Amplification of the oncogene HER2 in breast cancer: molecular basis and therapeutic significance

Journal of the Senologic International Society The World Society fo Breast Diseases Volume 1. Issue 2. 2012 REVIEW ARTICLE Amplification of the oncogene HER2 in breast cancer: molecular basis and therapeutic significance Francini Matos Lima Lin 1 ; Edmund Chada Baracat 2 ; Filomena Marino Carvalho 3 1 Graduate Student, Graduate Program in Obstetrics and Gynecology, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil; 2 Full Professor of Gynecology, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil; 3 Associate Professor, Department of Pathology, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil. Abstract The epithelial cells of the breast are under the influence of a range of hormones and growth factors. The HER2, a member of the family of growth factor receptors with tyrosine kinase activity, is amplified in approximately 15-20% of breast tumors, defining a highly aggressive subgroup. The breast cancers that overexpress HER2 inaugurated the era of targeted therapy in breast cancer and became the focus of numerous studies to enable better understanding and development of more efficient therapeutic strategies. Trastuzumab, a humanized monoclonal antibody that binds to the extracellular domain of HER2, causes inhibition of cell growth and increased apoptosis. However, response rates to monotherapy with this monoclonal antibody vary from 12-30%, and most of the responders patients develop resistance within one year. In this mini-review we present the major signaling pathways involved with HER2, as well as major mechanisms of resistance to trastuzumab and new potential targets. KEYWORDS: breast cancer, HER2, target therapy, prognosis, trastuzumab. 57

Introduction Breast cancer is the commonest malignancy in women and comprises 23% of all female cancers and 14% of all cancer deaths (1). Breast cancer represents a group of malignant epithelial tumors characterized by invasion of adjacent tissues as well as the tendency to metastases to distant sites. Breast cancer is a heterogeneous condition and includes different groups of disease, each one with particular clinical features and outcome. The high-throughput methods of examining simultaneously the expression of thousands of genes such as DNA microarray have suggested that the above mentioned heterogeneity of breast cancer can be explained, at least in part, by intrinsic differences in gene expression of primary tumors (2, 3). The use of high-throughput analysis of gene expression generated a molecular taxonomy for breast cancer in which carcinomas were grouped into five major prognostic and genetic classes that can be estimated by the immunohistochemical profile: luminal A, luminal B, HER2, basal-like and normal breast (4, 5). Among these types, HER2 group, characterized by the overexpression of genes in the ERBB2 locus at 17q22.24 including ERBB2 and GRB7, corresponds to a highly-aggressive group. The genetic category of HER2-carcinomas, corresponds to tumors that do not express any hormone receptor. Breast carcinomas that concomitantly express HER2 and estrogen receptor belong to another genetic category of tumors (2, 3, 5, 6) Besides, there are a subset of HER2-enriched gene expression group that are clinically triple negative, despite having the gene expression signatures of the HER2 tumors (7). The molecular era brought methods that allowed the development of profiles of gene expression implicated in determined behavior as recurrence risk, drug resistance, metastatic risk, therapeutic response, and others desirable endpoints. The methods of molecular studies coupled with the significance of gene expression groups carried a more biological understanding of breast cancer with significant impact on management. Thus, treatment strategies for breast cancer walk to more individualized approach and an increasing tendency for the development of targeted therapies, which take into account the presence or absence of specific tumor targets. In this context, the HER2 breast carcinomas constitute a special category in the sense that they have opened an era of targeted therapy in Senology. In this review we describe the family of growth factor receptors where HER2 is included, the clinical implications of HER2 amplification, the rational basis of targeted therapy, conjectures about some of the mechanisms underlying the 58

