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

Abstract

BackgroundTargeted 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.MethodsTo 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.ResultsWe 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.ConclusionWe 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.

Mechanisms Underlying the Action and Synergism of Trastuzumab and Pertuzumab in Targeting HER2-Positive Breast Cancer

“…The Fc receptor on immune effector cells, principally natural-killer (NK) cells, recognizes the Fc portion of trastuzumab in the targeted cancer cell and attacks the cancer cells. Action through ADCC has been supported by much evidence as a major mechanism of trastuzumab [ 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 ]. This mechanism is also supported by recent studies [ 101 , 104 ].…”

“…In our recent study with Chinese hamster ovary (CHO) cells stably expressing only HER2 (no other HER receptors), HER2 massively forms homodimers due to overexpression. However, trastuzumab does not inhibit the formation of HER2 homodimers [ 104 ]. In addition, trastuzumab does not block the phosphorylation of any of the major C-terminal tyrosine residues.…”

“…In addition, trastuzumab does not block the phosphorylation of any of the major C-terminal tyrosine residues. However, trastuzumab stimulates strong ADCC in the cells [ 104 ]. Thus, it is likely that pertuzumab, rather than trastuzumab, also inhibits the homodimerization of HER2.…”

Epigenetic downregulation of HER2 during EMT leads to tumor resistance to HER2-targeted therapies in breast cancer

“…Targeting HER2 by small molecule inhibitors and monoclonal antibodies is the current therapy for HER2-positive breast cancer that outcome significant tumor regression in the patients [11,12]. Lapatinib, a small molecule dual inhibitor of tyrosine kinase activity of HER2 and EGFR, and trastuzumab (Herceptin) and pertuzumab (Perjeta) which are anti-HER2 humanized monoclonal antibodies targeting ECD of HER2 approved by FDA to treat patients with early-stage and metastatic HER2-positive breast cancer as an adjuvant in combination with taxane therapy [11,13,14].…”

Trastuzumab in der Behandlung schwangerer Mammakarzinompatientinnen – ein Literaturüberblick

“…Es werden Wirkmechanismen sowohl auf extrazellulärer als auch auf intrazellulärer Ebene diskutiert. Auf extrazellulärer Ebene beruht die antineoplastische Wirkweise des Antikörpers nach derzeitiger Datenlage insbesondere auf zellvermittelter Zytotoxizität [38,39]. Die intrazellulären Wirkmechanismen von Trastuzumab werden hingegen kontrovers beschrieben [31].…”