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objective

Introduction

EGF signaling

EGF receptor and cancer therapy

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Introduction

Human EGF is a 6045-Da protein with 53 amino acid residues

Epidermal growth factors and their receptors are heavily involved in normal

development, differentiation, migration, wound healing and apoptosis

The discovery of EGF won Stanley Cohen and Rita Levi-Montalcini the Nobel

Prize in Physiology or Medicine in 1986.

EGF is a low-molecular-weight polypeptide first purified from the mouse

submandibular gland, but since then found in many human tissues

including submandibular gland, parotid gland.

Epidermal growth factor can be found in human platelets, macrophages,

urine, saliva, milk, and plasma.

Most EGF family proteins are produced as inactive membrane -anchored

proteins that require proteolytic cleavage either to achieve activity in

solution or bind to cell surface proteoglycans from where they can act as a

reservoir to be made available for receptor binding.

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The EGFR belongs to the ErbB family of receptor tyrosine kinases(RTKs).

Is an integral membrane protein

Is 170Kda and contains 1207 aa in humans

These receptors possess protein tyrosine kinase (PTK) activity and are found only in

metazoans.

The four receptor genes, encoding the EGF receptor (EGFR/erbB-1), c-erbB-2/HER2,c-

erbB3/HER3 and c-erbB4/HER4,

ErbB2, ErbB3 as well as ErbB1 are expressed in most epithelial cell layers,

While mesenchymal cells are a rich source of ErbB ligands, both neuregulins and

EGF-like ligands.

ErbB2 cannot be stimulated by any known ligand and ErbB3 is signalling defective

The 11 ligands currently identified for these receptors in mammals include EGF,

transforming growth factor-a (TGF-a), (HB–EGF), amphiregulin (AR),betacellulin (BTC),

epiregulin (EPR), epigen7, neuregulin 1-4, tomoregulin, epiglycan etc

In addition to HER-1, HB–EGF, BTC and EPR bind to and activate HER4

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Signaling in EGF A simple model to analyze signaling is by grouping it in to three layers

The initial, extracellular layer is composed of the ligands and will dimerise to

become active.

If the information in the first layer is sufficient to induce receptor dimerization

is achieved by the s1 domain, Vander walls, hydrophobic interactions.

consequently increase catalytic activity, will constitute the second

The third, intracellular layer of second messenger proteins can bind to

specific sites on the receptors and initiate the signals required to induce the

appropriate response.

it is now evident that most or all of the ErbB family of receptors further

aggregate into oligomers of several hundreds or a few thousand receptors

Erbb2 further has a higher catalytic activity than the rest furthermore when

the other EGFR combine with ErbB2 it wil increase their half life by

decreasing the interaction with c-cb22, AP2.

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The EGFR monomer possesses an

extracellular domain consisting of two

ligand binding subdomains (L1 and L2)

and two cysteine-rich domains (S1 and

S2), of which S1 permits EGFR

dimerization with a second ErbB

receptor. SH1 is the protein tyrosine

kinase domain and resides in the

cytoplasmic domain above the six

tyrosine residues available for

transphosphorylation.

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One thing to note is that the receptor may also oligomerize if there is enough ligand

and secondary signals forming an even stronger signal.

Also in the dimerization loop substitution of valine by glutamate results in continuous

dimerization causing transformation.

Cont’d The cytoplasmic region of the EGFR comprises three distinct domains:

1. the juxtamembrane domain, required for feedback by protein kinase C

(PKC), down regulation, epithelial basolateral polarity,

2. the noncatalytic carboxy-terminal tail, possessing the six tyrosine trans

phosphorylation sites mandatory for recruitment of adaptor/effector

proteins (e.g. Grb2 and phospholipase C (PLC) respectively) containing SH2

domains (src homology domain 2) or PTB (phosphotyrosine binding)

domains, plus the motifs necessary for internalization and degradation of

the receptor;

3. the central tyrosine kinase domain (src homology domain 1 (SH1)) that is

responsible for mediating transphosphorylation of the six carboxyterminal

tyrosine residues.

4. it has serine threonine domains when phosphorylated it gets

downgraded

This domain is initially inactive, but once ligand binds it will become active.

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ERBB3 further increases the signaling activity by forming dimer with ERBB2

and trans phosphorylating thus recruiting more second messengers or

adapter complexes.

Also stimulates enhanced and prolonged stimulation of the MAP kinase

(ERK) and c-Jun kinase (c-JNK) that would stimulate mobility, and cell cycle

regulators like Mcl-2

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the recruitment of the enzyme phospholipase C gamma (PLCγ). In its

inactive state, PLCγ is normally found in the cytosol.

Upon its phosphorylation PLCγ, relocates to the membrane, where it makes

contact with the substrate Phosphatidyl triphosphate and ultimately

generates the second messengers Inositol (1,4,5)P3 and diacylglycerol.

This activates calcium/calmodulin-dependent kinases and stimulation of

protein kinase C.

Protein kinase c that is activated by diacylglycerol phosphorylates NF-Kb

and the inhibitor part is I-kb when I-kb is phosphorylated the NF-Kb is

released and activates various transcriptional factors that would enhance

proliferation and epithelial- mesenchyme transformation

While through the influx of calcium calmodulin is activated and calmodulin

woud phosphorylate calcinurine, calcinurine intun dephosphorylate NFAT,

then NFAT would go to the nucleus and activate transcriptional factors.

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The MAPK and PI3K/Akt pathways promote cell proliferative and

survival/antiapoptotic signals via the activation of transcription factors and

up regulation of cyclin D1.

An increase in cyclin D1 that functions to sequester the cyclin kinase

inhibitor p27 and release Cdk2.

