The basic biology of erbB-2 and its participation in colorectal cancers

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Folia Biologica et Oecologica 2: 5-1 6

(Acta Univ. Lodz., Folia Biol. Oecol.)

MAGDALENA STAWIŃSKA1, MAG DALENA BRYŚ1, & W a n d a M . K r a j e w s k a 1

The basic biology of erbB-2 and its participation

in colorectal cancers

Abstract: ErbB-2 is one o f Tour cell surface growth factor receptors involved in transmis-sion o f signals controlling normal cell growth and differentiation. A range o f growth factors serve as ligands, but none is specific for the ErbB-2 receptor. Ligand binding to ErbB-1, ErbB-3 and ErbB-4 induces rapid receptor dimerization, with a marked preference for ErbB-2 as a dimer partner. When ErbB-2 is overexpressed multiple ErbB-2 hetero-dimers are formed and cell signalling is stronger, resulting in enhanced responsiveness to growth factors and malignant growth. This explains why ErbB-2 overexpression is an indicator o f poor prognosis in colorectal cancers and may be predictive o f response to treatment. ErbB-2 is a highly specific and promising target for new colon cancer treat-ments.

Key words: ErbB-2, colorectal cancers, signal transduction, tumour growth.

1. Introduction

The erbB-2 (neu, HER-2) oncogene is located on chromosome 17ql 1—q22 and encodes a 185 kDa transmembrane glycoprotein which has a structure consistent with a growth factor receptor, due to its similarity to the human epidermal growth factor receptor (EGFR) ( R u b in and Y a r d e n 2001). The

neu gene was originally identified in rat neuroectodermal tumours and later

its close human relative was isolated (Shih et al. 1981).

The ErbB-2 receptor has an important role in normal cell growth and differentiation. However, amplification of the erbB-2 gene leads to overex-pression of the receptor, which is linked to the development of many types

1 Department o f Cytobiochemistry, University o f Łódź, Banacha 12/16, 90-237 Łódź, E-mail:


of human cancers including breast, ovarian and those of the gastrointes-tinal tract ( H y n e s and S t e r n 1994; B r y ś et al. 2001; R u b i n and Y a r - d e n 2 0 0 1 ).

Adenocarcinomas of the colon and rectum are one of the major cause of morbidity and mortality for men and women. The pathologic stage of the tumor following surgical resection has long served as the cornerstone for predicting outcome for colorectal cancer ( g e l b and

SCHRO CK 1997; R O SS et al. 2001). Thus, considerable recent interest

has developed for the evaluation of new prognostic and predictive ma-rkers that could be applied to early stage lesions (indicating their pro-pensity for relapse) and the selection of these patients with relatively superficially invasive tumors to receive adjuvant therapy (R O SS et al.


In colorectal cancer ErbB-2 overexpression appears to be a significant adverse outcome indicator as judged by the current published literature. Either ErbB-2 protein overexpression or gene amplification is associated with a significant percentage of gastrointestinal adenocarcinomas. However, there are data suggesting that ErbB-2 overexpression or amplification is a significant prognostic factor for gastric and colorectal carcinomas. If this prognostic significance is confirmed, strategies designed to employ the marker in therapy selection appear to be warranted.

2. ErbB Family of Rectors and its Ligands

The ErbB family comprises four distinct receptors: EGFR/ErbB-1, ErbB-2/HER-2/neu, ErbB-3/HER-3, ErbB-4/HER-4. In general these cell surface receptors are composed of an extracellular ligand-binding domain, transmebrane part and a cytoplasmic region with tyrosine kinase activity (Fig. 1) ( U l l r i c h and S c h l e s s i n g e r 1990; K j le i n et al. 2001; T r o j a n e k

2002). This structure enables signals to be transmitted across the plasma membrane where they activate gene expression and ultimately induce cellular responses such as proliferation. The signal-transducing tyrosine kinase activity of the ErbB receptors is inactive when the receptors are in isolation (Ya r d e n 2001).

