Scientist discovering mechanism of herceptin cardiotoxicity

[Lani]

Research article
.
Anti-erbB2 treatment induces cardiotoxicity by interfering with cell survival pathways
Thea Pugatsch , Suzan Abedat , Chaim Lotan and Ronen Beeri
The Cardiovascular Research Center, Heart Institute, Hadassah-Hebrew University Medical Center, Jerusalem, Israel

Breast Cancer Research 2006, 8:R35 doi:10.1186/bcr1523

The electronic version of this article is the complete one and can be found online at: http://breast-cancer-research.com/content/8/4/R35

Received 22 March 2006
Revisions requested 12 June 2006
Revisions received 26 June 2006
Accepted 26 June 2006
Published 13 July 2006

© 2006 Pugatsch et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Cardiac dysfunction is among the serious side effects of therapy with recombinant humanized anti-erbB2 monoclonal antibody. The antibody blocks ErbB-2, a receptor tyrosine kinase and co-receptor for other members of the ErbB and epidermal growth factor families, which is over-expressed on the surface of many malignant cells. ErbB-2 and its ligands neuregulin and ErbB-3/ErbB-4 are involved in survival and growth of cardiomyocytes in both postnatal and adult hearts, and therefore the drug may interrupt the correct functioning of the ErbB-2 pathway.

Methods

The effect of the rat-anti-erbB2 monoclonal antibody B-10 was studied in spontaneously beating primary myocyte cultures from rat neonatal hearts. Gene expression was determined by RT-PCR (reverse transcription polymerase chain reaction) and by rat stress-specific microarray analysis, protein levels by Western blot, cell contractility by video motion analysis, calcium transients by the FURA fluorescent method, and apoptosis using the TUNEL (terminal uridine nick-end labelling) assay.

Results

B-10 treatment induces significant changes in expression of 24 out of 207 stress genes analyzed using the microarray technique. Protein levels of ErbB-2, ErbB-3, ErbB-4 and neuregulin decreased after 1 day. However, both transcription and protein levels of ErbB-4 and gp130 increased several fold. Calreticulin and calsequestrin were overexpressed after three days, inducing a decrease in calcium transients, thereby influencing cell contractility. Apoptosis was induced in 20% cells after 24 hours.

Conclusion

Blocking ErbB-2 in cultured rat cardiomyocytes leads to changes that may influence the cell cycle and affects genes involved in heart functions. B-10 inhibits pro-survival pathways and reduces cellular contractility. Thus, it is conceivable that this process may impair the stress response of the heart.



Outline Introduction



Human epidermal growth factor receptor (Her)-2 (ErbB-2), a 185 kDa transmembrane glycoprotein receptor with intrinsic tyrosine kinase activity, is thought to be one of the important mediators of cell growth [1-5]. It is overexpressed in 25–30% of human breast cancers, plays a role in its pathogenesis and is a predictive marker for poor prognosis in patients with metastatic disease [6-10].

Over recent years new therapies were developed to target tumour cells with Her-2 overexpression by blocking Her-2 on the cell surface of the tumour cells, thereby inhibiting their growth. The most widely used drug based on this principle is Trastuzumab (Herceptin®, Genentech Inc., San Francisco, CA, USA), a high-affinity humanized anti-Her-2 antibody that was shown to benefit patients with Her-2-overexpressing breast cancer, either as a single agent or in combination with chemotherapy [6,11,12]. In women who had received at least one prior chemotherapeutic regimen for metastatic disease, the response rate was found to be 15% and in previously untreated patients 23%.

One of the major side effects noted was cardiotoxicity [6]. Congestive heart failure linked to Herceptin therapy may be severe and has been associated with disabling symptoms. Of patients receiving Herceptin together with paclitaxel, 12% developed cardiac dysfunction, as compared with 1% receiving paclitaxel alone; the number increased further when Herceptin was administered in combination with anthracyclines (27%) compared with anthracyclines alone (7%) [6,13]. No data are yet available on identification of patients who are at risk for developing cardiac dysfunction when they receive anti-erbB2 therapy, although both prior cardiotoxic therapy (e.g. radiation to the breast area) and advanced age may play a role.

