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The effects of trastuzumab on HER2-mediated cell signaling in CHO cells expressing human HER2

Hamid Maadi¹, Babak Nami¹, Junfeng Tong¹

¹Department of Medical Genetics, and Signal Transduction Research Group, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, T6G 2H7, Canada.

|March 2, 2018

Abstract

Background:

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.

Methods:

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.

Results:

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.

Conclusion:

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.

Insights

Trastuzumab

Area of Science:

Oncology
Molecular Biology
Immunology

Background:

Trastuzumab is a key therapy for HER2-positive breast cancer, but its precise mechanism of action remains unclear.
Previous studies on trastuzumab's effects are often confounded by the diverse HER receptor expression profiles of different breast cancer cell lines.
A standardized cell model is needed to isolate the effects of trastuzumab on individual HER receptors.

Purpose of the Study:

To investigate the specific anti-cancer mechanisms of trastuzumab in a controlled cellular environment.
To elucidate whether trastuzumab's action is mediated by inhibiting HER2 signaling pathways or through other mechanisms like antibody-dependent cellular cytotoxicity (ADCC).

Main Methods:

Utilized Chinese Hamster Ovary (CHO) cell lines engineered to express single human HER receptors: EGFR, HER2, or HER3.
Conducted cytotoxicity assays, immunoblotting, indirect immunofluorescence, cross-linking studies, and ADCC assays.
Examined the effects of trastuzumab on HER receptor homodimerization, phosphorylation, and downstream signaling pathways (ERK, AKT).

Main Results:

Trastuzumab specifically bound to HER2 with high affinity but did not bind to EGFR or HER3.
In HER2-expressing cells (CHO-K6), trastuzumab did not inhibit HER2 homodimerization or phosphorylation, nor did it affect ERK/AKT phosphorylation or cell proliferation.
Trastuzumab demonstrated potent antibody-dependent cellular cytotoxicity (ADCC) in CHO-K6 cells.

Conclusions:

In the absence of other HER receptors, trastuzumab's primary anti-tumor effect is mediated by ADCC.
The findings suggest that inhibiting HER2 homodimerization and phosphorylation may not be the principal mechanism of trastuzumab's efficacy in this context.

Read full analysis

Keywords:

ADCC CHO cells Dimerization EGFR HER receptors HER2 HER3 Phosphorylation Trastuzumab

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