Anti-HER2 Antibody Trastuzumab Inhibits CDK2-Mediated NPAT and Histone H4 Expression via PI3K Pathway

Abstract

Anti-HER2 antibody trastuzumab (Herceptin®) has been used effectively to treat patients with breast cancer that overexpresses HER2. We have demonstrated that p27Kip1 upregulation is one of the key events that cause G1 arrest upon trastuzumab treatment. Here, we have examined the effect of trastuzumab on CDK2, Rb, E2F1, NPAT, histone H4, and their associated signaling pathway. Results showed that trastuzumab was able to dramatically inhibit the kinase activity and expression of CDK2, whereas the kinase activity and expression of CDK4 were not affected by trastuzumab treatment. Unlike the p27Kip1 upregulation that occurs primarily through post-translational mechanisms, CDK2 was mainly downregulated at a transcriptional level as shown by Northern blotting and real-time RT-PCR analyses. Trastuzumab decreased the kinase activity of cyclin E (because of CDK2 inhibition) although it had little effect on cyclin E protein level. Overexpression of wild-type cyclin E or its lower molecular weight forms did not influence the response to trastuzumab. Levels and activities of CDK6, cyclin A, and cyclin D1 were all suppressed by trastuzumab. As a result, Rb phosphorylations that associates with CDK2, cyclin E, CDK6, cyclin A, or cyclin D1 were substantially inhibited by trastuzumab. Trastuzumab decreased the DNA-binding activity of E2F1. Trastuzumab decreased the level of NPAT protein. Trastuzumab decreased the level of histone H4 mRNA. Blockade of PI3K pathway with LY294002 produced similar effects to trastuzumab treatment. Taken together, these results demonstrated that anti-HER2antibody trastuzumab inhibits CDK2, Rb phosphorylation, E2F1 activity, NPAT, and histone H4 via PI3K signaling pathway, which are essential to both DNA and histone synthesis during the progression of G1 phase to S phase of the cell cycle.