The document discusses epidermal growth factor (EGF) signaling and the EGF receptor. It notes that EGF is involved in normal cell processes like development, differentiation, and wound healing. The EGF receptor belongs to the ErbB family of receptor tyrosine kinases and plays a key role in signaling pathways regulating cell proliferation, survival, and apoptosis. Overexpression or abnormal activation of the EGF receptor and other ErbB family members is implicated in many epithelial cancers.

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Group 15

2. objective
 Introduction
 EGF signaling
 EGF receptor and cancer therapy
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3. 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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4.  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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5. 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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6. 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.

8. 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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9.  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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10.  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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11.  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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12.  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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15. 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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16. 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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17.  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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18. 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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19.  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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20.  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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21.  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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22.  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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23. 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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25. Science, August 20th 2004.
AG1478 can also do the same job

26. 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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27. Reference
 Study journal of cancer by stoscheck
 Science magazine
 www.kent.ac.uk/bio/gullick
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28. Q&A time
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