50P - Application and mechanism of tarloxotinib in HER2-positive breast cancer

Background

HER2 (Human epidermal growth factor receptor-2)-positive breast cancer occupies about 15%–20% of all breast cancer, the current treatment of which is still based on surgery and adjuvant targeted therapy with monoclonal antibody. Problems of drug resistance and cardiotoxicity hinder the use of monoclonal antibodies. And, TKIs (Tyrosine Kinase Inhibitors) have the advantages of multi-target, reduced drug resistance and lower cardiotoxicity. Tarloxotinib is a prodrug that harnesses tumor hypoxia to generate high levels of a potent, covalent pan-HER TKI, Tarloxotinib-effector (Tarloxotinib-E), within tumor microenvironment. This tumor-selective delivery mechanism was designed to minimize the dose-limiting toxicities.

Methods

HER2-positive breast cancer BT-474, SK-BR-3, HCC-1954 and JIMT-1 cells were treated with different concentrations of Tarloxotinib-E, Lapatinib and Tucatinib. The IC50 values were determined by CCK-8 assay. Then, we used Western Blot to detect the changes in the phosphorylation of EGFR, HER2, HER3, HER4, AKT and ERK and the protein expression of Cleaved Caspase-3, Caspase-7 and Parp1. Flow cytometry was used to determine the apoptosis rates and mitochondrial membrane potentials, and the mitochondrial superoxide was detected by the Immunofluorescence. Importantly, we used HPLC-TQ-MS to determine the ratio of Tarloxotinib-E released by Tarloxotinib. Finally, the subcutaneous xenograft mice were used to evaluate the antitumor effect and toxicity of Tarloxotinib in vivo.

Results

Tarloxotinib could be transformed into Tarloxotinib-E by breast cancer cells under hypoxia. Meanwhile, Tarloxotinib-E could effectively inhibit the level of HER family phosphorylation and downstream signaling in HER2-positive breast cancer cells, and superior to Lapatinib and Tucatinib. Mechanistically, Tarloxotinib-E induced apoptosis in HER2-positive breast cancer cells through a ROS-dependent manner, with significant decrease in the potential of mitochondrial membrane and increase in mitochondrial superoxide. During the in vivo experiment, Tarloxotinib significantly inhibited tumor growth in nude mice compared with Lapatinib.

Conclusions

Tarloxotinib released Tarloxotinib-E under hypoxic microenvironment of breast tumors, and inhibited the phosphorylation of HER2 dimers and downstream pathways to induce apoptosis in HER2-positive cells through a ROS-dependent manner. This lays a foundation for the further development of Tarloxotinib in HER2-positive breast cancer.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

Nanjing Municipal Science and Technology Bureau Grant the Medical Engineering Cooperation Project No. 202110032.The Second Hospital of Nanjing Grant the Talent Support Program No. RCMS23004 and the High-level Talents Program No. 202302. Key Research Program of Gusu School No. GSKY20220105.The Postgraduate Research and Practice Innovation Program of Jiangsu Province No. KYCX22_1927.

Disclosure

All authors have declared no conflicts of interest.