Trastuzumab-induced cardiac dysfunction: A ‘dual-hit’

In Canada, breast cancer is the leading cause of cancer-related death in women. Approximately 25% to 30% of breast cancers overexpress the human epidermal growth factor receptor 2. Trastuzumab (Trz), which is a monoclonal antibody against epidermal growth factor receptor 2, reduces recurrence and mortality by 50% and 33%, respectively. Other clinical studies have shown that 5% to 10% of patients who receive Trz following doxorubicin treatment develop cardiac dysfunction. This reviews discusses the various mechanisms that may account for doxorubicin and Trz-induced cardiac dysfunction.

Abstract

Trastuzumab (Trz) is a monoclonal antibody against the human epidermal growth factor receptor 2 that is found to be overexpressed in 25% to 30% of breast cancer patients. In spite of the therapeutic benefits of Trz, cardiotoxic side effects are still an issue. This effect is potentiated particularly when Trz is administered following doxorubicin (DOX) treatment. Among the various mechanisms that may account for DOX and Trz-induced cardiotoxicity, the role of oxidative stress has gained significant support. The present review discusses the evidence supporting the hypothesis that oxidative stress comes from multiple sources through an increase in the production of reactive oxygen species and/or a decrease in antioxidant defense systems. The adjuvant use of Trz can potentiate cardiomyocyte damage through a ‘dual-hit’ mechanism, which includes inhibition of the neuregulin-1 survival signalling pathway and angiotensin II-induced activation of NADPH oxidase, with the ability to further increase reactive oxygen species production. Preventive therapies for DOX- and Trz-induced cardiac dysfunction have eluded investigators, but may include the prophylactic use of angiotensin-converting enzyme inhibitors, beta-blockers and use of antioxidants. Thus, a better understanding of the mechanisms leading to this characteristic drug-induced cardiomyopathy, as well as potential cardioprotective strategies is required.

Breast cancer is the leading cause of cancer-related deaths among women in Canada. It is estimated that one in nine women will develop breast cancer in their lifetime (1). This translates to more than 23,000 women being newly diagnosed with breast cancer, and will account for the deaths of more than 5000 women in 2011 (2,3). Treatment for breast cancer includes surgical resection, radiation therapy, chemotherapy with an anthracycline-based agent, and the recent introduction of monoclonal antibodies (3). Approximately 25% to 30% of breast cancers overexpress the human epidermal growth factor receptor 2 (ErbB-2) (4–7). Trastuzumab (Trz), a monoclonal antibody against ErbB-2 (HER2), reduces breast cancer recurrence and mortality by 50% and 33%, respectively (8–10).

Although therapeutic treatment with doxorubicin (DOX) and Trz has demonstrated a significant reduction in morbidity and mortality in breast cancer patients, there are severe cardiac side effects that must be considered. Clinical studies have estimated that 5% to 10% of patients who receive Trz in the adjuvant setting of breast cancer develop cardiac dysfunction (11–13). However, in a retrospective study, it was demonstrated that the risk for developing asymptomatic cardiac dysfunction is actually as high as one in four when Trz is used in the adjuvant setting with DOX (9).

Despite the increasing number of clinical cases of Trz-induced cardiac dysfunction, little effort has been put into describing its underlying mechanism. In vivo studies of acute chemotherapy-induced cardiac dysfunction have linked Trz to altering antiapoptotic signalling pathways in cardiomyocytes that lead to congestive heart failure (14,15). Other studies have linked the renin-angiotensin system, including angiotensin II (ANG II) signalling, to Trz-induced cardiac dysfunction through the alteration of NADPH oxidase and mitogen-activated protein kinase (MAPK) signalling pathways (16,17). Furthermore, this alteration of HER2 signalling through NADPH oxidase and MAPKs has been associated with an increase in oxidative stress, leading to dilated cardiomyopathy (16). Studies using acute murine models of chemotherapy-induced cardiac dysfunction have demonstrated that antioxidants, including probucol and N-acetylcysteine, are cardioprotective against oxidative stress (15,18–20). The present review discusses the available evidence supporting DOX and Trz-induced cardiac dysfunction.