ErbB-2 forms heterodimers with other ErbB receptors (ErbB-3/ErbB-4) that can bind neuregulins, polypeptide growth factors that are known to promote survival and growth of cardiac myocytes [14,15]. Thus, blocking ErbB-2 with a monoclonal antibody may inhibit the myocardial adaptation to physiological stress and injury, such as that resulting from chemoradiotherapy, ultimately leading to cardiac failure in susceptible patients.

In order to elucidate the molecular mechanisms that lead to cardiotoxocity, we studied the effect of the rat-anti-erbB2 monoclonal antibody B-10 on spontaneously beating primary cultures derived from rat neonatal hearts. B-10 has previously been shown to have activity biologically similar to that of the humanized antibody that is used in the clinical setting [16].

I am lousy at creating links, but if you go to breast cancer research website the article is open access.

[Lani]

Discussion

Abstract
Introduction
Materials and methods
Results
Discussion
Conclusion
Abbreviations
Competing interests
Authors' contributions
Acknowledgements
References

Targeted monoclonal antibody therapy as a biological treatment for different cancers appears to have many advantages over conventional chemo/radiotherapy. In the case of ErbB-2 overexpressing breast cancer cells, it was postulated that antibody therapy would specifically eliminate only tumour cells without affecting any other ErbB-2 expressing cells [23-25]. Although erbB-2 is expressed ubiquitously, it was thought that those levels are sufficiently low not to be affected by anti-erbB-2 antibodies. However, ErbB-2 plays a vital role both in developing and in adult murine cardiomyocytes [14,26-28]. ErbB-2 null mutant mice exhibit an absence of trabecules in the heart ventricle and die at midgestation [14]. Adult mice carrying a conditional ErbB-2 mutation develop severe dilated cardiomyopathy, usually in the second month of life [28]. As shown by Özcelik and coworkers [29], loss of ErbB-2 in cardiomyocytes leads to physiological stress on the heart, which over time induces cardiac decompensation and dilated cardiomyopathy.

The effect of trastuzumab on cells on the molecular level has repeatedly been analyzed on human breast cancer cell lines [30,31]. However, to our knowledge very few molecular studies on the effect of the antibody on cardiomyocytes have been reported. In vitro cultures of primary human cardiomyocytes are not readily attainable for both ethical and technical reasons. Therefore, in our study we opted to use the generally accepted in vitro model of neonatal rat ventricular heart muscle cells. Trastuzumab, being a humanized monoclonal antibody, does not bind to rat cells, consequently we used B-10 instead, which was shown previously to have biologic activity similar to that of trastuzumab [16,32]. Our results reveal a substantial decrease in the level of all ErbB family members analyzed after treatment with B-10. ErbB-2 by itself has no tyrosine kinase activity and needs to form heterodimers with one of its ligands to be functional [33]. All the ligands tested in our system were downregulated, thereby affecting the central role played by ErbB-2 in the heart. Moreover, gp130, which is thought to bind to ErbB-2 after biomechanical stress (hypoxia, haemodynamic overload and myocardial injury, among other insults) and activates a survival pathway [34], is strongly affected by B-10. Our results imply that the turnover of gp130 is augmented, as reflected in the several fold increase in transcription; nevertheless, the decrease in gp130 cannot be completely compensated.

It was previously demonstrated that overexpression of calreticulin modulates protein kinase B/Akt signalling, promoting apoptosis during cardiac differentiation of cardiomyoblasts [35]. In our system B-10 induced an increase in both calreticulin and calsequestrin levels three days after antibody treatment. The high levels of both of these calcium storage proteins in the sarcoplasmic reticulum may modify calcium release and subsequently lead to reduced cell contractility, decreasing the percentage shortening by 40%. This finding corroborates the studies described in ErbB-2 mutant mice and calsequestrin overexpressing mice [36,37]. Sawyer and coworkers [38], when comparing anthracycline- and ErbB2-induced myofibrillar disarray, reported no effect of B-10 on either akt or p-akt in cultures of adult rat ventricular myocytes, which is consistent with our data.