ErbB receptors are activated by a number of ligands, referred to as EGF- related peptide growth factors. These include epidermal growth factor (EGF), amphiregulin (AR) and transforming growth factor a (TGFa) which bind specifically to ErbB-1, betacellulin (BTC), heparin-binding EGF (HB-EGF) and epiregulin (EPR), which exhibit dual specificity in that they


high affinity/ narrow specificity NRG-1: erbB-4>erbB-3 TGF-a: erbB-1 ligand extracellular domain (632 amino acids) ligand-binding site lo w affinity/ broad specificity NRG-1 : erbB-2>erbB-4-erbB-3>erbB-1 TGF-a: erbB-2>erbB-1>erbB-4>erbB-3 plasma membrane intracellular domain (580 amino acids) tyrosine kinase activity

Fig. 1. Structure of the ErbB family receptors and bivalence o f EGF-like ligands. The model refers to neuregulin-1 and transforming growth factor a and is based on results described in

Tz a h a r e t a l. (1997 )

bind both ErbB-1 and ErbB-4, and at least four subfamilies of neuregulins (N R G 14). NRG1 and NRG2 both bind ErbB-3 and ErbB-4, whereas NRG3 and NRG4 bind ErbB^ł (PELES and YARDEN 1993; KlRSCHBAUM

and Ya r d e n 2000; Ol a y i o y e et al. 2000).

Despite the large number of ligands so far identified for ErbB-1, ErbB-3 and ErbB-4, as well as intensive efforts, no direct ligand for ErbB-2 has yet been discovered. ErbB-2 appears to act as a ligand less receptor. It was stated that ligand binding to ErbB-1 (EGF), ErbB-3 (NRG1) or ErbB-4 (NRG1, BTC) induces the formation of receptor homodimers and ErbB-2 containing heterodimers. In fact, ErbB-2 is the preferred hetero- dimerization partner for all other ErbB family members and plays a role in the potentiation of ErbB receptor signalling (Fig. 2) (GRAUS-PORTA et al. 1995; Tz a h a r et al. 1996, 1997; Ol a y i o y e et al. 2000; Ya r d e n 2001).

From ten possible combinations, those heterodimers that contain ErbB-2 are more stable and their signalling is more potent than that of other receptor combinations (Tz a h a r et al. 1996; Pe n u e l et al. 2001).

Multiple ligands differ in their expression patterns and in the ability to form specific homo- and heterodimeric receptor complexes. Due to the latest hypothesis, this latter difference is caused by the existence of two receptor binding sites on each ligand. The model refers to neuregulin-1 and is based on results described by TZAHAR et al. (1997). Neuregulin-1 seems to be a bivalent molecule with two binding sites for ErbB receptors:


- Slow ligand dissocation - Relaxed ligand specifity - Rapid recycling Increased cell proliferation Increased cell m igration Resistance to apptosis

Fig. 2. Signalling by ErbB homodimers in comparison with ErbB-2-containing heterodimers. Binding o f a ligand (EG F or NR G) to the extracellular domain o f ErbB-1 or ErbB-3 (note inactive kinase marked by a cross) induces homodimer formation. When ErbB-2 is overexpressed, heterodimers form preferentially. Unlike homodimers, which are either inactive (ErbB-3 homodimers) or signal only weakly, ErbB-2-containing heterodimers have attributes that

prolong and enhance downstream signalling (white box) and their outputs (grey box)

a high affinity/narrow specificity site (N-terminal) and a low affinity with broad specificity site (С-terminal). By the high affinity site ligand binds to its specific receptor (Fig. 1). Once the low affinity site is effectively ‘immobilized’ at the plasma membrane, its operational affinity to potential partners of the dimer is increased. Importantly, the receptor that preferentaially binds to the immobilized low affinity arm of the ligand is ErbB-2. As a result of this heterodimerization, the ErbB-2 receptor is able to participate in signal transduction in the absence of a specific ligand. This preferential binding with ErbB-2 is also enhanced by the overexpression of ErbB-2 in human cancer cells (TZAHAR et al. 1997).