Anthracyclines

Since the late 1960s, DOX has been used in the cancer setting. It is highly effective against numerous cancers including breast cancer, childhood solid tumours, soft tissue sarcomas, and aggressive Hodgkin’s and non-Hodgkin’s lymphomas (21,22). DOX inhibits DNA/RNA synthesis by intercalating between the base pairs in DNA, as well as by binding and inhibiting topoisomerase II (23). Despite DOX’s ability to reduce solid tumour size and metastasis, its use within the clinical setting is limited due to its cardiac side effects. To minimize the risk of developing irreversible cardiac dysfunction, an empirical cumulative dose of DOX not exceeding 500 mg/m² has been suggested (24). Other risk factors contributing to DOX-induced cardiac dysfunction include age (older than 70 years), combination treatment (cyclophosphamide and actinomycin), radiotherapy and pre-existing cardiovascular disease (22). Clinical treatments for individuals who suffer from DOX-induced heart failure include the use of angiotensin-converting enzyme inhibitors (ACEi), angiotensin receptor blockers, beta-blockers and dexrazoxane (16,25,26); however, these treatments have been met with limited success.

There have been numerous attempts at developing an analogue for DOX that is as efficacious, but does not present with detrimental cardiac side effects. Several of these analogues include epirubicin, idarubicin and aclacinomycin A (27,28). To date, more than 2000 analogues have been produced; however, none have reduced the cardiotoxicity to an acceptable level while maintaining high efficacy against cancer cells (29,30). Epirubicin has shown to be promising, and is used regularly in cancer treatments; however, it too demonstrates dose-dependent cardiotoxicity (11).

The precise mechanism underlying DOX-induced cardiac dysfunction is unknown; however, oxidative stress has been suggested to play a role (22,30,31). Furthermore, DOX has been correlated with a decrease in cellular antioxidant enzymes (22). An accumulation of reactive oxygen species (ROS), a decrease in antioxidant reserve and some direct membrane effects illustrate the complexity of DOX-induced cardiomyopathy (23,30). Because cardiomyocytes are terminally differentiated cells, any loss of these cells through apoptosis and necrosis (23) can create a contractile deficit.

DOX use, even with the greatest care, carries a risk of developing cardiac dysfunction. To prevent these side effects, multiple approaches have been taken. The list includes dosage optimization of DOX, and the prophylactic use of vitamin E, iron chelation and ACEi (15,17,30). Optimization therapy was accomplished by extending the infusion time from 15 min to between 48 h and 96 h. Iron chelation with drugs, such as dexrazoxane provides cardiac protection and has demonstrated some success in reducing DOX-induced cardiac dysfunction (32). The underlying mechanism by which dexrazoxane prevents cardiac dysfunction is still relatively unknown. However, metal chelation and a reduction in ROS production have been suggested. Although dexrazoxane may be cardioprotective, it does limit the cytotoxic effects of DOX and is, thus, not used in standard clinical practice. Several studies have demonstrated some benefits from using ACEi and beta-blockers in the early treatment of DOX-induced cardiomyopathy in breast cancer patients (17,33).

Probucol, a lipid-lowering drug and antioxidant, has been shown to be beneficial in rat and mice models of DOX-induced cardiac dysfunction (5,18). Prophylactic treatment with probucol in mice and rats significantly reduced the cardiotoxicity and mortality that is commonly seen with DOX treatment. There was no effect on the cytotoxic efficacy of DOX (18,34). Probucol has been demonstrated to decrease cardiomyocyte apoptosis (18,34,35). These data provide compelling evidence that free radicals play a critical role in the pathogenesis of DOX-induced cardiotoxicity.

