Author: Danilo Nicosia
Date: 26/01/2015


What is Trastuzumab?

Trastuzumab, is a monoclonal antibody (trade name: Herceptin®). A monoclonal antibody is an antibody (a type of protein) that has been designed to recognise and attach to a specific structure (called an antigen) that is found on certain cells in the body. Trastuzumab has been designed to attach to HER2, which is overexpressed in about a quarter of breast cancers and a fifth of gastric cancers. By attaching to HER2, trastuzumab activates cells of the immune system, which then kill the tumour cells. Trastuzumab also stops HER2 producing signals that cause the tumour cells to grow. Trastuzumab is also being studied for the treatment of other cancers. It has been used with some success in women with uterine papillary serous carcinomas that overexpress HER2/neu.

Trastuzumab: History

The biotech company Genentech developed trastuzumab jointly with UCLA (University of California, Los Angeles) and gained FDA approval in September 1998. The drug was first discovered by scientists including Dr. Axel Ullrich and Dr. H. Michael Shepard. At UCLA's Jonsson Comprehensive Cancer Center, Dr. Dennis Slamon subsequently worked on trastuzumab's development. A book about Dr. Slamon's work was made into a television film called Living Proof, that premiered in 2008. (Trastuzumab, Wikipedia)

HER2: description

Receptor tyrosine-protein kinase erbB-2, also known as CD340 (cluster of differentiation 340), proto-oncogene Neu, Erbb2 (rodent), or ERBB2 (human) is a protein that in humans is encoded by the ERBB2 gene, which is also frequently called HER2 (from human epidermal growth factor receptor 2) or HER2/neu.
The HER receptors are proteins that are embedded in the cell membrane and communicate molecular signals from outside the cell (molecules called EGFs, epidermal growth factor) to inside the cell, and turn genes on and off. The HER proteins stimulate cell proliferation.

HER2: pathway

The HER2 pathway promotes cell growth and division when it is functioning normally. The EGF pathway includes the receptors HER1 (EGFR), HER2, HER3, and HER4; the binding of EGF to HER is required to activate the pathway. The pathway initiates the MAP Kinase pathway as well as the PI3 Kinase/AKT pathway, which in turn activates the NF-κB pathway. HER2 extends across the cell membrane, and carries signals from outside the cell to the inside. Signaling compounds called mitogens (specifically EGF in this case) arrive at the cell membrane, and bind to the extracellular domain of the HER family of receptors. Those bound proteins then link (dimerize), activating the receptor. HER2 sends a signal from its intracellular domain, activating several different biochemical pathways. These include the PI3K/Akt pathway and the MAPK pathway. Signals on these pathways promote cell proliferation and the growth of blood vessels to nourish the tumor (angiogenesis).

Normal cell division (mitosis) has checkpoints that keep cell division under control. Some of the proteins that control this cycle are called cdk2 (CDKs). Overexpression of HER2 sidesteps these checkpoints, causing cells to proliferate in an uncontrolled fashion. This is caused by phosphorylation by Akt.

HER2: over-expression

In cancer cells the HER2 protein can be expressed up to 100 times more than in normal cells (2 million versus 20,000 per cell). This overexpression leads to strong and constant proliferative signaling and hence tumor formation. Overexpression of HER2 also causes deactivation of checkpoints, allowing for even greater increases in proliferation.
The HER2 gene (also known as HER2/neu and ErbB2 gene) is amplified in 20-30% of early-stage breast cancers (the cell contains too many copies of HER2), which makes it overexpress epidermal growth factor receptors (EGFR) in the cell membrane. In some types of cancer, HER2 may send signals without growth factors arriving and binding to the receptor, making its effect in the cell constitutive.
Then, dysregulation of HER2 signaling in cancer involves an excess of signals that stimulate cancer cells to grow and spread. It is this excess of signals, rather than a mutation in the receptor itself, that results in the deleterious effects of HER2 in cancer. (Her2 dysregulation, Bio-oncology)