resistance to trastuzumab, and the role of androgen signaling pathways in HER2 carcinomas. HER family Breast epithelial cells have their functions regulated by a wide range of hormones, growth factors and other molecules. The growth factor receptors with tyrosine kinase receptors (TKRs) are among the most studied. Protein kinases are enzymes that catalyze the phosphorylation of proteins by transferring a phosphoryl group, usually from an ATP, to threonine, serine or tyrosine residues. Phosphorylation of these residues is responsible for transduction of extracellular stimuli into intracellular responses, providing a highly efficient mechanism for controlling the activity of proteins. Typically, the TKRs have three major domains: 1) Extracellular, ligand-binding, domain, which allows the docking of the extracellular, hormonal signal; 2) Transmembrane domain, which provides the anchorage of receptor in the cell membrane; 3) Intracellular kinase domain, portion C-terminal, which comprise the adenosine triphosphate (ATP)-linking position for receptor autophosphorylation and phosphorylation of respective substrates. Ligand binding to the extracellular domain leads to conformational changes that induce and stabilize receptor dimerization leading to increased kinase activity and autophosphorylation of tyrosine residues. The phosphorylation of a tyrosine provides a docking site for adaptor molecules that propagates a cascade of cellular events thorough a downstream signaling pathway (8, 9). Signaling pathways controlled by tyrosine kinases offer unique opportunities for pharmacological intervention. There are 20 RTK families that shares some structural and functional features (8). The family of receptors of epidermal growth factors (Human Epidermal Growth Factor Receptor), also termed HER or erbb, belongs to the RTK group. It comprises four receptors involved in cell signaling pathways that regulate the proliferative activity: EGFR or HER-1 (c-erbb1), HER-2/neu (c-erbb2), HER-3 (c- ErbB3) and HER-4 (c-erbb4) (Figure 1). 59

Figure 1: HER Family composed by four receptors: EGFR or HER-1 (c- ErbB1), HER-2/neu (c-erbb2), HER-3 (c-erbb3), and HER-4 (c-erbb4). The HER family receptors act either as homodimers, formed by two molecules of same type, or heterodimers, composed by two members of the same family. The ligands or growth factors for HER1, HER3 and HER4 have been identified. In contrast, there is no known ligant for HER-2, although it may suffer ligand-independent homodimerization in case of overexpression, as occurs in some breast carcinomas. Due to absence of intracellular catalitic domain, HER3 homodimers present no tyrosine kinase activity, although HER3 may transduce kinase activity if it binds to other HER family member as a heterodimer. HER family receptor molecules promote phosphorylation followed by activation of multiple cellular processes related mainly to the control of cell proliferation, promotion of angiogenesis and regulation of apoptosis. At least two major intracellular signaling pathways are recruited: the MAPK pathway (mitogen-activated protein kinase) involved in increased cell proliferation, and AKT / PKB (protein kinase B), implicated in increasing cell proliferation, inhibiting apoptosis and stimulating angiogenesis (Figure 2). 60

Figure 2: Activation of the two main intracellular signaling pathways, MAPK e AKT, following HER dimerization. HER2 HER2 is a 185kd glycoprotein encoded by a gene located on chromosome 17q21. This gene was described as amplified in approximately 15-30% of patients with breast cancer (10-13). Increased copy number of this gene leads to enhanced expression of HER2 in the cell membrane and release of oligopeptide generated by cleavage of extracellular portion of HER2 molecule into bloodstream. Therefore, amplification of gene for HER2 can be detected by direct assessment of gene by in situ hybridization methods, or by measurement of accumulation of transcripts for HER2, or by quantification of expression of protein in the membrane of breast cancer cells, and, possibly, also by the presence of HER2 peptide shed from breast cells (figure 3). 61

Figure 3: Identification of HER2 amplification: (1) gene amplification, (2) increase of transcripts, (3) membrane protein, and (4) plasmatic HER2 fragments HER family receptors activate MAPK pathway beginning by phosphorylating of the small intracellular G-protein RAS, followed by sequential activation of RAF and MEK kinase, leading to cell proliferation. The PI3K/AKT pathway has been considered the most important signaling pathway involved in HER2-carcinomas. This pathway is involved with the largest number of events related to the aggressiveness of breast malignancy cell: stimulation of proliferative activity, apoptosis inhibition, cell differentiation and angiogenesis (14). AKT family of serine/threonine-directed kinases regulates diverse biological processes through a cascade of phosphorylation, playing a key role in cancer progression by stimulating cell proliferation and inhibiting apoptosis (15). A disturbance of normal AKT signaling frequently occurs in various human tumors and this enzyme plays in important role in cancer progression and cell survival. AKT 62

