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

Abstract

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.

FIG. 1

Proliferation assays of BT474 and MKN7 cells treated with anti-ErbB2 antibodies. MKN7 and BT474 cells were seeded at a density of 2,000 cells/cm²; after 24 h of incubation, the medium was changed and either MAb FRP5 or MAb 4D5 was added to a final concentration of 10 μg/ml, or an equal volume of PBS was added. After 4 and 7 days of incubation, cells were trypsinized and total cell number was calculated (A), or after 4 days, cells were pulse-labeled with BrdU for 4 h and BrdU incorporation into nuclei was revealed by immunofluorescence (B). Shown are the percentages of nuclei labeled with BrdU during the pulse period.

FIG. 2

Screen of ErbB receptor protein and tyrosine phosphorylation levels and effects of anti-ErbB2 antibody treatment on tyrosine phosphorylation of the ErbB2 receptor in BT474 and MKN7 cells. Cells were seeded at a density of 3 × 10⁴ cells/cm². After 24 h of incubation, extracts were made and ErbB receptor protein (ErbB) and phosphotyrosine (Phosphotyr) levels were analyzed following immunoprecipitation of the appropriate receptor (A). Cells were seeded as described above, and either MAb FRP5 or MAb 4D5 was added to a final concentration of 10 μg/ml or an equal volume of PBS was added. Extracts were prepared, and ErbB2 protein and phosphotyrosine levels were assessed after 48 h of incubation (B) or at the times indicated (C) following immunoprecipitation (IP) with the ErbB2-specific polyclonal antibody 21N. Preimmune control precipitations are indicated (PI). In panels B and C, longer exposures were required for MKN7 cells due to the lower ErbB2 tyrosine phosphorylation levels in these cells than in BT474 cells. The exposures shown, therefore, do not represent a quantitative comparison of the two cell lines but were chosen to most clearly represent phosphorylation changes induced as a result of 4D5 treatment.

FIG. 3

In vivo [³²P]orthophosphate labeling of the ErbB2 receptor in BT474 and MKN7 cells after anti-ErbB2 antibody treatment; tryptic phosphopeptide mapping. Cells were seeded as in Fig. 2. Twice as many MKN7 cells as BT474 cells were seeded (see Materials and Methods). After 24 h of incubation, cells were deprived of phosphate for 12 h and then prelabeled with [³²P]orthophosphate followed by addition of MAb FRP5, MAb 4D5, or an equal volume of PBS for 1 h. Equal amounts of BT474 cell extracts (doubled in the case of MKN7 extracts) were immunoprecipitated with the anti-ErbB2 antibody 21N, and labeled proteins were identified by separation using SDS-PAGE (7.5% gel) (A). The labeled ErbB2 receptor protein was excised and analyzed by tryptic phosphopeptide mapping (B). Specific tryptic phosphopeptides are indicated by letters, and the origin is shown by a plus sign. Gels and tryptic phosphopeptide maps were analyzed with a phosphorimager. Although MKN7 samples were exposed for longer than BT474 samples, due to lower stoichiometry of phosphorylation, the same exposure is shown for each treatment protocol. The amount of label incorporated into the immunoprecipitated ErbB2 protein in panel A was quantified by scintillation counting. BT474 cells treated with MAb FRP5 and MAb 4D5 had 158 and 60% ³²P incorporation, respectively, compared to the control (PBS)-treated cells. MKN7 cells treated with MAb FRP5 and MAb 4D5 had 139 and 75% ³²P incorporation, respectively, compared to the control (PBS)-treated cells.

FIG. 4

Analysis of PKB and Erk1/2 phosphorylation after treatment of BT474 and MKN7 cells with anti-ErbB2 antibodies. Cells were seeded and treated with MAb FRP5 or MAb 4D5 as in Fig. 2. At the times indicated, cells were extracted and the protein levels (top) and phosphorylation (PO₄⁻) states of PKB (middle) and Erk1/2 (bottom) were evaluated by immunoblotting. Untreated cells (t = 0) were included as controls.

FIG. 5

Analysis of cell cycle distribution after treatment of BT474 cells with anti-ErbB2 antibodies. BT474 cells were seeded as in Fig. 2. After 24 h of incubation, the medium was changed, and MAb FRP5, MAb 4D5, or PBS was added as in Fig. 2. After 48 h, cells were harvested by trypsinization and nuclei were stained with propidium iodide. Shown is flow cytometry analysis of antibody-treated cells compared to PBS-treated controls (top). Percentages of cells in each cell cycle stage are indicated (bottom).