Trastuzumab: Mechanism of action

Trastuzumab binds to domain IV of the extracellular segment of the HER2/neu receptor. Cells treated with trastuzumab undergo arrest during the G1 phase of the cell cycle so there is reduced proliferation. It has been suggested that trastuzumab induces some of its effect by downregulation of HER2/neu leading to disruption of receptor dimerization and signaling through the downstream PI3K cascade. In addition, trastuzumab suppresses angiogenesis both by induction of antiangiogenic factors and repression of proangiogenic factors. It is thought that a contribution to the unregulated growth observed in cancer could be due to proteolytic cleavage of HER2/neu that results in the release of the extracellular domain. One of the most relevant proteins that trastuzumab activates is the tumor suppressor p27 (kip1), also known as CDKN1B. Trastuzumab activates p27 by simultaneously inhibiting PI3K/Akt, Mirk, hKIS, pathways. Trastuzumab has been shown to inhibit HER2/neu ectodomain cleavage in breast cancer cells. Experiments in laboratory animals indicate that antibodies, including trastuzumab, when bound to a cell, induce immune cells to kill that cell, and that such antibody-dependent cell-mediated cytotoxicity is another important mechanism of action. There may be other undiscovered mechanisms by which trastuzumab induces regression in cancer.

Trastuzumab: resistance

One of the challenges in the treatment of breast cancer patients by Herceptin® is the understanding towards herceptin® resistance. In the last decade, several assays have been performed to understand the mechanism of Herceptin® resistance with/without supplementary drugs. Recently, all this information has been collected and compiled in form of a database Herceptin®. This database is a collection of assays performed to test sensitivity or resistance of Herceptin® Antibodies towards breast cancer cell lines. This database provides comprehensive information about experimental data perform to understanding factors behind Herceptin® resistance as well as assays performed for improving Herceptin® sensitivity with the help of supplementary drugs. This is the first database developed to understand herceptin resistance that can be used for designing Herceptin® sensitive biomarkers. While trastuzumab and Lapatinib had been the mainstays of treatment in combination with chemotherapy, innate and acquired resistance to these therapies occur. More recently, two additional HER2-directed therapies have been approved for HER2-positive breast cancer: it has been found that the therapy with trastuzumab could be more efficient linking it to pertuzumab or to the cytotoxic agent mentarsine. (Recent advances in the development of anti-HER2 antibodies and antibody-drug conjugates, 2014)

1) Pertuzumab

Human epidermal growth factor receptor 2 (HER2) plays an important role in the development and maintenance of the malignant phenotype of several human cancers. As such, it is a frequently pursued therapeutic target and two antibodies targeting HER2 have been clinically approved trastuzumab and, pertuzumab. It is a humanized monoclonal antibody that binds to the extracellular portion of the receptor on a domain distinct from the binding site of trastuzumab. The addition of pertuzumab to trastuzumab results in synergistic tumor cell inhibition and has been shown to significantly improve clinical outcomes for patients with HER2-positive metastatic breast cancer (MBC) compared to trastuzumab plus chemotherapy alone. Since trastuzumab and pertuzumab were not co-developed, there may be potential for further optimizing HER2 targeting.

High affinity antibodies to all four extracellular domains on HER2 were identified and tested for ability to inhibit growth of different HER2 dependent tumor cell lines. An antibody mixture targeting three HER2 subdomains proved to be superior to trastuzumab, pertuzumab, or a combination in vitro and to trastuzumab in two in vivo models. Specifically, the tripartite antibody mixture induced efficient HER2 internalization and degradation demonstrating increased sensitivity in cell lines with HER2 amplification and high EGFR levels. When compared with individual and clinically approved mAbs, the synergistic tripartite antibody targeting HER2 subdomains I, II, and IV demonstrates superior anti-cancer activity. (Targeting Three Distinct HER2 Domains with a Recombinant Antibody Mixture Overcomes Trastuzumab Resistance, 2015)

