind phosphorylated tyrosine residues, the SH3 domains are composed of amino acid sequences that recognize sites rich in proline.

The SH3 domains of GRB-2 recognize proline-rich sequences present in the guanidine nucleotide exchange factor Sos (Son of Sevenless), leading to the formation of GRB-2/Sos complex being moved inside the membrane. Sos can activate the Ras protein, a small G protein, inducing the exchange of GDP for GTP. This causes, consecutively, activation of the Raf family of kinases (a-Raf, B-Raf and Raf-1), MEK (Mitogen activated extracellular signal regulated kinases) and subsequently of Erk1 and Erk2 (Extra-cellular signal-regulated kinases).

Finally, Erk1/2 kinases, members of the family of MAPK (Mitogen-Activated

Protein Kinases) positively regulate cell proliferation by activating major transcription

factors associated with cell proliferation such as c-Myc and isoforms of the RSK

(Ribosomal Subunit Kinase) family. In modulating the balance between cell

proliferation, apoptosis and senescence, the EGFR/PI3K/Akt pathway is crucial in

promoting an intense proliferation through EGFR/Ras/Raf/MEK/ERK cascade (Figure

7).

Introduction

Figure 7. EGFR signal transduction pathways

The C-terminal domain of EGFR contains the Y920 residue, which directly provides the site for the binding of p85 subunit of PI3K (phosphoinositol-3 kinase) either directly or indirectly through binding to GRB-2.

PI3K generates PIP3 (phosphatidyl inositol-3,4,5-triphosphate), which recruits and activates the protein kinase Akt, also known as protein kinase B (PKB), and PDK-1 (phospho inositol-dependent kinase) by binding their PH domain to PIP3.

Phosphorylated Akt protein is able to control the programmed cell death through

phosphorylation and consequent inhibition of Bad (a member of the family of pro

apoptotic Bcl-2) and Caspase 9 (an enzyme dependent on Fas receptor membrane

belonging to the extrinsic activation of apoptosis). The final result is the inhibition of

Introduction

relevant transcription factors as: HIF-1α, NFkB and CREB that cause an increasing of anti-apoptotic genes. In addition to survival mechanism regulated by Akt, EGFR may activate STAT (signal and activator of transcription) pathways through a JAK (Janus Kinase) -dependent or a JAK-independent mechanism.

Stimulation of EGFR induces phosphorylation of STAT1 and the beginning of the formation of the system STAT1 and STAT2 with JAK1 and JAK2. This allows STAT to be moved into the nucleus within only 15 minutes and this may contribute to cancer cell survival through effects on gene expression. A JAK independent mechanism of activation has been proposed for STAT5b which has a direct docking site at EGFR (Tyr845).

EGFR-mediated signal transduction also contributes to the regulation of both angiogenesis and metastasis. Angiogenesis, responsible for tumour progression, is mediated by EGFR through up-regulation of VEGF (Vascular Endothelial Growth Factor) and MMPs (metal protease). Furthermore, the phosphorylation of cytoplasmic domain of EGFR Tyr992, activating PLCγ (phospholipase Cγ) can directly influence the development of metastases. Tyr992 is indeed the binding site of PLCγ.

EGFR AND CANCER

The increased activity of EGFR is associated with many human cancers like Non

Small Cell Lung Cancer (NSCLC), colorectal adenocarcinoma, glioblastoma, Head and

Neck Squamous Cell Carcinoma (HNSCC) and tumours of the pancreas, breast, ovaries,

prostate and stomach, with a percentage that varies depending on the type of cancer

(Table 3). ^1,10

Introduction

Table 3. EGFR expression in human tumor

The oncogenic activation of EGFR can occur by various mechanisms: over- expression of ligand or receptor, activating mutations, failure of the mechanisms of inactivation or transactivation by dimerization of the receptor.

