Yang Lü

We previously reported that anti-epidermal growth factor (EGF) receptor monoclonal antibody (mAb) 225 can block receptor activation and inhibit proliferation of tumor cells bearing EGF receptors. To further explore the mechanism of mAb-mediated growth inhibition, we compared the capacities of bivalent 225 mAb and 225 F(ab')2, and monovalent 225 Fab' fragment to block ligand binding to EGF receptors, inhibit activation of receptor tyrosine kinase by exogenous and endogenous ligand, produce receptor dimerization, down-regulate receptors, and inhibit proliferation of cultured A431 squamous carcinoma cells. Unlike 225 mAb and 225 F(ab')2, 225 Fab' fragment was a poor inhibitor of A431 cell proliferation. The weak antiproliferative capacity of 225 Fab' was not due to depletion of active fragment from cultures. When cells were exposed to exogenous EGF, monovalent 225 Fab' remaining in conditioned culture medium could act as well as the bivalent forms of mAb to block binding and tyrosine kinase activation by exogenous EGF. However, unlike the bivalent forms, 225 Fab' fragment was unable to induce receptor dimerization and down-regulation, and it lacked the capacity to block autocrine activation of EGF receptors by endogenous ligand. These deficiencies were corrected by addition of rabbit anti-mouse IgG antibody, which also enabled 225 Fab' fragment to inhibit cell proliferation. We conclude that in A431 cells, inhibition of autocrine-stimulated proliferation by anti-EGF receptor mAbs requires antibody bivalency, which provides the capacity to produce EGF receptor dimerization accompanied by receptor down-regulation. These properties may explain the greater efficacy of bivalent mAb and F(ab')2, compared with monovalent Fab' fragment, in inhibiting proliferation of a variety of malignant and nonmalignant cultured cell lines.

HER2, a member of the human epidermal growth factor (EGF) receptor family, not only plays important roles in the progression of breast cancer tumorigenesis and metastasis, but may protect cancer cells from conventional cytotoxic therapies as well. In the current study, we evaluated the effect of targeting HER2 on radiosensitization of human breast cancer cells. Using six breast cancer cell lines with various levels of HER2 (BT474, SKBR3, MDA453, MCF7, ZR75B, and MDA468), we found that trastuzumab (Herceptin), a humanized monoclonal antibody that may inhibit breast cancer cell proliferation but does not induce apoptosis when used alone, enhanced radiation-induced apoptosis of the cells in a HER2 level-dependent manner. We furthered this study in MCF7 cells transfected for high levels of HER2 (MCF7HER2). Compared with parental or control vector-transfected MCF7 cells, MCF7HER2 cells showed increased phosphorylation of at least two important HER2 downstream molecules, protein kinase B/Akt and mitogen-activated protein kinase (MAPK), and increased resistance to radiotherapy, as shown by reduced induction of apoptosis and increased cell clonogenic survival after radiation. Exposure of the cells to trastuzumab down-regulated the levels of HER2 and reduced phosphorylation levels of Akt and MAPK in MCF7HER2 cells, and sensitized these cells to radiotherapy. When specific inhibitors of the phosphatidylinositol 3-kinase (PI3-K) and MAPK kinase (MEK) pathways were used, we found that exposure of MCF7HER2 cells to the PI3-K inhibitor LY294002 inhibited Akt phosphorylation and radiosensitized the cells, whereas the radiosensitization effect by the MEK inhibitor PD98059 was relatively weaker, albeit the phosphorylation of MAPK was reduced by PD98059 treatment. Our results indicate that the PI3-K pathway might be the major pathway for trastuzumab-mediated radiosensitization of breast cancer cells.