2) Trastuzumab emtansine: Kadcyla®

Trastuzumab emtansine (in the United States, ado-trastuzumab emtansine, trade name Kadcyla®) is an antibody-drug conjugate consisting of the monoclonal antibody trastuzumab (Herceptin®) linked to the cytotoxic agent mertansine (DM1). Trastuzumab alone stops growth of cancer cells by binding to the HER2/neu receptor, whereas mertansine enters cells and destroys them by binding to tubulin. Because the monoclonal antibody targets HER2, and HER2 is only over-expressed in cancer cells, the conjugate delivers the toxin specifically to tumor cells. The conjugate is abbreviated T-DM1. It is an effective treatment for HER2-positive breast cancer that has progressed on other HER2-directed therapies. Both pertuzumab and T-DM1 are relatively well tolerated. (Recent advances in the development of anti-HER2 antibodies and antibody-drug conjugates, 2014)

Trastuzumab: side effects

Since 1998, trastuzumab has been used to treat more than 450.000 women with breast cancer in the world. The studies conducted in the adjuvant have shown that, used singly or in combination with chemotherapy reduces the risk of recurrence of 50% and the risk of death by 33%. Unfortunately cardiotoxicity is an important side effect. The trastuzumab cardiotoxicity, attributed to the block of HER2 in cardiomyocytes, manifests as heart failure (HF) symptomatic or asymptomatic left ventricular dysfunction with reduced ejection fraction (EF). Clinical studies show that lapatinib on the cardiac safety of anti-HER2 is agent-specific; lapatinib in fact seems to determine lower cardiotoxicity compared to trastuzumab. For pertuzumab instead was reported HF and reduced EF in percentages similar to those of trastuzumab. The trastuzumab cardiotoxicity is different from that induced by anthracyclines. In particular, trastuzumab does not seem to cause loss of cardiomyocytes, the damage is dose-dependent and reversible. This cardiotoxicity is defined type II, to distinguish it from the type I induced by anthracyclines. In type I cardiotoxicity the initial damage is the myofibrillar disorganization and it is followed by apoptosis and necrosis of cardiomyocytes. When cardiac dysfunction occurs, the damage is irreversible. The disease can appear months or years after treatment and may be related to sequential cardiac stresses. In contrast, in the type II cardiotoxicity, myocytes appear histologically normal (for structural alterations can be viewed only in electron microscopy) the EF has the possibility of recovery and there is evidence that the drug re-administration after discontinuation is sufficiently safe. Unlike the damage caused by anthracyclines, in the type II toxicity there is a low probability of HF induced by sequential stresses.

Trastuzumab & cardiotoxicity

1) Risk factors

One of the main risk factors for cardiotoxicity associated with trastuzumab is the use of high cumulative doses of anthracyclines (>300 mg/m2). Other important risk factors include LV dysfunction, irrespective of anthracycline use, pre-existing systemic hypertension, body mass index >25 and advanced age. However, chest radiotherapy concomitantly with trastuzumab is clinically viable. Recent evidence shows that elderly cancer patients (aged over 70) with a history of heart disease and/or diabetes present a higher incidence of trastuzumab-related cardiotoxic effects in breast cancer treatment. (Cardiotoxicity associated with cancer therapy: Pathophysiology and prevention, 2013)

2) Pathophysiological mechanisms

The pathophysiological mechanism by which trastuzumab causes heart damage is not fully known. Neuroregulin-1, a member of the family of growth factors EGF-like, induces heterodimerization and transphosphorylation of HER2 and activates cardioprotection through the signaling of ERK1/2 and PI3K/AKT. In animal models it has been shown that the signaling of HER2 is important for embryonic development of the heart and for protection from cardiotossine. Transgenic mice with selective deletion of HER2 develop dilated cardiomyopathy and cardiomyocytes show increased susceptibility to cell death induced by anthracyclines. Serum levels of HER2 are usually increased in patients with heart failure. The pathway of HER2, required for the survival and the performance of cellular functions, seems to be stimulated by hemodynamic adverse events such as stress or treatment with anthracyclines.

