Introduction

The erbB2 (also known as HER2 or neu) gene encodes a 185-kDa transmembrane glycoprotein, which belongs to the epidermal growth factor receptor (EGFR) family. ErbB2 is a receptor tyrosine kinase with intrinsic tyrosine kinase activity. The mammalian EGFR family comprises four receptors (EGFR, ErbB2, ErbB3, and ErbB4), which are derived from a series of gene duplications early in vertebrate evolution and are 40%—45% identical.¹ ErbB2 is the only EGFR family member for which no ligand has been found. This may be explained by the unique structure of the ErbB2 extracellular domain, which is not favorable for ligand binding.^2,3 Since ErbB2 extracellular domain is always in the open conformation, ErbB2 is the preferred binding partner of all ErbB receptors even as a monomer.^2-4 The binding of ErbB2 to other ErbB receptors results in increased signaling potency of the dimerized receptors through several means, including increased ligand affinity, increased coupling efficiency to signaling molecules, and decreased rate of receptor internalization.^5-8

ErbB2 plays an important role in human malignancies. The erbB2 gene is amplified or overexpressed in approximately 30% of human breast cancers⁹ and in many other cancer types, including ovarian,⁹ stomach,¹⁰ bladder,¹¹ salivary,¹² and lung carcinomas.¹³ Overwhelming evidence from numerous studies indicates that amplification or overexpression of ErbB2 disrupts normal cell-control mechanisms and gives rise of aggressive tumor cells.¹⁴ Patients with ErbB2-overexpressing breast cancer have substantially lower overall survival rates and shorter disease-free intervals than patients whose cancer does not overexpress ErbB2. Moreover, overexpression of ErbB2 leads to increased breast cancer metastasis.^15-17 The important roles of ErbB2 in cancer progression render it a highly attractive target for therapeutic interventions of breast cancer.^18,19 The humanized ErbB2-targeting antibody trastuzumab (Herceptin, from Genentech)²⁰ was approved for the treatment of ErbB2-overexpressing breast cancers in 1998. In both phase II and phase III clinical trials, the antibody has shown remarkable therapeutic efficacy when given in combination with chemotherapeutic agents.^21,22 Trastuzumab represents an excellent example of the ErbB2-targeting therapies. Other efforts to develop ErbB2-targeting cancer therapies also have yielded promising results, such as E1A gene therapy,^23,24 single-chain antibodies,^25,26 and tyrosine kinase inhibitors,²⁷ just to name a few.

These novel targeted cancer therapies provide exciting new hope and opportunity for fighting breast cancers. However, the majority of patients with breast carcinoma still receive chemotherapy as a critical component of multimodality treatment.²⁸ Thus, understanding the effect and mechanisms of ErbB2 on chemosensitivity is important for anticancer agent selection and individualization of patient treatment, which are critical to the success of treatment of breast cancers.

ErbB2 and Chemoresistance

Although the role and the mechanisms of ErbB2 overexpression on chemosensitivity still require intensive investigation, findings of many clinical and laboratory studies suggest that ErbB2 overexpression leads to increased chemoresistance to certain chemotherapeutic agents.

The Existing Controversy

Despite the supporting evidence just described, current clinical and experimental data on the effect of ErbB2 overexpression on chemosensitivity remain controversial. Results of several clinical studies have suggested that ErbB2 overexpression does not necessarily lead to chemoresistance. One clinical study showed that ErbB2 expression does not predict response to docetaxel or sequential methotrexate and 5-fluorouracil in advanced breast cancers.⁵³ Another study showed that ErbB2 expression was not significantly associated with tumor response to neoadjuvant treatment with fluorouracil, adriamycin and cyclophoshamide (FAC) in 329 cases of breast cancer.⁵⁴ In yet another study, ErbB2-overexpressing breast cancers responded better to doxorubicin than did breast cancers expressing low levels of ErbB2.⁵⁵

