Effects of trastuzumab and afatinib on kinase activity in gastric cancer cell lines

Abstract

The molecular mechanism of action of the HER2‐targeted antibody trastuzumab is only partially understood, and the direct effects of trastuzumab on the gastric cancer signaling network are unknown. In this study, we compared the molecular effect of trastuzumab and the HER kinase inhibitor afatinib on the receptor tyrosine kinase (RTK) network and the downstream‐acting intracellular kinases in gastric cancer cell lines. The molecular effects of trastuzumab and afatinib on the phosphorylation of 49 RTKs and 43 intracellular kinase phosphorylation sites were investigated in three gastric cancer cell lines (NCI‐N87, MKN1, and MKN7) using proteome profiling. To evaluate these effects, data were analyzed using mixed models and clustering. Moreover, proliferation assays were performed. Our comprehensive quantitative analysis of kinase activity in gastric cancer cell lines indicates that trastuzumab and afatinib selectively influenced the HER family RTKs. The effects of trastuzumab differed between cell lines, depending on the presence of activated HER2. The effects of trastuzumab monotherapy were not transduced to the intracellular kinase network. Afatinib alone or in combination with trastuzumab influenced HER kinases in all cell lines; that is, the effects of monotherapy and combination therapy were transduced to the intracellular kinase network. These results were confirmed by proliferation analysis. Additionally, the MET‐amplified cell line Hs746T was identified as afatinib nonresponder. The dependence of the effect of trastuzumab on the presence of activated HER2 might explain the clinical nonresponse of some patients who are routinely tested for HER2 expression and gene amplification in the clinic but not for HER2 activation. The consistent effects of afatinib on HER RTKs and downstream kinase activation suggest that afatinib might be an effective candidate in the future treatment of patients with gastric cancer irrespective of the presence of activated HER2. However, MET amplification should be taken into account as potential resistance factor.

Caveolin-1 mediates cellular distribution of HER2 and affects trastuzumab binding and therapeutic efficacy

“…1b ). These observations are consistent with recent reports of HER2-positive gastric tumors having different HER2 densities at the cell membrane 38 . In cancer cells where CAV1 is absent, HER2 is exclusively present at the cell membrane (Fig.…”

Temporal Modulation of HER2 Membrane Availability Increases Pertuzumab Uptake and Pretargeted Molecular Imaging of Gastric Tumors

“…However, clinical studies have reported that 89 Zrlabeled antibodies do not always accumulate in HER2-positive tumors (25). Immunohistochemical staining of gastric tumors reveals nonuniform membrane expression of HER2 (15), which contributes to low accumulation of antibodies in these tumors (18,26,27). Moreover, endocytic trafficking mediates HER2 internalization and further reduces the availability of HER2 at the cell membrane, preventing binding with antibodies such as trastuzumab and pertuzumab and dampening their therapeutic efficacy (27)(28)(29)(30).…”

EPHA2 blockade reverses acquired resistance to afatinib induced by EPHA2‐mediated MAPK pathway activation in gastric cancer cells and avatar mice

“…The therapeutic effect of trastuzumab in HER2-positive patients is due to the reduction of phosphorylated HER2 and a triggering of antibody-dependent cell-mediated cytotoxicity. 24 We and other researchers 25 demonstrated that in addition to the direct inhibition of ErbB receptor activation, afatinib inhibited downstream signaling pathways, including PI3K/AKT/mTOR and MAPK, in vitro and in vivo. Afatinib also induced apoptosis and cell cycle arrest at the G1 phase, a result consistent with the findings in esophageal squamous cell carcinoma and colorectal cancer.…”