ErbB-2 Blockade and Prenyltransferase Inhibition Alter Epidermal Growth Factor and Epidermal Growth Factor Receptor Trafficking and Enhance ¹¹¹In-DTPA-hEGF Auger Electron Radiation Therapy

Abstract

The intracellular distribution of Auger electron-emitting radiopharmaceuticals is a determinant of cytotoxicity. However, the mechanisms by which these agents are routed through the cell are ill understood. The aim of this study was to investigate how trafficking of 111 In-labeled human epidermal growth factor ( 111 In-DTPA-hEGF) relates to that of the EGF receptor (EGFR) and whether coadministration of agents that modulate EGFR signaling alters the efficacy of 111 In-DTPA-hEGF. Methods: The spatiotemporal interaction between AlexaFluor488-EGF (AF488-EGF) and Cy3-conjugated anti-EGFR antibody (Cy3-anti-EGFR) was studied in the breast cancer cell line MDA-MB-468 using fluorescence resonance energy transfer and 2-photon fluorescence lifetime imaging. 111 In internalization and nuclear fractionation assays were performed to investigate the effect of the ErbB-2-blocking antibody trastuzumab and a prenyltransferase inhibitor, L-778,123, on the subcellular localization of 111 In-DTPA-hEGF in MDA-MB-468 (1.3 · 10 6 EGFR per cell; ErbB-2 negative) and 231-H2N (0.2 · 10 6 EGFR per cell; 0.4 · 10 5 ErbB-2 per cell) cell lines. The cytotoxicity of 111 In-DTPA-hEGF (0-64 nM) plus trastuzumab (0-50 mg/mL) or L-778,123 (0-22.5 mM) was measured using clonogenic assays in a panel of breast cancer cell lines that express different levels of EGFR and ErB-2. Clonogenic survival data were used to calculate combination indices. Tumor growth inhibition was measured in vivo in 231-H2N xenograft-bearing mice treated with 111 In-DTPA-hEGF plus trastuzumab or L-788,123. Results: Using fluorescence resonance energy transfer, we showed that EGF interacts with EGFR in the cytoplasm and nucleus after internalization of the ligand-receptor complex in MDA-MB-468 cells. Nuclear localization of 111 In-DTPA-hEGF is enhanced by trastuzumab and L-788,123. Trastuzumab and L-788,123 sensitized 231-H2N cells to 111 In-DTPA-hEGF. Nuclear localization and cytotoxicity of 111 In-DTPA-hEGF were significantly increased in 231-H2N xenografts by cotreatment with L-788,123 (P , 0.0001). Conclusion: The therapeutic efficacy of 111 In-DTPA-hEGF is increased through the coadministration of selected molecularly targeted drugs that modulate EGFR signaling and trafficking.

Molecular and cellular effects of Auger emitters: 2008–2011

“…Reilly et al (2006) point out that the radiation-absorbed dose in the nucleus is about 15 times higher when 111 In decays in the nucleus than when it decays in the cytoplasm and about 30 times higher when 111 In decays in the nucleus than when it decays at the cell surface. Good correlation has been found between EGFR expression and 111 In-DTPA-hEGF uptake, nuclear localization and DNA damage and cell killing (Cai et al 2008) and convincing evidence for nuclear localization of 111 In-DTPA-hEGF was shown by the fl uorescence energy transfer (FRET) imaging study of Cornelissen et al (2011b). Further, 111 In-DTPA-hEGF was found to inhibit growth of a breast cancer cell xenograft in athymic mice.…”

“…Realizing the therapeutic potential of epidermal growth factor signaling in cancer therapy, Reilly ' s group is using another Trojan horse approach to deliver an attack to a cancer cell nucleus in the form of human epidermal growth factor (hEGF) complexed to the Auger emitter, 111 In via DTPA (Cornelissen et al 2011b). In their investigations, the Auger emitter labeled hEGF was developed to target EGFR-overexpressing cancer cells.…”

“…In their investigations, the Auger emitter labeled hEGF was developed to target EGFR-overexpressing cancer cells. As discussed by Cornelissen et al (2011b), upon binding of 111 In-DTPA-hEGF to its receptor, the EGFR, the ligand-receptor complex clusters in the plasma membrane and is internalized into the cell via clathrinreceptor mediated endocytosis. Endocytosis transports the internalized 111 In-DTPA-hEGF-EGFR to early and then late endosomes.…”

“…Drugs that enhance accumulation of Auger emitter radiopharmaceuticals in the nucleus are metholtrexate, gelfi tinib (Bailey et al 2007), trastuzumab (Cornelissen et al 2011b) and the prenyltransferase inhibitor, L-788,123 (Cornelissen et al 2011b).…”

“…Th e use of trastuzumab has been found to increase accumulation of 111 In-DTPA-hEGF in breast cancer cells (Cornelissen et al 2011b). Trastuzumab treatment alone has limited duration of eff ectiveness due to development of resistance.…”