Mutated EGFR were identified: among them EGFR vIII (variant III) has been found in most human cancers and is characterized by the absence of the cysteine rich domain (Figure 2). ⁴

EGFR vIII presents a modified binding region and therefore is not able to interact with the ligand and to dimerize, so its kinase activity is constitutive.

Another EGF receptor devoid of the extracellular domain and of 32 amino acids in the carboxy-terminal portion is the product of the viral oncogene v-erb B, that is one of two oncogenes of avian erythroblastosis; also in this case the receptor is constitutively active. ¹¹

Kind Of Tumour

% Of EGFR Expression

NSCLC 40-80

Head and Neck 80-100

Colorectal 25-100

Stomach 33-81

Pancreas 30-50

Ovaries 35-70

Breast 15-37

Prostate 40-90

Glioma 40-92

Introduction

GATING EGFR ¹²

The function of all proteins is regulated by a mechanisms of attenuation or gating.

Upon activating of many tyrosine kinase, including EGFR, a rapid decrease both in the receptor number at the cell surface and in the cellular content of activated receptors occurs: this process is known as “down-regulation”.

The formation of the ligand-activated receptor complex, in fact, is followed by a membrane invagination that allows the incorporation of the complex itself into the cell (endocytosis).

During this process Cbl plays a key role, by direct interaction with EGFR or indirectly through the aforementioned Src. Their function is to facilitate the formation of vesicles incorporating the complex. The vesicles are coated with clathrin, a protein that after ligand-receptor interaction undergoes proliferation forming clathrin-coated pits that allow the entry of vesicles inside the cell; here they merge to form a primary endosome. Primary endosome maturation to late endosome allows the dissociation of the receptors from their ligands: some receptors are recycled and brought back to the plasma membrane, other, are delivered toward lysosomal degradation pathway (Figure 8). The above mentioned mechanism is altered in mutated receptors, they are in fact resistant to lysosomal degradation.

Figure 8. HER receptor endocytosis

Introduction

About this mechanism, it’s important to observe that exists an activated three- dimensional structure of HER2 that is ligand-independent: this receptor dimerizes preferentially with EGFR, determinig a reduction in EGFR endocytosis and, consequently, in its degradation .

Besides being involved in the mechanism of ligand-mediated endocytosis, EGFR undergoes a spontaneous metabolic turnover, with a half-life of approximately 10-14 hours both in fibroblasts and in epithelial cells and of 20-48 hours in transformed cells.

An understanding of the mechanisms governing negatively growth factors and endocytosis signals could lead to identification of new therapeutic targets for the treatment of cancer.

THE EPIDERMAL GROWTH FACTOR EGF

EGF is a 6kDa peptide, consisting of 53 amino acids, discovered in 1962 by Levi Montalcini and Cohen in studies on the factor responsible for the growth of nerve fibres (NGF, Nerve Growth Factor) (Figure 9). ¹³

Figure 9. EGF structure

It was noticed that the injection of extracts of submaxillary gland in newborn mice

induced the premature opening of eyelids and accelerating incisor eruption, effects

associated to stimulation of epithelial growth and differentiation.

Introduction

The epidermal growth factor plays a crucial role in promoting the transition of cells from G0 to G1 phase, in which the synthesis of the enzymatic and structural proteins kit necessary for subsequent DNA replication occurs (S phase) (Figure 10).

Figure 10. EGF effects and cell cycle

EGF not only induces increase in the number of sensitive cells but also modulates

cellular differentiation, with effects varying depending on the tissue. Structurally, the

fundamental characteristic of EGF is a pattern of 6 cysteine residues, highlighted in

yellow in Figure 6, located at definite intervals in a 40 amino acids framework sequence

that determine the formation of three intramolecular disulfide bridges, thus helping to

define the three-dimensional peptide structure. ¹¹ (Figure 11)

Introduction

Figure 11. Amino acid sequence of EGF

The disulfide bonds allow a protein folding that ensures interaction with the specific receptor. EGF is expressed by glandular and lining epithelia and is present in the oral cavity and gastrointestinal tract, where limits the gastric secretion, regulates tropism and mucosal integrity and promotes epithelial regeneration processes. It is also widely expressed in the mammary gland and its presence in the milk suggests an important role in the gastro-intestinal epithelium maturation of the newborn. EGF is present in neglectable concentrations in plasma and in significant amounts in platelets, where it is released after stimuli inducing agglutination and degranulation. Therefore it may be assumed that locally EGF could contribute to repair processes of wounds and injuries.

