帕妥珠单抗(Perjeta®),与HER2(人表皮生长因子受体2)的二聚化臂结合的人源化抗体,作为HER2过表达的恶性肿瘤的单一治疗剂失败。由于HER2与配体结合的EGFR(表皮生长因子受体)的分子相互作用已暗示有丝分裂信号传导和恶性增殖,我们假设这种相互作用,而不是单独的HER2表达和寡聚化,可能是帕妥珠单抗治疗疗效的潜在分子靶标和预测因子。因此,我们使用Förster共振能量转移(FRET)显微镜和荧光相关和交叉相关光谱(FCS/FCCS)研究了HER2+EGFR+SK-BR-3乳腺肿瘤细胞中存在和不存在帕妥珠单抗时,EGF刺激下HER2和EGFR分子之间的静态和动态相互作用.通过蛋白质印迹和MTT测定测量信号传导的相应激活和细胞增殖的变化。HER2扩散的自相关函数最好通过校正三重态形成的三分量模型拟合,在这些成分中,缓慢扩散的膜成分揭示了EGFR配体结合诱导的聚集,如光子计数直方图和共扩散分数所证明。帕妥珠单抗治疗有效地预防了这种聚集,这也抑制了刺激后EGFR和HER2的相互作用,如通过FRET效率的变化监测。总的来说,数据表明,帕妥珠单抗,通过阻碍EGFR和HER2之间的刺激后相互作用,抑制EGFR诱发的HER2聚集和磷酸化,并导致细胞增殖的剂量依赖性降低,特别是当存在较高量的EGF时。因此,我们提出,在预测帕妥珠单抗治疗结果时,可以将HER2阳性肿瘤上的EGFR表达作为一个潜在的生物标志物加以考虑.
Pertuzumab (Perjeta®), a humanized antibody binding to the dimerization arm of HER2 (Human epidermal growth factor receptor-2), has failed as a monotherapy agent in HER2 overexpressing malignancies. Since the molecular interaction of HER2 with ligand-bound EGFR (epidermal growth factor receptor) has been implied in mitogenic signaling and malignant proliferation, we hypothesized that this interaction, rather than HER2 expression and oligomerization alone, could be a potential molecular target and predictor of the efficacy of pertuzumab treatment. Therefore, we investigated static and dynamic interactions between HER2 and EGFR molecules upon EGF stimulus in the presence and absence of pertuzumab in HER2+ EGFR+ SK-BR-3 breast tumor cells using Förster resonance energy transfer (FRET) microscopy and fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS). The consequential activation of signaling and changes in cell proliferation were measured by Western blotting and MTT assay. The autocorrelation functions of HER2 diffusion were best fitted by a three-component model corrected for triplet formation, and among these components the slowly diffusing membrane component revealed aggregation induced by EGFR ligand binding, as evidenced by photon-counting histograms and co-diffusing fractions. This aggregation has efficiently been prevented by pertuzumab treatment, which also inhibited the post-stimulus interaction of EGFR and HER2, as monitored by changes in FRET efficiency. Overall, the data demonstrated that pertuzumab, by hindering post-stimulus interaction between EGFR and HER2, inhibits EGFR-evoked HER2 aggregation and phosphorylation and leads to a dose-dependent decrease in cell proliferation, particularly when higher amounts of EGF are present. Consequently, we propose that EGFR expression on HER2-positive tumors could be taken into consideration as a potential biomarker when predicting the outcome of pertuzumab treatment.