activation was positively associated with HER2 overexpression in breast carcinoma obtained from human material. AKT activation begins with the phosphorylation of PIP2 (phosphatidyl inositol biphosphate 3,4) to PIP3 (phosphatidyl inositol 3,4,5 triphosphate) through PI3K (phosphatidyl inositol 3 kinase). Alterations in PI3K pathway are common in cancer and aberrations in components of this pathway have been demonstrated in a variety of different human cancers (16). PI3K itself is a target of activated mutations (17-19) and the most common genetic alteration in breast cancer is a PIK3CA gene somatic mutation encoding the catalytic subunit p110a. In a study of samples of human breast cancer conducted to evaluate mutations in PIK3CA, 77 of 292 (26%) primary breast tumors and 14 of 50 (28%) cell lines of breast cancer showed a mutation in the PIK3CA gene (20). These results were consistent with a previous study in which the mutation rate was 22% in a group of 41 breast tumors (17). In both studies mutations were found clustered in two regions in exons 9 and 20 corresponding to the helic and catalytic domain p110a, respectively. The opposite reaction, ie, dephosphorylation of PIP3, is related to the action of PTEN (phosphatase and tensin homolog), a protein that acts on the road opposite to PI3K (Figure 2). The gene PTEN is located on chromosome 10, locus 10q23, and was initially identified through its mutation into a variety of tumor types. PTEN loss of function by mutation, loss of heterozygosity or epigenetic silencing was reported in approximately 50% of all breast cancers and also in many other cancers (21). Reduction or absence of PTEN protein expression has been recognized in 8-50% of all breast cancer tumors. A reduced expression of PTEN protein was shown to be associated with AKT activation (21). To date, the major substrate of AKT is mtor (mammalian target of rapamycin), a key molecule in cell growth. mtor is a serinethreonine kinase, belonging to the PI3K/AKT signaling pathway, which regulates cell cycle progression. Other substrates for AKT include proteins involved in the inhibition of proliferation such as p21 and p27, and molecules involved in the inhibition of apoptosis such as Bad and caspases (22-24). Cellular survival is largely influenced by AKT through stimulus to bcl2, an important apoptosis inhibitor, and also through inhibition of transcription factors that act activating apoptosis related genes (25-27). 63

Clinical implications of HER2 amplification HER2 gene amplification results in transmembrane protein overexpression and consequently, increased cell proliferative activity. Only a small fraction of protein overexpression (1-2%) is not related to gene amplification (10). HER2-positive tumors comprise a group characterized by aggressiveness (10) and resistance to certain chemotherapeutic agents (eg.: Cyclophosphamide, methotrexate and 5-fluorouracil) and hormones (tamoxifen), but with greater sensitivity to anthracyclines (28). Moreover, HER2 gene amplification correlated to worse disease-free survival and overall survival, either in the group with positive axillary lymph nodes, as in the group of negative lymph nodes (29-31). HER2-positive breast tumors are considered a poor prognosis entity, but they present a great diversity in clinical behavior and response to therapy. In a recent study of gene expression analysis in 58 breast carcinomas tissues with HER2 amplification, the authors identified three subgroups with different prognosis, including one with a higher survival rate and low mortality (32). In this study the behavior was associated to expression of genes related to immune response, invasion and metastasis, which also interfere with breast carcinomas of other molecular types, such as basal-like lesions (32). The clinical behavior of HER2-carcinomas depends on the influence of other genes activation and/or inactivation. The co-expression of steroids hormones, not only ER, but also androgen receptor, is one of the known factors that interfere in HER2-carcinomas behavior (33-35). The correct determination of HER2 positivity is of a great importance in the current treatment of breast cancer because it defines a group with specific clinical features with the possibility of specific therapeutic approaches. The recommendations for HER2 testing in clinical practice have been established by American Society of Clinical Oncologists (ASCO) and by College of American Pathologists (CAP) (36, 37). The immunohistochemistry (IHC) is the routine method of identification of the amplification. A HER2 IHC result is considered positive if the score is 3+ (uniform, intense and complete membrane staining of more than 30% of tumor cells) (figure 4), and it is considered negative if the score is 0 (no staining) or 1+ (weak, incomplete membrane staining in any number of tumor cells). A HER2 IHC result is considered equivocal if the score is 2+ (complete membrane staining that is either no uniform or weak in intensity, but with obvious circumferential 64

