ERBB2 Gene Amplification Plays a Role in Breast Cancer Development and Treatment

Introduction

Cancer can be defined as a heterogeneous group of genetic diseases characterised by unregulated clonal expansion of somatic cells brought about by multiple genetic and epigenetic changes [1].  Cancer development and progression in humans involves multi-step processes that usually take place over many decades.  During these processes, the cancer cells acquire multiple allelic mutations in genes such as proto-oncogenes, tumour suppressor (TS) genes and other genes that control cell proliferation [2].  These allelic mutations lead to the production of dysregulated proteins leading to the activation of oncogenes or the inactivation of TS genes; promoting abnormal regulation of signalling pathways involved in cell cycle regulation, genetic stability, apoptosis and cell differentiation.  This imbalance in cellular regulations drives the process of oncogenesis [2].

This essay aims to explore one of these molecular alterations: gene amplification of the proto-oncogene ERBB2, its roles in cancer development and how it has contributed to improve cancer treatment.  Although other types of cancers will be highlighted, the essay will focus on the role of ErbB2 in breast cancer (BC) development and treatment, and this will be discussed under the following headings.

Target identification

ErbB2 (also referred to as Her2, c-erbB2 or neu) is a 185KDa proto-oncogene that belongs to the transmembrane receptor tyrosine kinase family, of which there are other three receptor types, including epithelial growth factor (EGF) receptor 1 (Her1), Her3 and Her4 [3].  The ERBB2 gene is located in chromosome 17q11.2-12 [4], and its product is a glycoprotein comprising an N-terminal extracellular domain (ECD), a single 23 amino-acid transmembrane domain, and an intracellular tyrosine kinase domain [3].  The structure of the ECD reveals why ErbB2 is an orphan receptor (as opposed to others) as the ligand binding site is occluded by the direct contact between domains I and III of the ECD [5].

Binding of growth factors such as EGF or neuregulin to their cognate receptors, and subsequent heterodimerisation with their preferred dimerisation partner (ErbB2) regulates downstream processes such as cell growth, differentiation, and apoptosis via auto-phosphorylation or trans-phosphorylation of the tyrosines in their C-terminal domain [3].  The above processes are coordinated through at least three different pathways: phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase MAPK, and phospholipase C-γ as illustrated in Figure 1 below [3].  Although usually membrane localised, ErbB2 has been reported to translocate to the nucleus where it acts as a transcription factor for COX2 [6] and STAT3 [7] genes – key genes that promote tumourigenesis – suggesting a role for ErbB2 in transcription deregulation in tumours.

ErbB2 gene amplification and overexpression has been identified in 15-30% of human breast cancers [8], which is predictive of a more aggressive tumour with poor prognosis [3, 5, 9].  Other amplified ErbB2-associated malignancies include ovarian, gastric, and colon cancers [9]. In an attempt to understand the mechanisms of ERBB2 gene amplification, Marotta et al. (2012) proposed that a common copy-number breakpoint in a duplicated segment associated with the Keratin-associated protein (KRTAP) gene was the cause of ErbB2 gene amplification in primary BC due to this loci’s fragility.  In addition, Li et al. [10] showed that a macroH2A1.2 interacts with Her2 to induce Her2 transcription, and hence, increased cell proliferation and tumourigenicity in SKBR-3 breast cancer cells.  The amplified region in Her2 is located within chromosomes as homogeneously staining region [11], and telomeric deletions have been shown to be common in ERBB2 [12] indicating the involvement of DNA breaks in ERBB2 amplification.

Methods such as fluorescence in situ hybridisation (FISH), immunohistochemical (IHC) and immunocytochemical analyses, and PCR have been used to estimate and validate HER2 gene amplification and protein overexpression in breast cancer cells [13].  Results from this study show that FISH is the method of choice for Her2 status tests, and may be employed to confirm the results obtained from other methods above.  Moreover, Jonathan et al. [14] and Yeh, et al. [15] used cDNA microarray and array Comparative Genomic Hybridisation, respectively to show that ERBB2 gene was amplified and overexpressed in breast cancer.