The trastuzumab cardiotoxicity appears to be mediated by the binding of trastuzumab with the extracellular domain of HER2 on cardiomyocytes, blocking the signaling induced by dimerization HER2-HER4 pathway and inhibition of cell growth and cardioprotection. Interruption of treatment with trastuzumab is associated with the reactivation of the pathway of HER2 and the recovery of the ejection fraction. The mechanism proposed to explain the increase in the cardiac effects of trastuzumab, when administered in combination with anthracyclines, is the blocking of cellular repair mechanisms, which allows the oxidative damage induced by anthracyclines to progress freely. Experimental studies have shown that neuregulin-1 modulates the damage induced by doxorubicin in rat cardiomyocytes. Cardiotoxicity type II is exacerbated by the damage caused by the drugs responsible for tipe I cardiotoxicity through interference with the homeostatic mechanisms and pathways of cell survival. Scintigraphy with trastuzumab labeled with indium-111, performed immediately after anthracyclines and before trastuzumab, would allow the identification of patients more susceptible to the toxicity of trastuzumab and can be used to decide to postpone the start of therapy with trastuzumab until normalization of expression of HER2. These data are in agreement with the recommendations to avoid the concomitant use of trastuzumab and anthracyclines. Less exposure of the myocardium to anthracyclines can be achieved both by reducing the cumulative dose and using the liposomal anthracyclines (in particular the pegylated formulation), in which the chemotherapeutic agent is contained in the lipid particles, to enable the uptake in the tumor through the fenestrations of the capillary endothelium of the pathological tissues with minimal diffusion of anthracyclines in the myocardium. The combination of trastuzumab with liposomal anthracyclines, in particular with the pegylated liposomal anthracycline, showed a non-significant increase of cardiotoxicity compared to trastuzumab alone. Since the cardiac toxicity was attributed to the concomitant administration of anthracyclines and trastuzumab, trials involve cards treatment that avoids the concomitant administration. The trastuzumab cardiotoxicity is reduced when the administration is delayed compared to anthracyclines. (Cardio-oncologia 2013. La gestione del paziente oncologico prima, durante e dopo trattamenti con farmaci potenzialmente cardiotonici, 2013)

3) Studies

A study aimed to compare the long term cardiac effects of adjuvant trastuzumab therapies of HER2-positive breast cancer according to the treatment duration.
-Methods: Patients who completed adjuvant trastuzumab treatment at least 6 months before for the adjuvant setting in HER2 positive breast cancer were included to the study. A total of 164 patients were included to this study; 108 and 56 patients were treated with 9-weeks and 52-weeks trastuzumab, respectively. The main limitation of the study is due to the cross-sectional evaluation of cardiac biomarkers because the status of baseline cardiac biomarkers of this population can not be predicted.
-Results: The median follow-up of the study was 32 (10-95) months. The accompanying chronic diseases were similar in both two groups. Baseline left ventricular ejection fraction (LVEF) was 65.5±3.4% ve 67.1±4.5% in 9-weeks and 52-weeks trastuzumab treatment groups, respectively (P=0.13). Symptomatic heart failure was not observed during trastuzumab treatment in both groups. Trastuzumab induced cardiotoxicity (TIC) was observed in 2 (1.9%) and 17 (30.3%) patients of 9 and 52-weeks trastuzumab treatment groups, respectively (P<0.001). After median 24 months follow-up from the last dose trastuzumab, mean LVEF values were similar between two treatment arms (P=0.29). In the subgroup analyses, mean LVEF values were significantly lower in patients who developed TIC compared to who did not develop TIC (61.9±3.6% vs 64.4±2.6%, P=0.04). Average mean LVEF loss from baseline were significantly higher in patients who developed TIC compared to who did not develop TIC (10.0±6.0% vs 1.5±6.2%, P<0.001). Cardiac biomarkers were similar in both treatment groups. In the subgroup analyses serum Hs-CRP, and Pro-BNP levels were significantly higher in patients who developed TIC compared to who did not develop TIC.
-Conclusions: TIC was observed significantly higher in 52-weeks trastuzumab group. At the end of 32 months follow-up mean LVEF values and cardiac biomarkers were similar between two treatment groups. In the subgroup analyses, significant LVEF loss and higher cardiac biomarkers which show cardiac damage in patients who developed TIC suggests that TIC can be permanent in some of the patients and reversibility may underestimate long term cardiac damage. (Comparison of the long term cardiac effects associated with 9 and 52 weeks of trastuzumab in HER2-positive early breast cancer, 2015)