These contradictory clinical observations may be partly explained by intrinsic differences in the design of the studies. Since clinical studies are complicated processes, numerous factors may affect the outcome of the investigations. For example, amplification and overexpression of ErbB2 can be detected by fluorescence in situ hybridization, immunohistochemistry, or enzyme-linked immunosorbent assay on tumor tissue samples.⁵⁶ However, use of nonstandardized assay, subjectivity of the assay performer, limitations of techniques, differences in antibodies or DNA probes used, and differences in tissue treatment procedure have resulted in discordance in determining ErbB2 expression levels. In other words, the way in which the ErbB2 expression level was determined and defined is a very important factor that may affect the outcome of a study. Other factors that may affect outcome include the timing of treatment (neoadjuvant or adjuvant); the type of regimen (e.g., FAC or CMF); the treatment status of the patient (previously treated or untreated); patient's age (younger or older), menopausal status, and ethnic background; estrogen-receptor status of the tumor (positive or negative); and presence or absence of other genetic alterations that may interact with the ErbB2 receptor. Thus, any of these factors may lead to discordance in the outcomes of investigations on the role of ErbB2 in modulating chemosensitivity.⁵⁷

The controversy over the role of ErbB2 in chemosensitivity also exists in laboratory studies. For example, In a laboratory study in which erbB2-transfected breast and ovarian cancer cells were used to determine the effect of ErbB2 overexpression on chemosensitivity, ErbB2 overexpression was found to be insufficient to induce intrinsic resistance to drugs, including paclitaxel.⁵⁸ Although this study involved the use of a series of erbB2-transfected breast cancer cells, including erbB2-transfected MDA-MB-435 breast cancer cells, it yielded results that apparently disagreed with those of an earlier study showing that ErbB2 can confer paclitaxel resistance in erbB2-transfected MDA-MB-435 cells.⁴¹ The discrepancy may be explained partly by differences in ErbB2 expression levels between the erbB2-transfected MDA-MB-435 cells used in the two studies. In particular, the erbB2 transfectants that showed paclitaxel resistance expressed very high levels of ErbB2 protein, similar to those expressed in the SKBR3 breast cancer cell line, which was established from an ErbB2-overexpressing primary breast tumor.⁴¹ However, the erbB2 transfectants that showed no paclitaxel resistance produced less than one-third of the level of ErbB2 protein expressed by SKBR3 cells.^58,57 Based on the data from these two independent studies, we suggest that ErbB2 overexpression must reach a threshold level in breast cancer cells before they become resistant to paclitaxel. This might provide a reasonable explanation for the discrepancy between the findings of the two studies.

Molecular Mechanisms of ErbB2-Mediated Chemoresistance

The role of ErbB2 in chemoresistance is a problem of great clinical importance, and the observational data are abundant, as described above. Because of the controversy and the complexity of this problem, however, our knowledge on the molecular mechanisms of how ErbB2 confers chemoresistance is still limited. It is generally believed that breast cancer cells overexpressing ErbB2 are intrinsically resistant to DNA-damaging agents such as cisplatin as the result of an altered cell-cycle checkpoint, altered DNA repair mechanisms, and altered apoptosis responses.^59,60