ErbB-2 and Trz

The human epidermal growth factor receptors (ErbB) are some of the most extensively studied tyrosine-kinase receptors. Four isoforms have been documented in humans. They include ErbB1 (EGFR and HER1), ErbB2 (HER2), ErbB3 (HER3) and ErbB4 (HER4) (36,37). Under normal physiological conditions, these cell surface receptors mediate cell-cell interactions, cell proliferation and differentiation in the developing embryo and adulthood (38–40). Each receptor consists of a single transmembrane ligand-activated tyrosine kinase and a carboxyl-terminal regulatory domain. Interestingly, the HER2 receptor is the only receptor of the ErbB receptor family for which no high-affinity ligand is known (36,38). HER2 is the most important receptor of the ErbB receptor family because it plays a key role in cell survival, and is the receptor of choice for dimerization with the other receptor isoforms. Women who present with overexpressed HER2 breast cancer experience more aggressive tumours and a lower survival rate (4,41). Furthermore, these women experience a lower success rate for therapies that include cytotoxic agents and radiotherapy because cancer cells overexpressing HER2 receptors can develop resistance to these therapies (7).

Trz (Herceptin, Genentech, USA) is a recombinant, humanized monoclonal antibody directed against the extracellular domain of the HER2 protein (8,42). Presently, Trz is the only drug on the market that has been approved by the United States Food and Drug Administration for use in the breast cancer setting to specifically target the HER2 protein. Trz is a double-edged sword; while mitigating cancer, it potentiates cardiac dysfunction when used serially following anthracycline-based chemotherapy.

Mechanistic theories of Trz-related cardiac dysfunction

HER2 signalling is essential to cardiomyocyte survival because binding of neuregulin-1 (NRG-1) initiates cell survival pathways, which inhibit apoptosis and maintain cardiac function (43,44) (Figure 1). NRG-1 binds to and activates HER4, which is then primed for binding to HER2 (44). This results in tyrosine kinase activation and leads to the activation of a G-protein coupled receptor signalling pathway (45). The G-protein-alpha is able to activate a MAPK signalling pathway that results in the upregulation of ERK 1/2, a critical protein for signalling cell survival (44,45). ERK 1/2 has been documented as a mediator that activates downstream transcription factors and stimulates cell survival by stabilizing myofibril structure and inhibiting apoptosis (44) (Figure 1). Furthermore, NRG-1 signalling activates the phosphoinositide 3 kinase/AKT signal transduction cascade (Figure 1) (46). AKT is able to initiate a change in mitochondrial respiration, thereby decreasing the production of ROS and increasing cell survival (44,46,47). AKT can also alter the expression of BCL-2 family proteins, initiate glucose uptake and trigger activation of endothelial nitric oxide synthase. These changes have also been linked to a change in mitochondrial respiration and cell survival. A third pathway by which NRG-1 signalling is able to demonstrate cardioprotective properties is through the activation of focal adhesion kinases (FAKs) (Figure 1) (44,46). FAK is a well-known adaptor protein for Src that is capable of recruiting other adhesion proteins (48). The formation of this protein complex is essential because FAK signalling is critical in maintaining the structure and function of sarcomeres (44,48), as well as the survival of cardiomyocytes.

Figure 1).

Neuregulin signalling in cell survival. Binding of neuregulin to HER4 leads to its dimerization with receptor HER2. This dimerization activates cell survival pathways involving ERK 1/2, phosphoinositide 3 kinase (PI3K)/AKT and the focal adhesion kinase (FAK)/Src complex. ROS Reactive oxygen species

