Cetuximab is a monoclonal antibody developed to inhibit the binding of growth factors and the subsequent activation of epidermal growth factor receptor (EGFR). Triple-negative breast cancer (TNBC) is resistant to cetuximab treatment. The aim of the present study was to examine the partial agonistic properties of cetuximab, which not only blocks ligand binding, but also partially triggers EGFR activation, which may lead to cetuximab resistance in TNBC. The phosphorylation of growth factor receptors and their signalling pathways were evaluated by determining the phosphorylation of EGFR, insulin-like growth factor receptor (IGF-1R), vascular endothelial growth factor receptor (VEGFR)-2, Src kinase, phosphoinositide-3-kinase (PI3K), extracellular signal-regulated kinase (ERK1/2) and serine/threonine-specific protein kinase (Akt) and the degradation of EGFR, and by assessing the morphology and proliferation of MDA-MB-231 and MDA-MB-468 cells. Cetuximab treatment led to the phosphorylation of EGFR, VEGFR-2, IGF-1R and downstream signalling molecules, Src kinase and PI3K in these cells, as well as Akt in the MDA-MB-231 cells. The cetuximab-mediated phosphorylation of IGF-1R, VEGFR-2 and Akt was inhibited by the EGFR kinase inhibitor, AG1478, and the Src kinase inhibitor, PP2. Cetuximab treatment led to the degradation of EGFR. The cetuximab-induced phosphorylation and EGFR degradation were less prominent compared with those induced by EGF. Cetuximab partially inhibited EGF-mediated responses. Cetuximab, similar with EGF, altered cellular morphology in a serum-free medium. In both cell lines, the Src kinase inhibitor enhanced the cetuximab-induced anti-proliferative response. These results indicate that cetuximab exerts a partial agonistic effect on EGFR, which activates Src kinase and subsequently transactivates IGF-1R and VEGFR-2. This partial agonistic property is likely one of the mechanisms underlying the resistance of TNBC to cetuximab.

Triple negative breast cancer cell lines express high levels of β2-adrenergic receptor, which have a significant influence on the activity of extracellular signal‑regulated kinase (ERK)1/2. Therefore, it is important to understand the link between β2‑adrenergic receptor signaling and ERK1/2 activity in terms of cancer cell regulation and cancer progression. Although the molecular mechanisms are not completely clarified, β2‑adrenergic receptor stimulation appears to reduce the basal levels of phosphorylated (p)ERK1/2 in MDA‑MB‑231 breast cancer cells. The aim of the current study was to determine the mechanism of β2‑adrenergic receptor‑mediated ERK1/2 dephosphorylation by investigating the role of dual‑specificity phosphatase (DUSP)1/6 and protein phosphatase (PP)1/2, which are established regulators of ERK1/2 phosphorylation, in MDA‑MB‑231 and MDA‑MB‑468 breast cancer cell lines. (E)‑2‑benzylidene‑3‑(cyclohexyl​amino)‑2,3‑ dihydro‑1H‑inden‑1‑one (BCI) and calyculin A were employed as DUSP1/6 and PP1/PP2 inhibitors, respectively. Subsequently, the protein levels of DUSP1, PP1, pPP1, ERK1/2 and pERK1/2 were measured by western blot analysis. Cells were transfected with DUSP1 small interfering (si)RNA or PP1 siRNA to inhibit their expression. The results demonstrated that β2‑adrenergic receptor agonists led to the dephosphorylation of basal pERK1/2 in MDA‑MB‑231 and MDA‑MB‑468 cells. The DUSP1/6 inhibitor, BCI, and the PP1/PP2 inhibitor, calyculin A, antagonized the β2‑adrenergic receptor‑mediated dephosphorylation of ERK1/2. Furthermore, β2‑adrenergic receptor stimulation increased the protein expression level of DUSP1, with no effects on DUSP6, PP1 and PP2 expression, and enhanced the expression of the active form of PP1. Downregulation of the expression of DUSP1 or PP1 led to a decline in the β2‑adrenergic receptor‑mediated dephosphorylation of ERK1/2. The results of the present study indicate that β2‑adrenergic receptor‑mediated dephosphorylation of ERK1/2 may be associated with the activity of DUSP1 and PP1 in MDA‑MB‑231 and MDA‑MB‑468 triple negative breast cancer cell lines. The clinical importance of β2‑adrenergic receptor‑mediated inactivation of ERK1/2 as well as the activation of DUSP1 and PP1 should be carefully evaluated in future studies, particularly when β2‑adrenergic blockers are used in patients with triple negative breast cancer.