Apoptosis is a predominant mechanism by which cancer chemotherapeutic agents kill cells.^61,62 The failure of cancer cells to detect chemotherapeutic agent-induced damage and to activate apoptosis may lead to multidrug resistance. The results from our studies indicate that overexpression of ErbB2 renders human breast cancer cells resistant to paclitaxel.^39,41 While studying the molecular mechanisms of ErbB2-mediated paclitaxel resistance, we found that treatment of MDA-MB-435 breast cancer cells with paclitaxel caused them to undergo apoptosis, which was inhibited in their paired transfectants overexpressing ErbB2. Further investigation showed that paclitaxel-treatment induced a premature activation of p34^Cdc2 kinase, the mitotic serine/threonine kinase that binds to cyclin B and also plays an important role in cancer cell apoptosis.⁶³ The premature activation of p34^Cdc2 led to mitotic catastrophe, i.e., apoptosis. A chemical inhibitor of p34^Cdc2 and a dominant-negative mutant of p34^Cdc2 blocked paclitaxel-induced apoptosis in these cells, indicating that a premature Cdc2 activation is a prerequisite for paclitaxel-induced apoptosis in MDA-MB-435 cells. We demonstrated that overexpression of ErbB2 in MDA-MB-435 cells transcriptionally upregulates p21^Cip1, which associates with p34^Cdc2, inhibits paclitaxel-mediated p34^Cdc2 activation, delays cell entrance to G₂/M phase, and thereby inhibits paclitaxel-induced apoptosis. Therefore, upregulation of p21^Cip1 by ErbB2 inhibits p34^Cdc2 and deregulates G₂/M checkpoint that contributes to resistance to paclitaxel-induced apoptosis in ErbB2-overexpressing breast cancer cells.⁶⁴ In addition, we found that phosphorylation on tyrosine (Tyr)15 of Cdc2, the crucial inhibitory phosphorylation site of Cdc2, is elevated in ErbB2-overexpressing breast cancer cells and primary tumors independent of Wee1, Cdc25C, and p21^Cip1. We showed that ErbB2 binds to and colocalizes with cyclin B-Cdc2 complexes and can directly and specifically phosphorylate Cdc2 on Tyr15. Increased Cdc2-Tyr15 phosphorylation in ErbB2-overexpressing cells corresponds with delayed M phase entry and reduced sensitivity to paclitaxel-induced apoptosis. Expression of a kinase-defective ErbB2 in MDA-MB-435 breast cancer cells or expression of a nonphosphorylatable Cdc2Y15F mutant in ErbB2 gene transfectant of the MDA-MB-435 cells render the cells sensitive to paclitaxel-induced apoptosis. These data indicate that the increased Cdc2-Tyr15 phosphorylation by ErbB2 tyrosine kinase may be a pertinent cell-cycle checkpoint defect that is involved in paclitaxel resistance in ErbB2-overexpressing breast cancers. Taken together, these findings indicate that ErbB2 overexpression can confer breast cancer cell resistance to paclitaxel-induced apoptosis by inhibiting Cdc2 activation through at least two mechanisms: (1) ErbB2 kinase-independent p21 upregulation and (2) ErbB2 kinase-dependent direct phosphorylation of Cdc2 on Tyr15 (fig. 1). This model is further supported by results of a study on patient samples showing that ErbB2 overexpression is correlated with p21^Cip1 upregulation and with increased p34^Cdc2-Tyr15 phosphorylation in breast tumors.⁶⁵

Figure 1

Model of inhibition of p34^Cdc2 activation and apoptosis by ErbB2. P34^Cdc2 remains inactive without binding to cyclin B as it is phosphorylated on Thr14 by Myt1 and Tyr15 by Wee1. Activation of p34^Cdc2 occurs by accumulation and binding of cyclin B, dephosphorylation (more...)

Other molecular mechanisms may also underlie ErbB2-mediated paclitaxel resistance. In addition, the molecular mechanisms of ErbB2-mediated resistance to different chemotherapeutic agents could be different. For example, other molecular mechanisms of ErbB2-mediated chemoresistance may involve activation of the PI3K/Akt pathway by ErbB2, which leads to increased cancer cell survival,⁴⁶ the estrogen receptor-ErbB2 cross-talk in ErbB2-positive breast cancer cells,^66,67 and coexpression of ErbB2 with other ErbB family receptors.⁴⁴ Although hints of how ErbB2-overexpressing breast tumors evade chemotherapeutic agent-induced apoptosis and develop chemoresistance are beginning to surface, it is obvious that many questions remain to be answered.

Targeting ErbB2 to Overcome Chemoresistance

Numerous lines of laboratory and clinical evidence indicate that ErbB2-targeting agents can sensitize the response of tumor cells to chemotherapeutic agents; therefore, developing ErbB2-targeting strategies to improve the therapy of ErbB2-overexpressing breast cancer remains a high priority. During the last decade, several exciting techniques have been developed to target ErbB2. Although some are still under investigation, many studies have shown that these ErbB2-targeting techniques not only inhibit tumor growth, but also lead to chemosensitization of ErbB2-overexpressing cancer cells (Table 1).