Subsequently, Cdk2 becomes positively regulated via its association with

cyclin E and causes deregulation of the G1/S checkpoint such that the cell

cycle progression is promoted and leads to malignant transformation.

The downstream effectors of Akt also serve to sequester p27 such that the

constitutive activation of Akt that arises from c-erbB-2-overexpression is

thought to confer resistance to tumor necrosis factor induced apoptosis.

Anti apoptosis is further upregulated by the release of P21

Furthermore, Akt is known to stimulate endothelial nitric oxide synthase,

matrix metalloproteinase, and telomerase activity.

It also up regulates synthesis if MDM2

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Additional actions of c-erbB-2/c-erbB-3 signaling are the PLCγ pathway and

the JAK-STAT pathway.

In JAK-STAT pathway the JAK would phosphorylate STAT and and the STAT

would dimerize and move in to the nucleus activating transcription of

specific gene associated with cell survival.i

mportance of EGF can be shown by this table

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The new signaling pathway

discovered!

Recent reports also suggest that following EGF stimulation at the cell

surface, the full-length EGF receptors also migrate to the nucleus, where

they bind an AT-rich consensus sequence (ATRS) via an undefined domain

and enhance transcription via proline-rich region near their carboxy

terminal domain.

They also show that EGFRs associate with the promoter region of cyclin D1,

a protein that can play a key role in mitogenesis.

This changes the so called the “DOGMA” of the action of receptors

It has also been reported the activation of Erbb induces the activation of

more receptors

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EGF elicits cancer like phenotype

change

The addition of EGF to normal cells elicits certain responses which are

associated with neoplastic transformation. For example, EGF induces a

partial loss of density dependent inhibition of growth and the dependency

on serum for growth, an increase in the level of phosphotyrosine in proteins

and an increase in cellular metabolism including sugar and amino acid

transport, ATP turnover, and ornithine decarboxylase activity .

EGF induces the expression the mRNA of cfos and c-myc genes, cellular

proto-oncogenes . Cellular proto-oncogenes are the normal cellular

homologs of viral oncogenes.

Oncogenic viruses are thought to have acquired the oncogene from

normal cellular RNA.

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EGF also elicits certain responses which are associated with cancer such as

the loss of fibronectin, and an augmentation of the secretion of

plasminogen activator and metalloproteinase.

Growth of cells in soft agar, considered by many to be one of the best

assays for showing the tumorigenicity of a cell, is potentiated by EGF or

EGF-like factors

This potentiation by EGF is even greater for partially transformed cells and

tumor cells. Thus, EGF or EGF-like proteins contribute toward the malignant

phenotype of transformed cells in vivo.

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EGF and cancer

The impact of the EGFR signaling system on human neoplasia is shown by the following:

1. EGFR is overexpressed or activated by autocrine or paracrine growth factor loops in at least 50% of epithelial malignancies.

2. HER2 is amplified and dramatically overexpressed in approximately 20–30% of breast cancers and also cervical cancers.

3. HER3 is variably expressed in breast and colon, prostate, and stomach malignancies.

4. ErbB4 is overexpressed in breast cancer and granulosa cell tumours of the ovary.

Also, ErbB2 overexpression by itself can cause cellular transformation even in the absence of added ligand

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Also EGF is induced to be released from macrophages by the release of

CSF 1

Then the EGF will stimulate the epithelial to mesenchyme transformation of

the carcinoma,

This transformation aids in metastasis

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EGFR and ErbB2 have been showed to be overexpressed in a large

proportion of breast and ovarian tumours, primarily by gene amplification

In cervical cancers, HPV E5 is known to cause overexpression of EGFR.

Recently, ErbB2 was shown to cooperate with HPV viral oncoproteins E6 and

E7 to cause transformation.

The trans membrane receptor Notch1 protein has been shown to

overexpress ErbB2 and this along with the ability of Notch1 to activate the

PI3kinase PKB/Akt pathway and protect cells from apoptosis and to protect

cells from p53-induced cell death could play a major role in the progression

of many cancers like the cancer of the uterine cervix. where Notch is

known to be overexpressed.

It would be interesting to see whether Notch drives PI3kinase through

overexpression of ErbB2 in cervical cancers.

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The unaltered wild-type human ErbB2 is amplified or overexpressed in a

subset of breast cancers and this correlates with an aggressive tumour

phenotype, including tumour size, lymph node involvement, high

percentage of S-phase cells, aneuploidy and lack of steroid hormone

receptors

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The expression of EGFR protein has also been compared with tumor

differentiation grade.

The results appear to be controversial with some studies showing a link and

others reporting no relationship between tumor differentiation and EGFR

levels.

No significant correlation was demonstrated between patient gender and

patient age

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EGFR and colorectal cancer

Enhanced activity or overexpression of EGFR has been found to be associated with tumor progression and poor survival in various malignancies such as head and neck , lung , breast , gastrointestinal tract and bladder cancers.

It has been well documented that overexpression of EGFR in colon cancer may be linked to an advanced stage of the disease or may predict a potential metastatic risk.

However, the impact of EGFR expression on survival remains controversial and overexpression of EGFR is not uniformly associated with an unfavorable prognosis.

In most cases, immunohistochemical methods were used for the detection of EGFR in colorectal cancer. The variability of IHC is well known and thus EGFR overexpression in colorectal cancerranges from 25% to 82%.

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Science, August 20th 2004.AG1478 can also do the same job

Treatment cont’d

The treatment with antibody against EGF is out of the option due to the

fact that EGF is a small half life.

Herceptin has enjoyed a little success in treatment of breast cancer

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Reference

Study journal of cancer by stoscheck

Science magazine

www.kent.ac.uk/bio/gullick

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Q&A time

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