So, in physiological conditions, EGF plays an important functional role in regulating

growth and cellular differentiation. ¹¹

Introduction

EGFR INHIBITORS

Over twenty years ago, EGFR has been proposed as a target for cancer treatment for a number of reasons: first the fact that the receptor was over-expressed in various types of cancer cells; in addition, the growth of such cells was inhibited by a series of EGFR monoclonal antibodies both in vitro and in vivo. Preclinical and clinical studies confirmed the possibility of an EGFR-targeted therapy and the clinical activity of EGFR inhibitors was established and approved. There are two major classes of EGFR inhibitors: monoclonal antibodies, directed against the extracellular domain of the receptor, such as cetuximab and "small molecules", competitive inhibitors of the ATP binding site on the tyrosine kinase domain of EGFR, such as gefitinib and erlotinib.

Therapeutic options should also be remembered as the use of antisense oligonucleotides or ribozymes that decrease the expression of EGFR. ^9,14

The first approach to the inhibition of EGFR is represented by monoclonal antibodies; they are able to interact with the extracellular domain of the receptor by competing with its ligand and thus blocking activation. This process results in inhibiting cell growth.

Cetuximab (IMC-225), for example, is a G1 immunoglobulin (IgG1), that interacts with the domain III of EGFR, partially occluding the ligand binding region on this domain. Although ligand-binding to domain I is unaffected, growth factors must engage sites on both domains I and III for high-affinity binding, so blockade of either one is sufficient to prevent signalling. Cetuximab is also able to promote EGFR internalization. Once internalized, the receptor is then degraded without phosphorylation and/or activation, resulting in a down regulation from cell surface and consequent reduction of EGFR-dependent signalling pathways. ⁹

Another action of cetuximab is blocking the conformational change of the receptor, which is essential for dimerization and proliferation of pro-angiogenic factors.

Cetuximab has been shown to inhibit various human tumour cell lines, action due to cell

cycle arrest in G1 phase and/or apoptosis. This antibody has been evaluated in clinical

trials both as single agent and in combination with conventional chemotherapy and

radiation therapy. Because inhibition of EGFR and conventional therapies act

Introduction

therapies offers the potential advantage of synergism of activity, without overlapping of side effects. ^6,14

Moreover, there is another class of anti-EGFR monoclonal antibodies that prevent receptor dimerization, such as pertuzumab (2C4). This antibody binds the HER2 receptor and prevents its hetero-dimerization with other HER family members.

The second approach to cancer therapy involves the use of membrane-permeable small molecules that compete with ATP for binding to the tyrosine kinase portion of the receptor, thus blocking its catalytic activity. Some of them induce the formation of inactive EGFR homodimers or EGFR/HER2 heterodimers. Such molecules, in addition to the ability in inactivating of both receptors belonging to the HER family, also inhibit the mutated receptors avoid of the extracellular domain.

Monoclonal antibodies and small molecules have different mechanisms of action, although they lead to the same effects on signal transduction, effectively blocking EGFR signal transduction, including MAPK and PI3K/Akt and the Jak/Stat cascade. A significant difference between monoclonal antibodies and small molecules is that the former are selective inhibitors against EGFR, while the latter are less selective inside HER family members; in fact, if some cancer cells previously inhibited by small molecules are treated with monoclonal antibodies, the anti-tumour activity increases.