Table 1

Possible ways to overcome ErbB2-mediated chemoresistance.

One of the most successful example is Trastuzumab, which is a humanized antibody that binds to the extracellular domain of ErbB2.²⁰ Recent studies have shown that, in addition to inhibition of ErbB2 signaling, trastuzumab has other functions, such as activation of the PTEN tumor suppressor gene,⁶⁸ induction of p27^Kip1, and induction of G₁ cell cycle arrest.^69,70 Trastuzumab has demonstrated tumor-inhibitory and chemosensitizing effects for paclitaxel and several other chemotherapeutic agents in preclinical studies and in phase II and phase III clinical trials.^21,22,48,71 These results represent an excellent example of anti-ErbB2 antibody-mediated chemosensitization. On the basis of our understanding of the mechanisms of ErbB2-mediated paclitaxel resistance, we investigated the mechanisms by which trastuzumab enhances the antitumor effects of paclitaxel in vitro and in vivo. We found that treatment of ErbB2-overexpressing cells with trastuzumab can inhibit ErbB2-mediated Cdc2-Tyr15 phosphorylation and p21^Cip1 upregulation, which allows effective p34^Cdc2 activation and induction of apoptosis upon paclitaxel treatment.^49,65

In addition, the past 15 years have witnessed the development of several effective ErbB2-tageting strategies. These include, but not limited to, adenovirus type 5 E1A protein (please refer to the chapter by Liao and Hung for more details),^51,72-75,76 ErbB2-specific tyrosine kinase inhibitors such as emodin,⁵⁰ HKI-272,²⁷ and GW572016,⁷⁷ anti-ErbB2 intracellular single-chain antibodies (sFv),^25,26,78,79 ErbB2-targeting antisense oligonucleotide,^47,80-83 rationally designed anti-ErbB2 peptide mimetics (AHNP),^84-86 all-trans retinoic acid (ATRA) and fenretinide (4-HPR).⁸⁷ These ErbB2-tageting strategies either have been shown to have chemosensitization effect or are currently under testing for chemosensitization in ErbB2-overexpressing breast cancer cell lines, animal models, or clinical trials.⁵⁷

These approaches discussed above have already shown promise in overcoming ErbB2-mediated chemoresistance in either laboratory or clinical studies. Other new technologies are also under development. For example, ErbB2-targeting small interfering RNAs (siRNAs) silence erbB2 mRNA by using double-stranded RNA oligonucleotides.^88-90 One study showed that ovarian cancer cells and breast cancer cells infected with a retrovirus expressing anti-ErbB2 siRNA exhibited effective downregulation of ErbB2 expression and slower proliferation, increased apoptosis, increased G₀/G₁ arrest, and decreased tumor growth in mouse models.⁸⁹ On the basis of these results, ErbB2-targeting siRNA may have great potential in overcoming ErbB2-mediated chemoresistance in ErbB2-overexpressing breast cancer cells. Additionally, attempts are being made to modulate existing chemotherapeutic agents so that they can overcome resistance.⁹¹ These combined efforts held great potential to improve the therapies for patients with ErbB2-overexpressing breast tumors.

Future Investigation

Understanding the role of ErbB2 in chemoresistance is important and has significant clinical relevance. Although many studies have been done during the last decade, our current knowledge on the role of ErbB2 in cancer chemosensitivity is still limited. To develop more effective treatment for patients with ErbB2-overexpressing breast cancers, scientists and physicians need to team up to pursue the following and other related issues.

Acknowledgements

We apologize to those whose work could not be cited owing to space limitations. We thank Katherine Hale for editorial assistance. D. Yu's laboratory is supported by NIH grants RO1-CA60488, RO1-CA109570, 1PO1-CA099031-1 project 4, and the Department of Defense grant DAMD17-02-1-0462.

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