Finally, apoptosis may be a factor in the loss of ventricular function that is part of the cardiotoxic side effect profile of anti-erbB2 monoclonal antibody therapy. In our system B-10-induced apoptosis was three times greater than that recently described by Grazette and coworkers [39]. However, those investigators used a different antibody (clone 9G6), which may account for the difference in intensity.



Conclusion


In this molecular analysis of the effects of B-10 on neonatal cardiomyocytes, we demonstrated that the rat-anti-erbB2 monoclonal antibody B-10 severely affects ErbB-2 and its major ligands ErbB-3/4, neuregulin and gp130. In addition, the drug promotes increased expression of calreticulin and calsequestrin, which are involved in the sarcoplasmatic reticulum calcium pump and decrease cell contractility, which in vivo may lead to dilated cardiomyopathy in mice and humans as described previously [22,28,34,40]. Despite being present at a low level, in cardiomyocytes ErbB-2 plays a central role in the correct functioning of the cell. Blocking ErbB-2 by a monoclonal antibody disrupts the cell survival pathways and the ErbB-signalling network that depends on the relative stoichiometry between all of the players involved. We therefore speculate that this process may impair the stress response of the heart.

Problems with this study, among others:

done in mice(may not necessarily apply to humans)
done in neonatal mice heart-cells (adult cells may not rely on her2 to as great an extent)
didn't particularly look at mitochondrial function (as articles cited by Dr. Dennis Slamon did)
other mechanisms of toxicity may also apply

[jhandley]

Lani
Can you send the links re this and herceptin please. I am interested re. Q10 which acts on the mitochrondria.
Jackie

[Lani]

1: J Am Coll Cardiol. 2004 Dec 7;44(11):2231-8. Links

Inhibition of ErbB2 causes mitochondrial dysfunction in cardiomyocytes: implications for herceptin-induced cardiomyopathy.

Grazette LP, Boecker W, Matsui T, Semigran M, Force TL, Hajjar RJ, Rosenzweig A.

Program in Cardiovascular Gene Therapy, Cardiovascular Research Center, Boston, Massachusetts, USA.

OBJECTIVES: We investigated the effects of erbB2 inhibition by anti-erbB2 antibody on cardiomyocyte survival and mitochondrial function. BACKGROUND: ErbB2 is an important signal integrator for the epidermal growth factor family of receptor tyrosine kinases. Herceptin, an inhibitory antibody to the erbB2 receptor, is a potent chemotherapeutic but causes cardiac toxicity. METHODS: Primary cultures of neonatal rat ventricular myocytes were exposed to anti-erbB2 antibody (Ab) (7.5 mug/ml) for up to 24 h. Cell viability, mitochondrial function, and apoptosis were measured using multiple complementary techniques. RESULTS: ErbB2 inhibition was associated with a dramatic increase in expression of the pro-apoptotic Bcl-2 family protein Bcl-xS and decreased levels of anti-apoptotic Bcl-xL. There was a time-dependent increase in mitochondrial translocation and oligomerization of bcl-associated protein (BAX), as indicated by 1,6-bismaleimidohexane crosslinking. The BAX oligomerization was associated with cytochrome c release and caspase activation. These alterations induced mitochondrial dysfunction, a loss of mitochondrial membrane potential (psi) (76.9 +/- 2.4 vs. 51.7 +/- 0.1; p < 0.05; n = 4), a 35% decline in adenosine triphosphate levels (p < 0.05), and a loss of redox capacity (0.72 +/- 0.04 vs. 0.64 +/- 0.02; p< 0.01). Restoration of Bcl-xL levels through transactivating regulatory protein-mediated protein transduction prevented the decline in psi mitochondrial reductase activity and cytosolic adenosine triphosphate. CONCLUSIONS: Anti-erbB2 activates the mitochondrial apoptosis pathway through a previously undescribed modulation of Bcl-xL and -xS, causing impairment of mitochondrial function and integrity and disruption of cellular energetics.