This suggests a possible combined therapy of the two different types of receptor inhibitors. ¹⁴

The first small molecule to be approved by FDA in 2003 for the treatment of non- small cell lung cancer (NSCLC) is gefitinib (Iressa®, ZD1839).

Gefitinib, a 4-anilinoquinazoline derivative (molecular weight 447), is active when administered orally and is a reversible competitive type, potent and selective EGFR inhibitor. ^6,15

In vitro, this molecule is able to inhibit receptor kinase activity with an IC 50 of 23nM, but in vivo, because of high intracellular concentration of ATP, higher doses are needed that lead to inhibition of other tyrosine kinase as HER2. In fact, gefitinib inhibits HER2 autophosphorylation with an IC 50 of 1µM in breast cancer cells in which this receptor dimerizes with EGFR. ⁶

In vitro effects of gefitinib as a single agent were mainly cytostatic, although

cytotoxic effect have been observed in a few cases. It has been suggested that gefitinib

Introduction

could favour several mechanisms involving pro-apoptotic members of Bcl2 protein family such as Bad. ¹⁶

Gefitinib did not confer an overall survival advantage; however, gefitinib treated patients survived longer than those placebo-treated in two specific patient subsets:

individuals of Asian origin (median, 9.5 versus 5.5 months, respectively) and never- smokers (median, 8.9 versus 6.1 months, respectively). ¹⁷

Gefitinib appears to indirectly inhibit angiogenesis, the growth of cancer cells in human colon, breast, ovaries and stomach, in vivo accompanied by reduced production of VEGF and TGF-α. Treatment with gefitinib is associated with a rapid regression of the mentioned tumours, but if the administration is discontinuous the tumour grows, so a long-term drug administration is required to maintain a response in patients. ⁶

Gefitinib has been combined with chemotherapy with a variety of cytotoxic agents, resulting in enhanced anti-tumour effects in cultured cells and in vivo models, except in combination with gemcitabine. In addition, gefitinib treatment resulted in synergistic effects in combination with radiation. Importantly, sequence-dependent effects were reported in cells treated with combinations of gefitinib with radiation or chemotherapy (cisplatin and/or 5-fluorouracil), with the best results observed when gefitinib was administered before radiation and before or during chemotherapeutic treatments;

whereas some antagonistic effects were observed when gefitinib was given after cytotoxic treatments. ¹⁶ Side effects of this molecule are: diarrhoea, nausea, vomiting and rash.

Erlotinib (OSI-774, Tarceva®), lapatinib (GW572016) and canertinib (CI-1033) are other examples of 4-anilinoquinazoline derivatives with potent EGFR inhibitory activity. Erlotinib is active when administered orally and has a reversible high selectivity towards EGFR, with IC 50 2nM. ¹⁶

Although the mechanism of action is equal to that of gefitinib, erlotinib appears to be

more active because it is dosed at its maximum tolerable dose (MTD), while the dose of

gefitinib is one third of the MTD. ¹⁷ Studies have demonstrated its inhibitory activity on

many cancers such as renal carcinoma, colorectal carcinoma, NSCLC, pancreatic

cancer. Prolonged treatment of patients with this drug leads to a reduction of metastases

by 30%. Side effects are similar to those caused by gefitinib, suggesting that these

Introduction

Lapatinib, instead, is a potent inhibitor of EGFR and of HER2 (IC 50 11 nM and 9.2 nM respectively) and is devoid of activity against other kinases. ¹⁸ EGFR and HER2 have homologous domains and their simultaneous inhibition represents a viable therapeutic opportunity for patients with cancer. An over-expression of HER2 was observed in breast cancer and a co-over-expression of both receptors in patients with ovarian cancer. ¹⁹

Finally, canertinib is an irreversible inhibitor of EGFR (IC 50 1.5 nM) and HER2 and HER3 kinase activity. ²⁰ Canertinib acts by alkylation of cysteine residues in the binding pocket of ATP in EGFR and other HER family members, blocking phosphorylation.

