Department of Medical Genetics, and Signal Transduction Research Group, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, T6G 2H7, Canada.
BMC Cancer. 2018 Mar 1;18(1):238. doi: 10.1186/s12885-018-4143-x.
Targeted therapy with trastuzumab has become a mainstay for HER2-positive breast cancer without a clear understanding of the mechanism of its action. While many mechanisms have been suggested for the action of trastuzumab, most of them are not substantiated by experimental data. It has been suggested that trastuzumab functions by inhibiting intracellular signaling initiated by HER2, however, the data are very controversial. A major issue is the different cellular background of various breast cancer cells lines used in these studies. Each breast cancer cell line has a unique expression profile of various HER receptors, which could significantly affect the effects of trastuzumab.
To overcome this problem, in this research we adopted a cell model that allow us to specifically examine the effects of trastuzumab on a single HER receptor without the influence of other HER receptors. Three CHO cell lines stably expressing only human EGFR (CHO-EGFR), HER2 (CHO-K6), or HER3 (CHO-HER3) were used. Various methods including cytotoxicity assay, immunoblotting, indirect immunofluorescence, cross linking, and antibody-dependent cellular cytotoxicity (ADCC) were employed in this research.
We showed that trastuzumab did not bind EGFR and HER3, and thus did not affect the homodimerization and phosphorylation of EGFR and HER3. However, overexpression of HER2 in CHO cells, in the absence of other HER receptors, resulted in the homodimerization of HER2 and the phosphorylation of HER2 at all major pY residues. Trastuzumab bound to HER2 specifically and with high affinity. Trastuzumab inhibited neither the homodimerization of HER2, nor the phosphorylation of HER2 at most phosphotyrosine residues. Moreover, trastuzumab did not inhibit the phosphorylation of ERK and AKT in CHO-K6 cells, and did not inhibit the proliferation of CHO-K6 cells. However, trastuzumab induced strong ADCC in CHO-K6 cells.
We concluded that, in the absence of other HER receptors, trastuzumab exerts its antitumor activity through the induction of ADCC, rather than the inhibition of HER2-homodimerization and phosphorylation.
曲妥珠单抗的靶向治疗已成为 HER2 阳性乳腺癌的主要治疗方法，但对其作用机制仍缺乏明确的认识。虽然已经提出了许多曲妥珠单抗作用的机制，但其中大多数都没有实验数据的支持。有人认为曲妥珠单抗通过抑制由 HER2 启动的细胞内信号而发挥作用，但数据存在很大争议。一个主要问题是这些研究中使用的各种乳腺癌细胞系具有不同的细胞背景。每个乳腺癌细胞系都具有各种 HER 受体的独特表达谱，这可能会显著影响曲妥珠单抗的作用。
为了克服这个问题，在这项研究中，我们采用了一种细胞模型，可以在没有其他 HER 受体影响的情况下，专门研究曲妥珠单抗对单个 HER 受体的影响。使用了三种稳定表达人表皮生长因子受体（CHO-EGFR）、HER2（CHO-K6）或 HER3（CHO-HER3）的 CHO 细胞系。本研究采用细胞毒性测定、免疫印迹、间接免疫荧光、交联和抗体依赖性细胞毒性（ADCC）等多种方法。
我们表明，曲妥珠单抗不与 EGFR 和 HER3 结合，因此不影响 EGFR 和 HER3 的同源二聚化和磷酸化。然而，在没有其他 HER 受体的情况下，CHO 细胞中 HER2 的过表达导致 HER2 的同源二聚化和 HER2 上所有主要磷酸酪氨酸残基的磷酸化。曲妥珠单抗特异性地、高亲和力地与 HER2 结合。曲妥珠单抗既不能抑制 HER2 的同源二聚化，也不能抑制大多数磷酸酪氨酸残基上 HER2 的磷酸化。此外，曲妥珠单抗不能抑制 CHO-K6 细胞中 ERK 和 AKT 的磷酸化，也不能抑制 CHO-K6 细胞的增殖。然而，曲妥珠单抗在 CHO-K6 细胞中诱导了强烈的 ADCC。
我们得出结论，在没有其他 HER 受体的情况下，曲妥珠单抗通过诱导 ADCC 发挥其抗肿瘤活性，而不是抑制 HER2 同源二聚化和磷酸化。
