Original articleIn vitro effects of Trastuzumab Emtansine (T-DM1) and concurrent irradiation on HER2-positive breast cancer cells
Introduction
Anti-HER2 (Human Epidermal Growth factor Receptor 2) drugs have become standard treatment for HER2-positive breast cancer patients and are routinely used in adjuvant and neoadjuvant therapy, and for metastatic disease [1], [2]. Trastuzumab emtansine (T-DM1, Kadcyla®), is an ADC (Antibody-drug conjugate) which combines trastuzumab (an anti-HER2 monoclonal antibody) covalently linked via a non-reducible linker to the maytansinoid DM1, a powerful mitotic spindle inhibitor [3]. T-DM1 improves overall survival in patients with HER2-positive metastatic breast cancer [4], [5], [6]. T-DM1 binds the extracellular domain of HER2 via the trastuzumab component and enters into the cell. The HER2-T-DM1 complex is then internalized and cleaved by lysosomal degradation, allowing intracytoplasmic release of the cytotoxic agent DM1, a potent inhibitor of tubulin polymerization [7].
In vitro, down-regulation or over-expression of HER2 in breast cancer cells induces changes in the radio-response. In HER2 transfected cells, the mechanisms of radioresistance are complex and have not been fully elucidated, but the PI3-K/Akt (Phosphatidylinositol-3-Kinase/Protein kinase B) pathway appears to play a major role in radioresistance by deregulating the cell cycle, accelerating DNA repair mechanisms, leading to resistance to apoptosis [8], [9], [10], [11], [12].
It has been previously reported that anti-HER2 therapies, and more specifically Trastuzumab and Lapatinib (inhibitor of tyrosine kinase activity in the intracellular domain of HER2), are specific radiosensitizing agents for HER2-positive cells [8], [9], [13]. These findings have led clinicians to deliver radiation concurrently with these anti-HER2 drugs [14].
We therefore conducted an in vitro preclinical study in HER2-positive breast cell lines to evaluate the potential radiosensitizing effect of T-DM1.
Section snippets
Cell lines
Five HER2-positive cell lines expressing different levels of HER2 amplification and one triple-negative (MDA-MB-231) human breast cancer cell line (TNBC) were used in this study (see characterics of these cell lines in supplementary table TS1). All cell lines, originally derived from the ATCC®, were kindly provided by Drs L. De Koning and T. Dubois (Institut Curie). Cells were routinely subcultured every 5 days and grown (37 °C, 5% CO2) as monolayers in RPMI (HCC1954 and ZR-75-1), DMEM/F12
T-DM1: a long and major cytotoxic effect depending on the level of HER2 expression (except for BT474)
The HER2 protein expression of cells lines was first demonstrated by western blots, confirming different levels of HER2 expression for the various cell lines (Fig. 1A and B). Cell lines were classified as follows: high (HER2 3+: HCC1954, SKBr3, BT474), moderate (HER2 2+: MDA-MB-453) and low (HER2 1+: ZR-75-1) HER2 expression. The triple-negative cell line MDA-MB-231 was used as negative control. The three HER2 3+ cell lines showed approximately the same levels of HER2. HER2 expression by
Discussion
This in vitro study provides three take home messages: The first is that T-DM1 presents a delayed toxic effect on HER-2 positive cell lines. The second is the linear correlation between intrinsic HER2-positive status and radioresistance (D10). The third is that concurrent combination of T-DM1 allowing cell survival with radiation does not radiosensitize HER2-positive breast cancer cells.
Despite improvement of the outcome associated with the use of anti-HER2 drugs, between 10 and 15% of patients
Conclusion
Our work indicated that:
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within the first 24 hour-incubation time, the main determining factor is the presence of HER-2 receptors: the higher, the better (except for BT-474). For the moderate and low HER-2 expressing cell lines, T-DM1 uptake increased more slowly over time;
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the G2/M blockade observed on the triple-negative (HER-2 negative) cell line leads us to think about a HER-2 independent trapping for T-DM1, even if we have no clear evidence of this mechanism;
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the effects of T-DM1 on BT-474
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Disclosure of interest
The authors declare that they have no competing interest.
Funding
Academic study. This work was supported by financial aid from the Institut Curie, INSERM and CNRS.
Authors’ contributions
FM and FMC conducted the experiments. FM and FMC analyzed the data. PV, MPTF and YK read and approved the final manuscript.
Acknowledgements
The authors thank Genetech, USA Inc. (San Francisco, CA) for providing T-DM1 under Material Transfer Agreement (# OR-215963) and Drs Nora Ady-Vago and Fanny Bouquet (Roche Institute, France) for their help and continuous support. Members of the Institut Curie RadeXp platform is acknowledged. We want to thank Emilie Brun (Institut Curie, Genomics Platform) for DNA sequencing, Leanne de Koning (Institut Curie, RPPA Platform) and Thierry Dubois (Institut Curie, Breast Cancer Biology Group,
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