Heterodimer formation is especially important between ErbB-2 and ErbB-3, not only because a high-affinity ligand-binding site is formed but also because ErbB-2 provides an active tyrosine kinase (EC for the catalytically impaired ErbB-3 (Gu y et al. 1994). Homodimerization of

ErbB-3 or ErbB-4 results in the formation of a low-affinity neuregulin binding site, whereas heterodimerization between ErbB-2 and either ErbB-3 or ErbB-4 is sufficient for the formation of a high-affinity binding site


3. Mechanism of action of ErbB family receptors

Ligand-induced receptor dimerization leads to transactivation and tyrosine autophosphorylation at site in the cytoplasmic carboxyl terminus. When phosphorylated, these residues can act as docking sites for SH2 (Src homology 2) or PTB (phosphotyrosine binding) domains containing proteins for activation of signalling pathways. The functions of a number of a substrates recruited by ErbB-2 receptor overlap, e.g. She (Src homology collagen homology) Grb-2 (growth factor receptor bound) and Chk (chec-kpoint kinase) interacts with most if not all ErbB dimeric species. Other receptors interacts with selected substrates like phospholipase С у (PLCy), RasGAP (Ras GTPase activating protein) and Grb-7 (RICCI et al. 1995; D a n k o r t et al. 1997; Z r ih a n - L ic h t et al. 1998; O la y io y e et al. 2000, 2001). All ErbB family members activate the МАРК (mitogen-activated protein kinase) pathway. Activation of Ras МАРК pathway is regulated by association of Grb-2 or She and phosphorylated ErbB-2. Activation of phosphatidylinositol-3 kinase is regulated by its association with YXXM motifs in the carboxy terminus of ErbB-3 and Cbl (Casitas B-lineage lymphoma) that specifically associates with EG FR, all of which contribute to cell growth and survival. The kinetics and potency of MAP kinase activation exhibit significant dependency on the identity of receptor dimers. Heterodimers are more sustained activators than homodimers, and hetero-dimers containing ErbB-2 are especially potent in this regard ( T z a h a r and

Y a r d e n 1998; P e n u e l et al. 2001). ErbB-2-containing heterodimers display increased ligand affinity due to a deacelerated off rate that can be correlated with prolonged activation of downstream signalling pathways (G R O U S-PO R T A

et al. 1995).

Heterodimers containing ErbB-2 remain at the cell surface for a lon-ger period of time, undergoing endocytosis at lower rate than homo- dimers do. Furthermore, once the activated complex is internalized ErbB- 2/ErbB-l heterodimers are targeted for recycling, while ErbB-1 homo-dimers are destined for degradation. The recycling pathway returns recep-tors to the cell surface, ready for another cycle of activation and au-gments growth-factor signalling ( L e n f e r i n k et al. 1998; P e n u e l et al.

2001; Y a r d e n 2001).

Furthermore, biological responses such as proliferation, morphological differentiation and migration/ invasion are enhanced in cells expressing ErbB-2 ( G r o u s - P o r t a et al. 1995; K a r u n a g a r a n et al. 1996; S p e n c e r

et al. 2000; O l a y i o y e et al. 2001).

ErbB heterodimerization is a means not only for signal amplification but also for signal diversification. The subset of SH2- and PTB-binding


signalling molecules recruited to an activated receptor are defined by the pattern of phosphorylated tyrosine residues in the C-terminus of the receptor. Based on the finding that the Cbl protein coupled only to EGF- but not to NRG-activated ErbB-1 it is speculated that signal diversifica-tion arises at one level by differential transphosphoryladiversifica-tion of a given receptor in distinct ErbB dimers (Gr o u s-Po r t a et al. 1995; Ol a y i o y e et

al. 2000).

Signalling diversity emanating from the ErbB family is generated by the repertoire of ErbB ligands and the combinatorial properties of induced receptor dimers. With the exception of EGF, which is found in many fluids, ErbB ligands generally act over short distances as autocrine or paracrine growth factors. The availability of a specific ligand is, therefore, one way to control its signalling ability. In this respect, ErbB ligands demonstrate distinct expression patterns that are organ- and developmental stage-specific

(Ol a y i o y e et al. 2000).