Target validation

Identification of ERBB2 as the gene overexpressed in cancer enabled scientists to demonstrate that it does play a critical role in breast cancer development and progression.  To effectively target this molecular defect, the role of ErbB2 overexpression should be investigated and validated.  To achieve this, several functional studies have been carried out on ErbB2-induced cancer cells.  To validate this, Vaughn et al. [16] used antisense DNA to specifically downregulate overexpressed ErbB2 in breast cancer cells.  This caused a shift in cell cycle profile, with a significant time in the G1-phase.  In a similar study, siRNA was used to silence the Her2/neu gene, resulting in the induction of apoptosis and inhibition of proliferation in SKBR3 BC cell lines [17].  Similarly, transformation of primary murine mammary epithelial cells with ErbB2 overexpression results in the destabilisation of the cdk inhibitor p27 and concomitant increased expression of cyclin D1 leading to increased proliferation [18].  Results from these independent experiments show that ErbB2 overexpression is associated with tumourigenesis, and could serve as potential therapeutic target in breast cancer (and other cancer type where ErbB2 is overexpressed).

Drug discovery

Knowledge of the genetic basis of cancer at the molecular level has contributed to the development of novel targeted therapies [19].  Drugs targeting the ErbB2 receptor selectively attack tumour cells, thereby discriminating, to some extent, against normal cells – which is the ultimate aim of cancer therapy [19].  Drugs that target two domains: ECD and the cytoplasmic tyrosine kinase domain of the ErbB2 receptor have been developed [19].

Drebin et al. [20] first used monoclonal antibodies (mAbs) to specifically target and immunoprecipitate the p^185/her2/neu from a DNA donor rat neuroblastoma.  Further to this was the finding that specific mAbs (such as mAb7.16.4) could actually induce the reversible downregulation of ErbB2 receptors, leading to a growth inhibition in athymic mice overexpressing the receptor [21, 22].  These studies paved way for the use of mAbs for the treatment of human malignancies and further validated oncogenes as targets for therapeutic agents [19].  Sequel to these developments, recombinant humanised Antibody rhumAb4D5 was engineered, and later renamed trastuzumab or Herceptin (developed by Genetech Inc.) and was approved for BC treatment by the FDA in 1998 [19, 23].

Herceptin targets domain IV of the ECD of ErbB2 and studies have shown that when combined with standard chemotherapy, it reduces the risk of cancer recurrence as compared with chemotherapy alone as indicated in Figure 2 below [24-26].  Herceptin mechanisms of action have been proposed as follows:

1. ErbB2 internalisation and degradation via the activity of ubiquitin ligase c-Cbl [27].
2. Antibody dependent cellular cytotoxicity (ADCC) via the recruitment of natural killer cells [28]
3. Inhibition of the MAPK and PI3K/Akt pathways leading to cell growth suppression, cell cycle arrest and PTEN (a negative regulator of PI3K/Akt and STAT-3) overexpression by interfering with ErbB2 dimerisation [29]

Pertuzumab is another mAb that binds to domain II of the ECD, hindering ErbB2 interactions with cognate receptors; and also mediates ADCC [30, 31].  Lapatinib is a tyrosine kinase inhibitor that causes potent inhibition of both EGFR and ErbB2 activation, hence indirectly inhibiting the activity of downstream effectors MAPK and Akt in breast cancer cells in vivo and in vitro [19].

Clinical application

Clinical application of trastuzumab and lapatinib is limited since only about 20% of BC show ErbB2 overexpression.  Therefore, diagnostic tests such as FISH, IHC are performed to detect gene amplifications [32].  Adjuvant therapies that include trastuzumab and chemotherapy are now seen as the standard of care for patients having ErbB2-induced BC [24, 25].  Recently in a clinical trial, it was shown that trastuzumab plus lapatinib was effective in the treatment of metastatic breast cancer than lapatinib alone, again showing the benefit of combined therapy in the treatment of BC [33].

Although there has been great success in the use of trastuzumab in combination with other drugs, it is presented with resistance by the cancer cells and with some potential side effects [34].  This resistance could be inherent or acquired and it has been shown that approximately 70% of patients develop secondary resistance [34].  Some tumour suppressor proteins and oncogenes, including PTEN and src have been implicated to play critical roles in the mechanisms of primary or secondary resistance [34].  Several mechanisms of resistance in BC have been proposed including alterations in Her2 receptor complex, downstream activation of PI3K signalling pathway, alteration in other receptors such as insulin-like growth factor receptor-1 and Met receptor [32, 34].  This is further summarised in table 1 below.

Conclusion

In conclusion, cancer treatments have received great attention in recent times due to the molecular understanding of the genetic basis of cancer development and progression and the recent completion of the sequencing of the human genome.  Identification of ErbB2 as the protein overexpressed in some breast cancer patients have paved way for the development of novel therapies for the treatment of breast cancer.  Although these novel therapies have been clinically useful, more future research is expected to overcome the challenge of drug resistance and other potential side effects associated with them.

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