Yamashita-Kashima Yoriko, Shu Sei, Osada Masahiro, Fujimura Takaaki, Yoshiura Shigeki, Harada Naoki, Yoshimura Yasushi
Product Research Department, Chugai Pharmaceutical Co., Ltd., 200 Kajiwara, Kamakura, Kanagawa, 247-8530, Japan.
Cancer Chemother Pharmacol. 2020 Nov;86(5):641-654. doi: 10.1007/s00280-020-04138-5. Epub 2020 Sep 30.
Trastuzumab emtansine (T-DM1) is the standard treatment in the current second-line therapy of human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. However, a useful therapy after T-DM1 resistance has not been established. In this study, we established two different HER2-positive T-DM1-resistant cancer cells and evaluated the antitumor effect of trastuzumab in combination with pertuzumab (TRAS + PER).
Single-cell-cloned OE19 and BT-474 cells were cultured with increasing concentrations of T-DM1 to generate T-DM1-resistant OE19bTDR and BT-474bTDR cells, respectively. HER2 expression was assessed by immunohistochemistry. Multidrug resistance proteins (MDR1 and MRP1) were evaluated by real-time polymerase chain reaction and western blotting. Intracellular trafficking of T-DM1 was examined by flow cytometry and immunofluorescence staining. Efficacy of TRAS + PER was evaluated by cell proliferation assay, HER3 and AKT phosphorylation, caspase 3/7 activity, and antitumor activity.
HER2 expression of both resistant cells was equivalent to that of the parent cells. Overexpression of MDR1 and MRP1 was observed and affected the T-DM1 sensitivity in the OE19bTDR cells. Abnormal localization of T-DM1 into the lysosomes was observed in the BT-474bTDR cells. In BT-474bTDR cells, TRAS + PER inhibited the phosphorylation of AKT involved in HER2-HER3 signaling, and apoptosis induction and cell proliferation inhibition were significantly higher with TRAS + PER than with the individual drugs. TRAS + PER significantly suppressed tumor growth in the OE19bTDR xenograft model compared with each single agent.
The results suggest that the TRAS + PER combination may be effective in T-DM1-resistant cancer cells where HER2 overexpression is maintained.
曲妥珠单抗-恩美曲妥珠单抗（T-DM1）是目前人表皮生长因子受体2（HER2）阳性转移性乳腺癌二线治疗的标准疗法。然而，T-DM1耐药后的有效治疗方法尚未确立。在本研究中，我们建立了两种不同的HER2阳性T-DM1耐药癌细胞，并评估了曲妥珠单抗联合帕妥珠单抗（TRAS+PER）的抗肿瘤作用。
将单细胞克隆的OE19和BT-474细胞用浓度递增的T-DM1培养，分别生成T-DM1耐药的OE19bTDR和BT-474bTDR细胞。通过免疫组织化学评估HER2表达。通过实时聚合酶链反应和蛋白质印迹法评估多药耐药蛋白（MDR1和MRP1）。通过流式细胞术和免疫荧光染色检测T-DM1的细胞内转运。通过细胞增殖试验、HER3和AKT磷酸化、半胱天冬酶3/7活性以及抗肿瘤活性评估TRAS+PER的疗效。
两种耐药细胞的HER2表达均与亲本细胞相当。观察到MDR1和MRP1过表达，且影响了OE19bTDR细胞对T-DM1的敏感性。在BT-474bTDR细胞中观察到T-DM1异常定位到溶酶体中。在BT-474bTDR细胞中，TRAS+PER抑制了HER2-HER3信号通路中涉及的AKT磷酸化，与单药相比，TRAS+PER诱导的凋亡和细胞增殖抑制显著更高。与每种单一药物相比，TRAS+PER在OE19bTDR异种移植模型中显著抑制肿瘤生长。
结果表明，TRAS+PER联合用药可能对维持HER2过表达的T-DM1耐药癌细胞有效。