The inhibitory effect is prolonged by its ability to bind irreversibly to the site. This molecule also interferes with the process of dimerization between EGFR and HER2. ⁹ Canertinib also shows a positive synergistic effect when combined with other cytotoxic agents or ionizing radiation. ²¹

NEWS ON THE CLINICAL USE OF EGFR INHIBITORS

EGFR inhibitors are a dynamic field of research and development, as they have characteristics that make them particularly versatile in combating tumour progression.

So we can list the clinical benefits that can be derived by this new class of anti-cancer drugs:

• Effectiveness in many types of solid tumours.

• Utility for long-term treatment.

• Possibility to use either alone or in combination with other drugs.

• Good tolerability.

• Absence of haematotoxicity.

• Capability to reduce resistance to radiotherapy or hormone therapy.

• Improvement of the quality of life with respect to other types of anticancer drugs.

• Pre-medication or monitored dosage are not requested.

Table 4 shows the description of some drugs used in the testing. ¹⁹

Introduction

Drug Action Target Tumour

Type Clinical Stage Gefitinib

IRESSA ZD1839

Reversible inhibitor, ATP-competitive

EGFR NSCLC Approved

(2003)

Erlotinib TARCEVA

OSI-774

Reversible inhibitor, ATP-competitive

EGFR

NSCLC, pancreatic

cancer

Approved (2004)

Lapatinib GW572016

TYKERB EGFR, ErbB2

Advanced ErbB2 positive breast cancer, NSCLC,

HNSCC

Approved (2007) Phase II

Canertinib CI-1033

Irreversible inhibitor

EGFR,ErbB2, ErbB3

NSCLC and

breast cancer Phase I / II

Cetuximab Erbitux IMC-225

Chimeric human-

murine mAb EGFR

CRC and pancreatic

cancer

Approved (2004) Phase II

Pertuzumab rhumAb2C4 Omnitarg

Humanized mAb ErbB2 dimerization

Breast, prostate,

ovarian cancers Phase II / III

Table 4. EGFR inhibitors in clinical development

RESISTANCE TO "EGFR-TARGETING" THERAPIES

Differences have been observed in the responses of individual patients treated with EGFR inhibitors. The first difference emerged was a close dependence of therapeutic efficacy on the "contents" of EGFR, that is its expression rate in cells. Other differences in the response to therapy are concerned with the presence of mutations: these, depending on their location, may influence the effect of some EGFR inhibiting agents:

- The presence of mutations at the extra-cellular level affect therapy with monoclonal antibodies;

- Mutations affecting amino acids in the ATP-pocket on the intracellular tyrosine kinase

Introduction

respond much better to therapy, at least in the initial phase of treatment. Indeed it was noted that in some patients, who initially responded to administration of gefitinib, after a prolonged treatment EGFR acquires a second mutation that leads to a high resistance to these substances; ^14,22

- Mutations at intermediate factors, such as k-Ras, Raf, PI3K are elements that can influence the activity of EGFR-inhibitors.

This different sensitivity to various treatments depending on the factors listed above

requires a careful selection of patients to proceed with an appropriate therapeutic

protocol.

Introduction

NEW TREATMENT GUIDELINES ¹⁹

In the attempt to improve the effectiveness of cancer therapy more and more combination therapies are emerging, which may be essentially of two types:

- Combination therapies aimed at the same target, either using irreversible or reversible inhibitors, characterized by different spectrum of resistance induced by mutation. A clinical trial with lapatinib and trastuzumab has led to encouraging results;

- Therapy directed towards more different receptors; there are many tyrosine inhibitors very poorly selective against an unique receptor. This perspective can be exploited by the selection of agents targeting more kinases involved in tumour progression.

