The effects of trastuzumab on HER2-mediated cell signaling in CHO cells expressing human HER2.
The effects of trastuzumab on HER2-mediated cell signaling in CHO cells expressing human HER2.
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DOI：
10.1186/s12885-018-4143-x
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发表时间：
2018-03-01
期刊：
影响因子：
3.8
通讯作者：
Wang Z
中科院分区：
文献类型：
--
作者：
Maadi H;Nami B;Tong J;Li G;Wang Z
曲妥珠单抗靶向治疗已成为her2阳性乳腺癌的主要治疗手段，但对其作用机制尚不清楚。虽然曲妥珠单抗的作用机制被提出了许多，但其中大多数都没有得到实验数据的证实。有人认为曲妥珠单抗通过抑制HER2启动的细胞内信号传导发挥作用，然而，这些数据非常有争议。一个主要问题是这些研究中使用的各种乳腺癌细胞系的细胞背景不同。每个乳腺癌细胞系都有不同HER受体的独特表达谱，这可能会显著影响曲妥珠单抗的效果。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，使我们能够特异性地检查曲妥珠单抗对单个HER受体的作用，而不受其他HER受体的影响。使用三种仅稳定表达人EGFR （CHO-EGFR）、HER2 （CHO- k6）或HER3 （CHO-HER3）的CHO细胞系。本研究采用了细胞毒性测定、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。我们发现曲妥珠单抗不结合EGFR和HER3，因此不影响EGFR和HER3的同二聚化和磷酸化。然而，在缺乏其他HER受体的情况下，CHO细胞中HER2的过表达会导致HER2的同二聚化和HER2在所有主要pY残基上的磷酸化。曲妥珠单抗与HER2特异性和高亲和力结合。曲妥珠单抗既不能抑制HER2的同二聚化，也不能抑制HER2在大多数磷酸酪氨酸残基上的磷酸化。此外，曲妥珠单抗没有抑制CHO-K6细胞中ERK和AKT的磷酸化，也没有抑制CHO-K6细胞的增殖。然而，曲妥珠单抗在CHO-K6细胞中诱导强ADCC。我们得出结论，在缺乏其他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her2二聚体化和磷酸化。
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.
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