Modulation of p27/Cdk2 complex formation through 4D5-mediated inhibition of HER2 receptor signaling.

The molecular mechanisms mediating the anti-proliferative effects of the murine anti-HER2 monoclonal antibody (4D5) were investigated in HER2-overexpressing human carcinoma cell lines. Treatment with 4D5 resulted in a dramatic accumulation of BT-474 breast carcinoma cells in G1; concomitant with reduced expression of proteins involved in sequestration of the cyclin E/Cdk2 inhibitor protein p27, increased association of p27 with Cdk2 complexes and Cdk2 inactivation. No equivalent effects were observed in BT-474 cells treated with a control, non-inhibitory HER2 monoclonal antibody (FRP5) or in a HER2-overexpressing cell line insensitive to 4D5 treatment (MKN7 gastric carcinoma cells), confirming the relationship between these molecular changes and 4D5-mediated inhibition of proliferation. Increased p27 expression was also observed in 4D5-treated BT-474 cells; however an antisense approach demonstrated that this increase was not required for Cdk2 inactivation or establishment of the G1 block. These data suggest that 4D5 interferes with HER2 receptor signaling, resulting in downregulation of proteins involved in p27 sequestration. This causes release of p27, allowing binding and inhibition of cyclin E/Cdk2 complexes and inhibition of G1/S progression. This model was confirmed using a second 4D5-sensitive. HER2-overexpressing breast tumor line (SKBR3), and suggests that the dependency of a given tumor cell on elevated HER2-receptor signaling for the maintenance of p27 sequestration proteins may determine the clinical response to treatment with the humanized anti-HER2 monoclonal antibody Herceptin (trastuzumab).

ErbB2 potentiates breast tumor proliferation through modulation of p27(Kip1)-Cdk2 complex formation: receptor overexpression does not determine growth dependency.

Overexpression of the ErbB2 receptor, a major component of the ErbB receptor signaling network, contributes to the development of a number of human cancers. ErbB2 presents itself, therefore, as a target for antibody-mediated therapies. In this respect, anti-ErbB2 monoclonal antibody 4D5 specifically inhibits the growth of tumor cells overexpressing ErbB2. We have analyzed the effect of 4D5-mediated ErbB2 inhibition on the cell cycle of the breast tumor cell line BT474. 4D5 treatment of BT474 cells resulted in a G(1) arrest, preceded by rapid dephosphorylation of ErbB2, inhibition of cytoplasmic signal transduction pathways, accumulation of the cyclin-dependent kinase inhibitor p27(Kip1), and inactivation of cyclin-Cdk2 complexes. Time courses demonstrated that 4D5 treatment redirects p27(Kip1) onto Cdk2 complexes, an event preceding increased p27(Kip1) expression; this correlates with the downregulation of c-Myc and D-type cyclins (proteins involved in p27(Kip1) sequestration) and the loss of p27(Kip1) from Cdk4 complexes. Similar events were observed in ErbB2-overexpressing SKBR3 cells, which exhibited reduced proliferation in response to 4D5 treatment. Here, p27(Kip1) redistribution resulted in partial Cdk2 inactivation, consistent with a G1 accumulation. Moreover, p27(Kip1) protein levels remained constant. Antisense-mediated inhibition of p27(Kip1) expression in 4D5-treated BT474 cells further demonstrated that in the absence of p27(Kip1) accumulation, p27(Kip1) redirection onto Cdk2 complexes is sufficient to inactivate Cdk2 and establish the G(1) block. These data suggest that ErbB2 overexpression leads to potentiation of cyclin E-Cdk2 activity through regulation of p27(Kip1) sequestration proteins, thus deregulating the G(1)/S transition. Moreover, through comparison with an ErbB2-overexpressing cell line insensitive to 4D5 treatment, we demonstrate the specificity of these cell cycle events and show that ErbB2 overexpression alone is insufficient to determine the cellular response to receptor inhibition.

Stat5a serine phosphorylation. Serine 779 is constitutively phosphorylated in the mammary gland, and serine 725 phosphorylation influences prolactin-stimulated in vitro DNA binding activity.

The activity of transcription factors of the Stat family is controlled by phosphorylation of a conserved, carboxyl-terminal tyrosine residue. Tyrosine phosphorylation is essential for Stat dimerization, nuclear translocation, DNA binding, and transcriptional activation. Phosphorylation of Stats on specific serine residues has also been described. We have previously shown that in HC11 mammary epithelial cells Stat5a is phosphorylated on Tyr(694) in a prolactin-sensitive manner, whereas serine phosphorylation is constitutive (Wartmann, M., Cella, N., Hofer, P., Groner, B., Xiuwen, L., Hennighausen, L., and Hynes, N. E. (1996) J. Biol. Chem. 271, 31863-31868). By using mass spectrometry and site-directed mutagenesis, we have now identified Ser(779), located in a unique Stat5a SP motif, as the site of serine phosphorylation. By using phospho-Ser(779)-specific antiserum, we have determined that Ser(779) is constitutively phosphorylated in mammary glands taken from different developmental stages. Stat5a isolated from spleen, heart, brain, and lung was also found to be phosphorylated on Ser(779). Ser(725) in Stat5a has also been identified as a phosphorylation site (Yamashita, H., Xu, J., Erwin, R. A., Farrar, W. L., Kirken, R. A., and Rui, H. (1998) J. Biol. Chem. 273, 30218-30224). Here we show that mutagenesis of Ser(725), Ser(779), or a combination of Ser(725/779) to an Ala had no effect on prolactin-induced transcriptional activation of a beta-casein reporter construct. However, following prolactin induction the Ser(725) mutant displayed sustained DNA binding activity compared with that of wild type Stat5a. The results suggest that Ser(725) phosphorylation has an impact on signal duration.

ErbB receptor-induced activation of stat transcription factors is mediated by Src tyrosine kinases.

Epidermal growth factor (EGF) binding to its receptor, ErbB1, triggers various signal transduction pathways, one of which leads to the activation of signal transducer and activator of transcription (Stat) factors. The mechanism underlying ErbB1-induced Stat activation and whether Stats are downstream targets of other ErbB receptors have not been explored. In this report we show that ErbB2, ErbB3, and ErbB4 do not potentiate Stat5 phosphorylation by EGF. However, neu differentiation factor-induced heterodimers of ErbB2 and ErbB4 activated Stat5. In A431 cells, Stat1, Stat3, and Stat5, were constitutively complexed with ErbB1 and rapidly phosphorylated on tyrosine in response to EGF. Neither mutation of the conserved tyrosine residue (Tyr694) nor inactivation of the Stat5a SH2 domain disrupted this association. However, an intact SH2 domain was necessary for EGF-induced Stat5a phosphorylation. In contrast to prolactin, which induced only Tyr694 phosphorylation of Stat5a, EGF promoted phosphorylation on Tyr694 and additional tyrosine residue(s). Janus kinases (Jaks) were also constitutively associated with ErbB receptors and were phosphorylated in response to EGF-related ligands. However, we provide evidence that EGF- and neu differentiation factor-induced Stat activation are dependent on Src but not Jak kinases. Upon EGF stimulation, c-Src was rapidly recruited to Stat/ErbB receptor complexes. Pharmacological Src kinase inhibitors and a dominant negative c-Src ablated both Stat and Jak tyrosine phosphorylation. However, dominant negative Jaks did not affect EGF-induced Stat phosphorylation. Taken together, the experiments establish two independent roles for Src kinases: (i) key molecules in ErbB receptor-mediated Stat signaling and (ii) potential upstream regulators of Jak kinases.