Resveratrol: anti HER2-cancer & cardioprotective agent

Resveratrol is a type of natural phenol, a stillbenoid. Is a 3,5,4'-trihydroxy-trans-stilbene, found in grapes, red wine (it contain 0.1-14.3 mg/l of Resveratrol), purple grape juice, peanuts, and some berries. Resveratrol is a fat-soluble compound that occurs in a trans and a cis configuration. Its synthesis from p-coumaroyl-CoA and malonyl-CoA (these substrates are present in all plants but most plants do not contain the enzyme, resveratrol synthase, necessary for the biosynthesis of the polyphenolic stilbene structure of resveratrol) is induced by stress, injury, infection or UV-irradiation, and it is classified as a phytoalexin anti-fungicide conferring disease resistance in the plant kingdom. The first mention of Resveratrol was in a Japanese article in 1939 by Michio Takaoka, who isolated it from the poisonous, but medicinal, Veratrum album, variety grandiflorum. Its name probably comes from the fact that is a resorcinol derivative coming from a Veratrum species. (Resveratrol and benefit, 2013)
In breast cancer cells, over-expression of human epidermal growth factor receptor 2 (HER2) increases the translation of FASN (fatty acid synthase) by altering the activity of PI3K/Akt signaling pathways. Earlier studies showed a role for resveratrol in the inhibition of FASN, alleviating the PI3K/Akt/mTOR signaling by down-regulation of Akt phosphorylation and up-regulation of PTEN expression. This concurrently causes a prominent up-regulation of PEA3. (The Ets protein PEA3 suppresses HER-2/neu overexpression and inhibits tumorigenesis, 2000)

PEA3 it is a DNA-binding protein, encoded by a gene of the ETS family, that specifically targets a DNA sequence on the HER-2/neu promoter and downregulates the promoter activity. Expression of PEA3 results in preferential inhibition of cell growth and tumor development of HER-2/neu-overexpressing cancer cells. This is a new approach to targeting HER-2/neu overexpression and also provides a rationale to the design for repressors of diseases caused by overexpression of pathogenic genes. (Resveratrol suppresses the proliferation of breast cancer cells by inhibiting fatty acid synthase signaling pathway, 2014)

Studies data show that resveratrol-mediated down-regulation of FASN and HER2 genes synergistically induce apoptotic death in SKBR-3 cells (it is a cell line over-expressing the HER2 gene product).
These findings suggest that resveratrol alters the cell cycle progression and induce cell death via FASN inhibition in HER2 positive breast cancer.
Resveratrol, furthermore, has been reported to play a cardioprotective role in diseases associated with oxidative stress: it protects against DOX-induced oxidative stress through changes in mitochondrial function, specifically the Sirt1 pathway leading to cardiac cell survival. (Resveratrol prevents doxorubicin cardiotoxicity through mitochondrial stabilization and the Sirt1 pathway, 2009)

Author: Danilo Nicosia
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