Recently new multi-target agents have been developed, including inhibitors of EGFR and other HER family members (as canertinib, inhibitor of EGFR, HER2 and HER3), or multi-target inhibitors of EGFR tyrosine kinases belonging to different groups.

INDIRECT AND DIRECT TARGETING OF ANGIOGENESIS PATHWAYS

The development of a vascular supply is essential not only for organ development

and differentiation during embryogenesis but also for wound healing and reproductive

functions in the adult. Angiogenesis is also implicated in the pathogenesis of a variety

of disorders: proliferative retinopathies, age-related macular degeneration, rheumatoid

arthritis, and psoriasis and demonstrated to be a fundamental event in the process of

tumour growth and metastatic dissemination. Hence, the molecular basis of tumour

angiogenesis has been of keen interest in the field of cancer research. The vascular

endothelial growth factor (VEGF) pathway is well established as one of the key

regulators of this process. ²³ The EGFR and VEGFR pathways seem to be closely linked

in solid tumours, particularly with respect to angiogenesis. Simultaneous inhibition of

EGFR and VEGFR pathways might provide improved efficacy or anti-angiogenesis

actions over blocking either pathway alone as a result of indirect and direct down-

regulation of angiogenic pathways and direct tumour targeting with EGFR antagonists. ⁵

Introduction

VASCULAR ENDOTHELIAL GROWTH FACTORS AND RECEPTORS VEGF/VEGFR

VEGF was isolated and cloned in 1989 by Ferrara, Plouet and co-workers ²³ and belongs to a family of growth factors characterized by high amino acidic homology, inclunding VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and Placenta Growth Factor (PIGF-1 and PIGF-2). The various members of VEGF family have overlapping abilities to interact with a set of cell-surface receptors: VEGFR-1/Flt-1 (fms-like tyrosine kinase) and VEGFR-2/KDR/Flk (fetal liver kinase), originally identified on endothelial cells but also expressed on various hematopoietic cell lineages in the adult;

both receptors are primarily involved in angiogenesis. A third membrane receptor is VEGFR-3/Flt-4 expressed in lymphatic vessels, for which it seems to be critical. ²⁴

VEGFR-1 and VEGFR-2 consists of 1338 and 1356 amino acids, respectively. They are structurally similar, consisting of an extracellular ligand-binding domain with a seven-immunoglobulin (Ig)-like motif, a single transmembrane domain, a juxtamembrane domain, a kinase domain split by a kinase insert, and a carboxyl terminus. Overall, there is 43.2% sequence homology between VEGFR-1 and VEGFR- 2. The extracellular domain of VEGFR-1 and VEGFR-2 displays 33.3% homology and the cytoplasmic region 54.6%. The kinase domains of VEGFR-1 and VEGFR-2 represent the most conserved region with 70% homology. In contrast, the carboxyl terminus represents the most divergent region with only 28.1% sequence homology. ²⁵

The VEGF/VEGFR axis triggers a network of signalling processes that promote endothelial cell growth, migration, and survival from pre-existing vasculature. ²⁶

The predominant member of VEGF family is VEGF-A, a 45 kDa glycoprotein composed of 165 amino acids that interacts with VEGFR-1 and VEGFR-2, playing an essential role in angiogenesis. VEGF-A also exists in other isoforms characterized by a different number of amino acids.

The VEGF-B protein through its binding to VEGFR-1 exert a mitogenic effect on

endothelial cells and moreover may link VEGF-A, increasing its action. VEGF-C and

VEGF-D are structurally similar and may be considered as members of a subfamily

(Figure 12). These proteins, interacting with VEGFR-2, induce a slight mitogenic effect

on endothelial cells, while binding VEGFR-3 regulate lymphangiogenesis. At last,

Introduction

VEGF-E, the most recently identified factor, is specific only for endothelial cells of endocrine glands.