distribution in at least 10% of tumor cells). For such cases the in situ hybridizations for identification of gene amplification is indicated. Figure 4: Invasive ductal carcinoma with strong and diffuse membrane expression of HER2 (socre 3+) (Sp3 antibody immunohistochemical reaction --, original magnification 400X) Target therapy cancer Targeted therapy cancer refers to a set of drugs that blocks the growth of neoplasic cells by interfering with specific molecules involved with carcinogenesis and tumor growth. The knowledge of the main signaling pathways in breast tumors that overexpress HER2 allowed the development of drugs, such as monoclonal antibodies, tyrosine-kinase inhibitors and mtor antagonists, capable of blocking the growth of tumors that utilize the growth factor pathway. Trastuzumab is a humanized monoclonal antibody that binds to the extracellular domain of HER2 with consequent inhibition of signal transduction, followed by proliferation blocking and increased apoptosis. A basic effect of trastuzumab is inhibition of phosphorylation of AKT by PI3K, but other mechanisms contribute to its inhibitory action. For example, the neoplasic cell bound to the 65

antibody becomes a target for NK cells (natural killers) (38). Trastuzumabe was the first HER2 target therapy to be approved for use in clinical practice (39), and it was initially evaluated in patients with metastatic breast cancer with any level of HER2 expression (40). Later, a strong correlation between the degree of HER2 expression and the clinical response was demonstrated (41). Therefore, trastuzumab should be used in patients with score 3+ HER2 overexpression detected by immunohistochemistry, or with confirmed HER2 gene amplification detected by an in situ hybridization method, such as fluorescence in situ hybridization (FISH) (36). Trastuzumab revolutionized breast cancer therapy; however, not all women whose tumors overexpresses HER2 are good responders. Response rates to monotherapy with this monoclonal antibody vary from 12 to 30% (42). Moreover, even patients who respond may develop resistance after one year (43). Thus, new strategies are presented, such as association with inhibitors of tyrosine kinase activity. Pertuzumab represents a new generation of monoclonal antibodies. It acts on the extracellular domain of the molecule by preventing the dimerization between HER2 and HER1 or HER3 (44). As the region of HER2 molecule that binds to the antibody is different from that which binds to trastuzumab, it is possible that the drug may act in case of resistance (45). Lapatinib is a tyrosine kinase inhibitor that acts on the intracellular domains of the receptors EGFR (or HER1) and HER2. In some cases, it has been effective in treating patients resistant to trastuzumab therapy. Blackwell et al, in a study of 296 patients with metastatic breast carcinomas, HER2-positive, who have experienced disease progression with trastuzumab, were randomly assigned to receive lapatinib alone or in combination with trastuzumab. In this study combined treatment was associated with increased disease-free survival and higher pathological response (46). Another potential therapeutic target is the mammalian Target of Rapamycin (mtor). Rapamycin is a specific antagonist of mtor that stabilizes the cell cycle in G1 by inducing apoptosis and inhibiting angiogenesis (47-49). The principal rapamycin derivatives are everolimus (RAD001) and temsirolimus (CCI-779). In a phase II study involving 270 postmenopausal patients whose breast tumors were positive for hormone receptors, the group that received everolimus (RAD001) in combination with letrozole as adjuvant therapy, showed a higher reduction of tumor size than the group receiving everolimus in combination with placebo (68% versus 59%, p = 0.0616) (50). Temsirolimus (CCI-779) was evaluated in a 66