4. ErbB-2 receptor and cancer

ErbB-2 is a protein expressed in a variety of tissues of epithelial origin and it plays a fundamental role in cellular proliferation and differentiation during fetal development. In adults, the erbB-2 gene is present as a single copy and protein overexpression is seen in various cancers including breast, ovarian, colon, uterine, gastric, prostate and adenocarcinoma of the lung

(Be r n h a r d et al. 2002).

ErbB-2 overexpression triggers ligand-independent activation of the kinase domain, apparently as a result of spontaneous dimmer formation. Homodimerization of ErbB-2 can be achieved by mutating a single amino acid residue in the transmembrane domain, leading to constitutive dimerization and activation of this receptor (Ba r g m a n n et al. 1986; Ol a y i o y e et al.

1998; Pe n u e l et al. 2001). Although ErbB-2 homodimers alone may

contribute to malignancy, a number of observations suggest that ErbB-2 does indeed cooperate with other ErbB receptors during tumor development

(Ha r a r i and Ya r d e n 2000; Ol a y i o y e et al. 2000). Many human tumors

that contain ErbB-2 also exhibit autocrine stimulation of ErbB-1 via expression of one of its numerous ligands (SALOMON et al. 1995; DlGlOVAN-

NA et al. 1998). The ability of ErbB-2 to potentate ErbB-1 signalling would provide tumor cells with a more potent growth stimulus and could lead to the activation of additional intracellular pathways. Such cooperation would contribute to the maintenance of increased proliferation rates associated with tumor development.


ErbB-2 and ErbB-4 are also coexpressed in more than 50% of child-hood medulloblastomas, and the ErbB-4 ligand NRG1 is found in a sig-nificant proportion of the same tumors. Coexpression of ErbB-2 with ErbB-4 can enhance the effects of autocrine or paracrine NRG1 signalling

( O l a y i o y e et al. 2000). A nother important observation pertaining to

ErbB heterodimer collaboration during tumor development is that expres-sion of ErbB-3 is seen in many of the same tumor types that overexpress ErbB-2, including breast, bladder and melanoma ( G i l b e r t s o n et al.


ErbB-2 and ErbB-3 function together to stimulate mitogenic signalling networks. This in turn contributes to uncontrolled tumor cell proliferation by a mechanism involving deregulation of the G l-S transition through modulation of the activation status of the essential G l-S regulator cyclin E-dependent kinase 2 ( N e v e et al. 2000; O l a y i o y e et al. 2000).

The relatively low, normal expression o f ErbB-2 level is enhanced in several types o f carcinomas. For exam ple 25 -r- 35% o f ovarian carcinomas show overexpression o f ErbB-2 and survival o f patients with ErbB-2 overexpression is significantly shorter than survival o f non-overexpressors

(TZAH AR and Y a r d e n 1998). Lung cancer is another example. Unlike

squamous lung carcinom as, where the significance o f overexpression is still un settled, 30 h- 35% o f lung adenocarcinom as display overexpression, which correlates with shorter patient survival time (K e r n et al. 1990; T a t e i sh i 1991; L O PE Z -G U E R R E R O et al. 1999). Overexpression o f ErbB-2 due to gene amplification occurs in about 20 h- 30% o f breast tumors but particularly ( ~ 9 0 % ) in com edo forms o f ductal carcinom a in situ (DC IS), a malignant ductal carcinoma that has not progressed beyond the basement membrane barrier (B A R N E S et al. 1992). Even after progression to invasive disease, a correlation with the D CIS subtype and ErbB2 overexpression is maintained. Importantly, high ErbB-2 level predicts lower disease-free and overall survival in both lymph node negative and particularly in lymph node positive tumors, indicating a functional role o f ErbB-2 in breast cancer ( B a r n e s et al. 1992; Liu et al. 1992; T z a h a r and Y a r d e n 1998; D o w s e t t M. 2000; H a r a r i and Y a r d e n 2000).