Figure 12. Representation of vascular growth factors and their receptor interaction

Introduction

VEGFR SIGNALLING ²⁶

The mechanism by which VEGFR-1 and VEGFR-2 receptors transduce signal have been extensively studied at the molecular level. A ligand mediated dimerization of receptor momomers occurs, followed by transphosphorylation of the intracellular domain by dimerized receptors which in turn catalize the phosphorylation of cytosolic substrate proteins and downstream cellular events (Figure 13).

Activation of the MAPK pathway in response to VEGF has been observed in many types of endothelial cells. The PLC-γ-PKT pathway has also been implicated in the mitogen action of VEGF. VEGFR-1 interacts with the PLC-γ SH2 domain, inducing the phosphorylation and activation of PLC-γ leading to the hydrolysis of phosphatidylinositol-4,5-bisphosphate to diacylglycerols and inositol-1,4,5- trisphosphate. Inositol-1,4,5-trisphosphate is likely responsible for the increase in intracellular Ca ²⁺ after VEGF stimulation, whereas diacyl-glycerol, in turn, activates PKC isoforms expressed in the target cells.

VEGF also activate PI3-K that activates Akt, a serine kinase involved in antiapoptotic signaling. Akt has also been reported to directly activate endothelian nitric oxide synthase, suggesting that Akt may regulate the increased production of nitric oxide in response to VEGF stimulation. STATs are latent cytoplasmic transcription factors. STAT activation by the VEGFRs has been studied in transient transfection assay. All three receptors were shown to be strong activators of STAT3 and STAT5, whereas STAT1 was not activated by the VEGFRs. However, the role of this pathway in endothelian cell biology is unknown.

Although very little is known about the specific signal transduction of VEGFR-3 in the lymphatic endothelium, mutations in VEGFR-3 have been linked with hereditary lymphedema, an autosomal dominant disorder of the lymphatic system.

Endothelial cell proliferation and survival in response to VEGF may require the

association of VEGFR-2 with cell surface adhesive proteins. Activated VEGFR-2 was

found in a complex with integrin αvβ3, an adhesion molecule specifically expressed on

angiogenic endothelium; αvβ3 has been shown to be involved in the regulation of the

cell cycle and the survival of endothelial cells. VE-cadherin, an endothelium-specific

cell-adhesion protein, has also been implicated in molecular interactions with the

Introduction

Figure 13. VEGFR pathway

VEGF/VEGFR IN ANGIOGENESIS ²⁵

The different roles of VEGFR-1 and VEGFR-2 have been extensively studied.

VEGFR-1 is a kinase-impaired TK receptor and may act in the context of receptor

heterodimer. VEGFR-1 may play both negative and positive roles in angiogenesis by

modulating the availability of VEGF for VEGFR-2. In contrast, VEGFR-2 is highly

kinase active receptor and leads to diverse biological responses. VEGFR-2 by activation

of PI3 kinase promotes endothelial proliferation cell survival, through PLCγ mediates

tubulogenesis/angiogenesis, and linking Src regulates endothelial cell survival and

permeability. Expression of VEGF and of its receptors correlates with the degree of

vascularisation of many experimental and clinical tumours as detected by in situ

hybridization and immunohistochemistry, and both have been used as prognostic

indicators of an increased metastatic risk. Although the detailed molecular mechanism

of the “angiogenic switch” by which quiescent endothelium becomes activated is

Introduction

combination with the locally increased VEGF concentrations, up-regulates VEGFR-1 and VEGFR-2 on tumor endothelial cells. VEFGR-3 is up-regulated in tumor angiogenesis in general, for example, in breast carcinomas. In addition, VEGFR-3 has been shown to be increased in the endothelium of lymphatic vessels in metastatic lymph nodes and in lymphangiomas.