Trz binds to HER2 with high affinity, thereby eliminating its ability to dimerize with other HER receptors. By inhibiting HER2’s ability to dimerize and signal cell survival through MAPK/ERK 1/2, phosphoinositide 3 kinase/AKT and FAK-dependent pathways, cardiomyocytes are unable to cope with the added stress (46,48,49) (Figure 2). Because cardiomyocytes are constitutively active cells that have high-energy demands, ATP is always needed. As a result, there is a large demand for ATP production from the mitochondria, which is prone to the generation of ROS. Fortunately, endogenous antioxidants are able to scavenge most ROS. However, there is a limited antioxidant reserve. By blocking HER2 signalling, cardiomyocytes are unable to activate cell survival pathways that cope with the excess ROS. Therefore, blockage of HER2 enables the accumulation of ROS within the cardiomyocytes, which leads to the development of cardiac dysfunction by stimulating cardio-myocyte apoptosis (43,50,51) (Figure 2).

Figure 2).

Schematic representation of potential mechanisms involved in doxorubicin/trastuzumab-induced cardiotoxicity. Binding of trastuzumab to HER2 inhibits its dimerization with the neuregulin-induced HER4 receptor. Binding of angiotensin II to its receptor AT1 leads to NADPH oxidase activation, production of a superoxide radical and an increase in oxidative stress. Adjunct treatment with doxorubicin also increases oxidative stress. Such an increase in oxidative stress leads to the activation of ASK-1 and p38/jun N-terminal kinase (JNK)-associated pathways, leading to apoptosis and heart failure

When Trz is used in conjunction with DOX, the development of cardiac dysfunction is potentiated. This could be due to the inherent ability of DOX to increase oxidative stress (23). Primary treatment with DOX leads to cardiac dysfunction through ROS-dependent pathways (23). The sequential treatment with DOX and Trz potentiates this, because it blocks key receptors in pathways that regulate cell survival (43) (Figure 2).

Furthermore, the binding of Trz to HER2 has been correlated with a significant change in the ratio between antiapoptotic and proapoptotic proteins. After binding to HER2, there is an immediate downregulation of BCL-XL, an antiapoptotic protein, and an immediate upregulation of BCL-XS, a proapoptotic protein (14). The ratio of the antiapoptotic to proapoptotic proteins from the BCL protein family is crucial because they are key mediators in mitochondrial function and apoptosis. Therefore, a shift in the ratio toward proapoptotic proteins is correlated with mitochondrial dysfunction, which leads to cardiomyocyte death (14,50).

Combination therapy with DOX and Trz leads to the formation of ROS and a reduction in antioxidants, thus causing oxidative stress; the latter contributes to cardiac dysfunction. This results in increased stress on the heart that leads to the upregulation of circulating ANG II. Upregulation of ANG II has two detrimental effects on the heart. First, ANG II is a potent inhibitor of NRG. Therefore, ANG II is able to downregulate and prevent NRG-1 from binding to its HER receptors (Figure 2). This prevents NRG from signalling through other HER receptors to initiate cell survival pathways. Furthermore, inhibition of HER signalling may contribute to the oxidative stress in the heart, because more ROS will accumulate due to the inhibition of the essential MAPK/ERK 1/2 cell survival pathway. The second detrimental effect that ANG II has on the heart is that it leads to the activation of NADPH oxidase (16,17). ANG II binds to the AT1 receptor, a well-known G-protein coupled receptor that also activates NADPH oxidase through a protein kinase C-dependent action (16) (Figure 2). NADPH oxidase produces superoxide – a potent ROS. Stimulation of the mitochondria also produces more ROS leading to mitochondrial dysfunction and cell death. This initiates a vicious cycle, because ROS increase electron leakage from the mitochondria, specifically the electron transport chain, further fueling the production of superoxide and leading to mitochondrial dysfunction. Furthermore, AT1 signalling is correlated with activation of apoptosis signal-regulating kinase 1, which is a member of the mitogen-activated protein kinase family (52). It activates p38 and jun N-terminal kinase pathways – both of which have been demonstrated to be active during DOX treatment and to participate in apoptosis and cardiac dysfunction (53) (Figure 2).