PMID: 15582322 [PubMed - indexed for MEDLINE]

As you see it affects the mitochondria in several ways (and these may be only some of even more that aren't described yet!) and I am not sure they understand coQ' s effects well enough to know if it reverses or potentiates
these effects. Plus this is in mice as most people don't allow cardiac biopsies just to study the effects of drugs on their bodies. Many drugs and natural compounds have different effects when given in different doses (sometimes opposite effects at high and low doses). So you are getting into a lot of unknowns. I encourage others who have researched more on coQ to send in their links--but I can assure you, there is no simple answer when so much is unknown.

PS I have yet to find the article Dr. Slamon used in his talks but will try to send it on when I do

[Lani]

It discusses how tumors (and in this case her2 breast cancer) when faced with lack of oxygen because they overgrow their blood supply change their metabolism to "burn" glucose for energy without oxygen instead of with oxygen. The article discusses that reactive oxygen species/antioxidants
might actually select for those cells which don't need oxygen and encourage growth of those cancer cells which don't need oxygen to utilize glucose and grow. Another "wrinkle" in the dilemna of what to do!

Here is the info on the study. The full article is in my opinion a milestone
in thinking about how to treat cancer--


Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance


Valeria R. Fantin1, 3, 4, Julie St-Pierre2, 3, 5 and Philip Leder1, ,

1Department of Genetics, Harvard Medical School and Howard Hughes Medical Institute, Boston, Massachusetts 02115
2Dana-Farber Cancer Institute and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115

Received 21 November 2005; revised 27 February 2006; accepted 24 April 2006. Published: June 12, 2006. Available online 12 June 2006.


Referred to by: Cancer's sweet tooth, Cancer Cell, Volume 9, Issue 6, 13 June 2006, Pages 419-420
Thi Bui1 and Craig B. Thompson1, ,
SummaryPlus | Full Text + Links | PDF (202 K)

[Lani]

1: J Clin Oncol. 2005 Nov 1;23(31):7820-6. Links

Reversibility of trastuzumab-related cardiotoxicity: new insights based on clinical course and response to medical treatment.

Ewer MS, Vooletich MT, Durand JB, Woods ML, Davis JR, Valero V, Lenihan DJ.

Department of Cardiology, The University of Texas MD Anderson Cancer Center, Houston, 77030, USA. mewer@mdanderson.org

PURPOSE: Trastuzumab is an important biologic agent with significant activity in breast cancers that overexpress the HER2/neu marker. However, trastuzumab is associated with cardiotoxicity that has not yet been fully explored. We present our experience with patients who developed trastuzumab-related cardiotoxicity. PATIENTS AND METHODS: Over a 4-year period, 38 patients with HER2/neu-positive breast cancer were referred for suspected trastuzumab-related cardiotoxicity. All patients had previously received anthracycline-based chemotherapy. Results After doxorubicin but before trastuzumab, the mean (+/- standard deviation) left ventricular ejection fraction (LVEF) was 0.61 +/- 0.13, and the LVEF decreased to 0.43 +/- 0.16 after trastuzumab (P < .0001). After withdrawal of trastuzumab, the LVEF increased to 0.56 +/- 0.11. Mean time to recovery of LVEF was 1.5 months and was temporally associated with medical treatment in 32 (84%) of the 38 patients but occurred without treatment in six patients (16%). Increases in LVEF were noted in 37 of the 38 patients. Twenty-five of these patients were re-treated with trastuzumab; three patients had recurrent left ventricular dysfunction, but 22 patients (88%) did not. All re-treatment patients continued on their therapeutic regimen for heart failure when rechallenged with trastuzumab. Nine patients underwent endomyocardial biopsy. Ultrastructural changes were not seen. CONCLUSION: Patients who develop cardiotoxicity while receiving trastuzumab therapy generally improve on removal of the agent. The mechanism of trastuzumab-related cardiac dysfunction is different from that of anthracycline cardiotoxicity, in part, demonstrated by the absence of anthracycline-like ultrastructural changes. Reintroducing trastuzumab may be appropriate for some individuals who previously have experienced trastuzumab-related cardiac dysfunction.

PMID: 16258084 [PubMed - indexed for MEDLINE]

"ultrastructural changes" refers to findings such as changes in subcellular organelles ( including abnormal appearance of mitochondria)