RELATIONSHIP BETWEEN EGFR AND VEGF SIGNALLING PATHWAYS ²⁸

In solid tumours, the VEGF and EGFR pathways seem to be linked, particularly with respect to angiogenesis. EGF and TGF-α both induce VEGF expression via activation of EGFR in cellular culture models and have pro-angiogenic properties. It is likely that the EGFR pathway modulates angiogenesis by up-regulation of VEGF or other key mediators in the angiogenic process. In preclinical models, the use of the anti-EGFR monoclonal antibody cetuximab determined a down-regulation of pro-angiogenic mediators, resulting in a reduction of microvessel density and of metastasis. Similar results have been reported for the small-molecule EGFR inhibitor gefitinib. However, EGFR inhibition is not sufficient to block VEGFR, thereby allowing tumour angiogenesis to continue.

VEGFR AND DUAL VEGFR/EGFR TARGETED THERAPIES

Approaches to disrupting the VEGF/VEGFR signalling cascade has led to the rational design and development of agents that selectively target this pathway, ranging from biological agents (soluble receptors, anti-VEGF and anti-VEGFR-2 antibodies, and VEGF transcription inhibitors) to small molecule ATP-competitive VEGFR inhibitors.

The most important anti-VEGF recombined humanized immunoglobulin G1 monoclonal antibody is bevacizumab, that acts by binding to all VEGF isoforms, thus removing VEGF from the circulation and preventing activation of VEGFRs.

Bevacizumab, when used in combination with 5-fluorouracil (5-FU)- and irinotecan-

based chemotherapy, was shown to significantly improve survival and response rates in

Introduction

patients with metastatic colorectal cancer; in fact, the US FDA approved bevacizumab in February 2004 for use with any 5-FU based first-line regimen and approval in Canada (September 2005) was received for a similar indication. European Union approval (January 2005) was limited to use with a first-line irinotecan-based regimen.

Toxicities included malaise, hypertension and proteinuria. ²⁹

Moreover, bevacizumab was used in phase III clinical trials associated with paclitaxel in metastatic breast cancer, showing improvement in response rate and survival. ²⁸

Although the latter clinical results seem to validate VEGF pathway inhibitors as an important new treatment modality in cancer therapy, the real risk-benefit profile of bevacizumab is not established at all.

Encouraging results have been seen for bevacizumab plus erlotinib in metastatic breast cancer and recurrent or metastatic head and neck squamous cell carcinoma.

Examples from the above mentioned intracellular target therapy with small- molecules VEGFR inhibitors, that are currently in clinical development, include compounds from distinct chemical classes such as: indolin-2-ones, anilinoquinazolines, anilinophthalazines, isothiazoles, indolo- and indenocarbazoles. ³⁰

Inside the class of indolin-2-ones, sunitinib (or SU11248) is a novel, oral, multitarget receptor tyrosine kinase inhibitor. ²⁴ This drug inhibits angiogenesis and tumour growth in different xenograft models and has been tested in clinical trials on patients with metastatic renal cancer, gastrointestinal stromal tumours and metastatic breast cancer resistant to the other therapies. In January 2006, it was approved by FDA for the treatment of gastrointestinal and kidney cancer.

Valatanib (or PTK787/ZK222584) belongs to anilinophthalazine derivatives and is one of the most potent and selective first-generation VEGFR kinase inhibitors. This compound is currently in phase III clinical trials for metastatic colorectal cancer.

Combining targeting of angiogenic pathway and tumour growth offers the potential

for improved efficacy versus targeting each pathway alone. A drug that joins the two

actions with a dual pharmacological profile is vandetanib (ZD6474, AstraZeneca), an

orally active tyrosine kinase inhibitor that exhibits potent agonist activity toward

VEGFR-2 and, to a lesser extent, to EGFR. Its administration in mice leads to inhibition

Introduction

of VEGF signal, of angiogenesis, of neuro-vascularization induced by the tumour, and the tumour growth.

ZD6474 is undergoing phase I clinical testing in patients who have advanced solid tumours. ^31,32

Preclinical and clinical trials will determine whether compounds with dual activity

will provide more benefit than single-agent therapy or combination therapy that targets

multiple pathways.