phase II study with 109 patients refractory to multiple chemotherapy treatments. A partial response rate of 9.2% and stable disease for more than 24 weeks in 4.6%, conferred a clinical benefit of 13.8% (51). Mechanisms of resistance to trastuzumab Trastuzumab resistance mechanisms can be grouped into three categories: blocking of antibody binding to HER2, stimulation of the AKT signaling pathway, and activation of alternative pathways. The blockage of the binding between the antibody and the HER2 can occur through the MUC4, a cell surface mucin, overexpressed in breast cancer cells and related to improving phosphorylation of HER2 molecule (52). The generation of truncated forms of the HER2 receptor that lack the trastuzumab-binding domain can determine the resistance of tumors that harbor the truncated receptor (53). Persistent activation of PI3K/AKT pathway can be related to PTEN gene loss of function. PTEN is an important suppressor gene product that is activated in cases of trastuzumab response. Fujita et al. observed that PI3K/AKT pathway was highly active in cells that were resistant to the drug, and, in such cases, PTEN levels were low (42). Mutations in the gene encoding the PI3K, the PIK3CA, were detected in breast cancer, independent of clinical stage (54). Although there are no studies investigating the relationship between these mutations and trastuzumab response, it does not exclude the possibility that this altered kinase may explain the resistance. Finally, cell cycle is stimulated by AKT through mtor pathway, a kinase implicated in the transition from G1 to S phase (55). PI3K/AKT pathway activation plays a crucial role in the initiation and progression of human breast cancer. The PI3K activation can occur in response of diverse extracelular signals and characterizes another possible mechanism of trastuzumab resistance, that of alternative signaling pathways, for example, the heterodimerization of HER2 with the Insulinoid growth factor-1r (IGF-1R). From the above we conclude that HER2-positive breast carcinomas have part of their biological aggressiveness dependent of activation 67

of several cellular signaling pathways related to cell cycle control, including the PI3K/AKT pathway. On the other hand, judging by the differences in responsiveness to the monoclonal antibody trastuzumab, these tumors must be heterogeneous, as confirmed by their genetic profile and prognosis differences (32). In this scenario, PTEN and PI3K are important players for the aggressiveness of HER2-positive breast carcinomas, both involved in the mechanisms of trastuzumab resistance. However, the exact mechanisms affecting the expression of these molecules is unclear. Androgens and HER2 carcinomas Androgens receptor are expressed in most human breast cancer, both estrogen-receptor positive and negative (56), (35), (33). The apocrine molecular subtype is characterized by ER-negative and ARpositive (57). In ER-negative tumors, functional cross talk of AR with the HER-2 signaling pathway has been shown in vitro and in genetic studies (58, 59). The role of AR in HER2-carcinomas as a therapeutic molecular target, as well as, how AR and HER-2 signaling pathway are related is not well known (34, 35). In an elegant in vitro study, Ni et al. proposed a model where the AR mediates liganddependent activation of Wnt and HER2 signaling pathways through direct transcriptional induction of WNT7B and HER3. The tumor cell growth can be impaired targeting AR, Wnt or HER3, suggesting potential therapeutic approaches for apocrine molecular carcinomas (34). Conclusion HER2-positive breast carcinomas comprise a clinically heterogeneous aggressive group. The knowledge of the signaling pathways involved in the activity of membrane receptor allowed the development of several specific therapeutic approaches, such as the monoclonal antibody trastuzumab, a drug that has changed the history of these breast carcinomas. However, in this duel some cancers are able to progress despite treatment and others develop drug resistance. In this scenario two important fronts emerge, that of identification of mechanisms of resistance, and the newer one, the understanding of the complex HER2-signaling pathways in order to identify potential new targets. At this time, lapatinibe, pertuzumab and mtor inhibitors are a reality, but new drugs can emerge, such as AR and Wnt blockers. 68

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