Other types of carcinomas that show variable overexpresion are those of the kidney ( S e l l i et al. 1997), bladder ( I m a i et al. 1995; R a j k u m a r

et al. 1996; T A N N APFEL et al. 1996), salivary gland ( S k a l o v a et al. 2001),

prostate ( A g u s et al. 2000; S a n C H E Z et al. 2002) and the pancreas ( S a t o h

et al. 1993; K O K A et al. 2002). However, the prognostic significance of

overexpression in these tumors is not as clear as in breast, ovarian and lung cancers.

The other reason of ErbB-2 changes observed in cancers seems to be connected with polymorphism at codons 654 and 655. E h s a n i et al. (1993)


described a G-to-A polymorphism of the erbB-2 gene resulting in a varia-tion from valine to isoleucine. The ile-ile, ile-val and val-val configura-tions of codons 654 and 655 had frequencies of 0.782, 0.206 and 0.012, respectively. In a population-based case control study of the val655-to-ile polimorphism X lE et al. (2000) found that the val allele was associated

with an increased risk of breast cancer, particularly among younger woman.

Recently there has been a flurry of interest in the relationship between ErbB-2 and colon cancer. Normal colonic mucosa is negative for ErbB-2, but significant number of benign lesions and adenocarcinomas overexpressed this protein. Adenocarcinoma were significantly more positive than benign lesions. The ErbB-2 rate expression show significant correlation with the epithelial abnormality degree and clinical parameters including Dukes’ classification and relapse-free and postoperative survival period ( K a p i t a n o v i c

et al. 1997). Colon cancer metastatic to liver or lymph nodes has higher levels of ErbB-2 (SA E K I et al. 1995).

In our studies erbB-2 mRNA expression was identified in 51.5% of colorectal carcinoma samples (17/33) and in 29% of normal colonic mucosa (6/21) (Fig. 3). Obtained results suggest also that the erbB-2 mRNA expression in colorectal cancer cells is correlated with the progression of the disease. The expression of the erbB-2 mRNA was found in 1/2 of colon cancer cases at stage A, in 2/11 at stage B, in 7/13 at stage С and 7/7 at stage D. However no differences were found in the level of erbB-2 mRNA expression and tumor size.

ÖP M 1 2 3 4 5 6 7 a

Fig. 3. Representative R T-PC R analysis o f the erbB-1 expression in colorectal cancer (lanes 1 -j- 5) and normal colonic mucosa (lanes б-т-8). Total R N A was isolated from normal and tumor specimens according to Ch o m c z y n s k i and Sa c c h i (1978), reverse transcribed using R N A PCR Kit ver.2.1 (Takara Shuzo C o., Ltd., K yoto, Japan) and amplified with specific set оГ primers (5’- A AC TG CA CC C AC TC CT G TG T-3’;

5’-CAGGGATCCAGATGCCCTTG-3’) according to Wo r t h y l a k e et al. (1997). Amplification

products were separated on the 1.5% TBE-agarose gel and visualized by ethidium bromide. M, molecular weight markers; bp, base pairs


5. Conclusions

ErbB-2 is being investigated as a target for cancer therapy since is frequently overexpressed in human tumors and often confers a more aggressive clinical course. Its localization at the cell surface makes it an easy target to access. A wide range of therapeutic strategies targeting many tumors that overexpress ErbB-2 have been investigated, including tyrosine kinase inhibitors, antisense approaches designed to downregulate expression of the erbB-2 gene, and immunization to actively boost anti-ErbB-2 responses. In addition, selective targeting can be achieved using monoclonal antibodies directed against the extracellular domain of the ErbB-2 protein (De POTTER

et al. 1995). Therefore, monoclonal antibodies are the potential greatest advance in the treatment of tumors overexpressiong ErbB-2. Elucidation of the role of ErbB-2 in cell growth should allow the mechanism of action of monoclonal antibodies to be determined and should help optimize treatment of aggressive ErbB-2-positive tumors.

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