Genes Affecting the Cell Cycle, Growth, Maintenance, and Drug Sensitivity Are Preferentially Regulated by Anti-HER2 Antibody through Phosphatidylinositol 3-Kinase-AKT Signaling

Abstract

The molecular mechanisms by which the anti-HER2 antibodies trastuzumab and its murine equivalent 4D5 inhibit tumor growth and potentiate chemotherapy are not fully understood. Inhibition of signaling through the phosphatidylinositol 3-kinase (PI3K)-AKT pathway may be particularly important. Treatment of breast cancer cells that overexpress HER2 with trastuzumab inhibited HER2-HER3 association, decreased PDK1 activity, reduced Thr-308 and Ser-473 phosphorylation of AKT, and reduced AKT enzymatic activity. To place the role of PI3K-AKT in perspective, gene expression was studied by using Affymetrix microarrays and real time reverse transcription-PCR. Sixteen genes were consistently down-regulated 2.0 -4.9-fold in two antibody-treated breast cancer cell lines. Fourteen of the 16 genes were involved in three major functional areas as follows: 7 in cell cycle regulation, particularly of the G 2 -M; 5 in DNA repair/replication; and 2 in modifying chromatin structure. Of the 16 antibody-regulated genes, 64% had roles in cell growth/maintenance and 52% contributed to the cell cycle. Direct inhibition of PI3K with an inhibitor markedly reduced expression of 14 genes that were also affected by the antibody. Constitutive activation of AKT1 blocked the effect of the anti-HER2 antibody on cell cycle arrest and on eight differentially expressed genes. The antibody enhanced docetaxel-induced growth inhibition but did not increase the fraction of apoptotic cells induced with docetaxel alone. In contrast, the antibody plus docetaxel markedly down-regulated two genes, HEC and DEEPEST, required for passage through G 2 -M. Thus, anti-HER2 antibody preferentially affects genes contributing to cell cycle progression and cell growth/maintenance, in part through the PI3K-AKT signaling. Transcriptional regulation by anti-HER2 antibody through PI3K-AKT pathway may potentiate the growth inhibitory activity of docetaxel by affecting cell cycle progression.The human epidermal growth factor receptor 2 (HER2, 1 also known as c-Neu or ErbB-2) encodes a 185-kDa transmembrane tyrosine kinase growth factor receptor. The ligand that binds to the homodimers of HER2 has not yet been identified. Rather, HER2 functions as a preferred co-receptor to form heterodimers with HER1 (epidermal growth factor receptor), HER3, or HER4. Of these heterodimers, HER2-HER3 is particularly important for intracellular signaling (1). HER2 signaling has been linked to a variety of cellular responses to growth factors under both normal and pathophysiological conditions. HER2 signaling is required not only during normal development of the mammary gland but also during development of the glia, neurons, and heart (1, 2). Amplification of the HER2 gene and overexpression of HER2 protein have been documented in ϳ30% of breast and 15% of ovarian cancers (3). In many (but not all) reports, HER2 overexpression has been associated with a more aggressive course of disease. Although the underlying mechanisms for this association are still not well characterized, HER2 overexpression...

HER2 signaling modulates the equilibrium between pro- and antiangiogenic factors via distinct pathways: implications for HER2-targeted antibody therapy

“…HER2 siRNA treatment of HER2-overexpressing BT474 cells also increases TSP-1 expression (Yang et al, 2004). Affymetrix microarray analysis indicated that treatment of BT474 cells with trastuzumab elevates TSP-1 expression (Le et al, 2005a). In this report, we showed that HER2 signaling closely regulated TSP-1 expression (Figure 3), which supports the notion that trastuzumab-induced TSP-1 expression (Figures 4 and 6) may contribute to tumor growth inhibition.…”

“…We next examined possible signaling pathways that could regulate expression of these angiogenic factors. One was PI3K-AKT, which is important for HER2 signaling, and inhibition of HER2 signaling with trastuzumab significantly inhibits PI3K-AKT signaling (Le et al, 2000(Le et al, , 2005a. It is also well documented that the PI3K-AKT pathway plays an important role in angiogenesis (Dimmeler and Zeiher, 2000;Jiang et al, 2000).…”

“…In other cell systems, delayed activation of the p38 MAP kinase pathway also has a negative impact on cell proliferation by inducing apoptosis (Daly et al, 1999;Takekawa et al, 2002;Bulavin and Fornace, 2004;Naito et al, 2004). Treatment of BT474 and SKBr3 cells with anti-HER2 antibodies for 72 h does not induce significant apoptosis (Le et al, 2003(Le et al, , 2005a. Instead, trastuzumab-induced TSP-1 expression (Figures 3 and 4), which was mediated by the delayed activation of p38 MAP kinase.…”

“…Generation of dominant-positive AKT1 stable transfectants SKBr3 subclones that stably express AKT1 were established as described previously (Le et al, 2005a).…”

“…Total RNA isolation and RT-PCR were performed according to the methods described previously (Le et al, 2005a). One internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and one gene of interest (VEGF, IL-8 or TSP-1) were analysed in the same tube.…”