We believe that Trz-induced cardiomyopathy is the result of a ‘dual-hit’ mechanism. First, Trz directly inhibits antiapoptotic pathways. Second, Trz upregulates ANG II, which leads to an increase in ROS production and inhibition of NRG signalling (12).

Early detection of Trz-mediated cardiac dysfunction using cardiac imaging

Early detection of left ventricular (LV) systolic dysfunction using non-invasive cardiac imaging would be useful for addressing the cardiac safety profile of Trz, potentially avoiding the detrimental effects of heart failure. Noninvasive cardiac imaging includes the use of multigated acquisition scans, echocardiography and cardiac magnetic resonance imaging (51). Multigated acquisition scans and two-dimensional transthoracic echocardiography are routinely used to monitor serial ejection fraction (EF) in breast cancer patients (51). Once the LVEF drops to less than 40%, overt cardiac dysfunction has occurred. The use of tissue Doppler imaging (TDI) in detecting early LV systolic dysfunction before any observable changes in the traditional LVEF was recently evaluated (2) (Figure 3). In addition, the cardiotoxic effects of Trz, DOX and the combination of both agents in an acute murine model of chemotherapy-induced cardiomyopathy were evaluated (15). It was demonstrated that TDI results were abnormal in mice receiving either DOX or a combination of Trz and DOX as early as 24 h after treatment, and were predictive of ensuing LV systolic dysfunction and increased mortality. Whereas TDI changed acutely on day 1, LVEF values decreased by only day 3 of the study (Figure 3). Whether TDI using echocardiography could be used in the clinical setting of early detection of chemotherapy-induced cardiac dysfunction requires further study.

Figure 3).

Endocardial velocity using tissue Doppler imaging in a C57Bl/6 mouse receiving a combination of doxorubicin and trastuzumab at baseline (upper panel), which decreased from 3.0 cm/s at baseline to 1.4 cm/s at 24 h (lower panel). This acute change in tissue Doppler imaging values precedes the drop in left ventricular ejection fraction, which occurs at day 3 following the administration of doxorubicin and trastuzumab

We recently evaluated whether cardiac biomarkers, tissue velocity and strain imaging, and cardiac magnetic resonance imaging could predict early LV dysfunction in HER2-positive breast cancer patients treated with Trz in the adjuvant setting (54). Of 42 patients (mean age 47±9 years) prospectively followed between 2007 and 2009, 10 (25%) developed Trz-mediated cardiomyopathy. Troponin T, C-reactive protein and brain natriuretic peptide did not change over time. Within three months of adjuvant therapy with Trz, there was a significant decrease in the lateral systolic annular velocity (S’) between the normal cohort and those patients who developed LV systolic dysfunction (9.1±1.6 cm/s and 6.4±0.9 cm/s, respectively, P<0.05). Similarly, the peak global longitudinal and radial strain decreased as early as three months in the Trz-mediated cardiotoxicity group. The LVEF subsequently decreased at six months of follow-up in all 10 patients, necessitating discontinuation of the drug. All 10 patients demonstrated delayed enhancement of the lateral wall of the LV within the mid-myocardial portion, consistent with Trz-induced cardiomyopathy. Both TDI and strain imaging were able to detect preclinical changes in LV systolic function, before conventional changes in LVEF, in patients receiving Trz in the adjuvant setting (54). The clinical use of TDI parameters for the early detection of Trz-mediated cardiac dysfunction has been validated in other recent studies (55–57).

CONCLUSIONS

Our current understanding of Trz-induced cardiac dysfunction is incomplete. It is likely that the accumulation of ROS and subsequent oxidative stress plays a major role in the development of cardiac dysfunction. In the present study, we have provided evidence that the renin-angiotensin system and NADPH oxidase are also likely major contributors of oxidative stress through the production of superoxide-free radicals. By understanding the molecular mechanisms of Trz-induced cardiac dysfunction, we should be able to develop pharmacological approaches to mitigate the adverse effects of this beneficial drug.