FIG. 6

Analysis of G₁ regulators in anti-ErbB2 antibody-treated BT474 cells. BT474 cells were seeded and treated with MAb FRP5, MAb 4D5, or PBS as in Fig. 2. After 48 h, cells were extracted and the protein levels of G₁ regulators were evaluated by immunoblotting (A and B). Additionally, p27^Kip1 association with Cdk2 complexes, and Cdk2 activity, was assessed through immunoprecipitation (IP) of Cdk2 followed by immunoblotting for associated p27^Kip1 protein or in vitro histone H1 kinase assay (C). Preimmune control precipitations are indicated (PI).

FIG. 7

Time course of the effect of MAb 4D5 treatment on the cell cycle distribution and the levels of cell cycle markers in BT474 cells. BT474 cells were seeded and treated with MAb FRP5 or MAb 4D5 as in Fig. 2. At the times shown, cells were trypsinized, and half of the sample was either stained with propidium iodide and analyzed for cell cycle distribution using flow cytometry (A) or extracted for immunoblot analysis of the proteins indicated (B). Untreated cells (t = 0) and cells treated for 48 h with MAb FRP5 were included as controls.

FIG. 8

Time course of Cdk2 inactivation and Cdk2-p27Kip1 complex formation in MAb 4D5-treated BT474 cells. BT474 cells were seeded and treated with MAb FRP5 or MAb 4D5 as in Fig. 2. At the times indicated, cell extracts were prepared and immunoprecipitated with Cdk2-specific or cyclin E-specific antibodies followed by in vitro histone H1 kinase assay (A). Additionally, the levels of p27^Kip1 and Cdk2 protein in the same extracts were either analyzed directly by immunoblotting (WB) or after immunoprecipitation (IP) with Cdk2-specific antibodies (B). Untreated cells (t = 0) and cells treated with MAb FRP5 for 48 h were included as controls. Cdk2 kinase activity is expressed as percentage of control (t = 0) cells.

FIG. 9

Time course of MAb 4D5-induced effects on Cdk/p27^Kip1 association and levels of proteins involved in p27^Kip1 sequestration in BT474 cells. BT474 cells were seeded and treated with MAb FRP5 or MAb 4D5 as in Fig. 2. At the times indicated, cell extracts were prepared and analyzed by immunoblotting (WB) either directly or after immunoprecipitation (IP) with Cdk2-specific antibodies (A). Additionally, p27^Kip1 association with Cdk2 and Cdk4 was analyzed by immunoblotting after immunoprecipitation (IP) with Cdk2- or Cdk4-specific antibodies (B). Untreated cells (t = 0) and cells treated with MAb FRP5 for 24 h were included as controls.

FIG. 10

Effects of anti-ErbB2 MAb treatment on SKBR3 cell proliferation and the expression and activity of G₁ regulators. SKBR3 cells were seeded as in Fig. 2. (A, Expt. 1; B and C) or at half the density (A, Expt. 2). After 24 h of incubation, PBS, MAb FRP5, or MAb 4D5 was added as in Fig. 2, and cells were treated as follows: (A) incubated for 4 days and trypsinized, after which total cell number was calculated; (B) incubated for 24 h, trypsinized, and treated with propidium iodide, after which cell cycle distribution was analyzed by flow cytometry; (C) incubated for 24 h, after which cell extracts were prepared and the protein levels of G₁ regulators were evaluated by immunoblotting, or p27^Kip1 association with Cdk2 complexes, and Cdk2 activity, was assessed through immunoprecipitation (IP Cdk2) of Cdk2 followed by immunoblotting for associated p27^Kip1 protein or in vitro histone H1 kinase assay. Cdk2 activity is indicated as a percentage of that in control (PBS)-treated cells.

FIG. 11

Time course of the effects of MAb 4D5 treatment on G₁ regulators in MKN7 cells. MKN7 cells were seeded at a density of 3 × 10⁴ cells/cm². After 24 h of incubation, the medium was changed and MAb FRP5 or MAb 4D5 was added to a concentration of 10 μg/ml for the times indicated. Cell extracts were prepared and analyzed by immunoblotting (WB) either directly or after immunoprecipitation (IP) with Cdk2-specific antibodies. Untreated cells (t = 0) and cells treated with MAb FRP5 for 24 h were included as controls.

FIG. 12

Effect of antisense-mediated inhibition of MAb 4D5-induced p27^Kip1 accumulation on the cell cycle of BT474 cells. BT474 cells were seeded at a density of 3 × 10⁴ cells/cm². After 24 h of incubation, cells were treated with LipofectAMINE alone (Lipo), p27^Kip1 antisense oligonucleotide (AS), or a mismatch control oligonucleotide (MM) as outlined in Materials and Methods. Cells were subsequently refed with normal growth medium; after 3 to 5 h, MAb 4D5 (+ 4D5) was added (10 μg/ml) for 24 or 36 h. After these times, cell extracts were prepared and p27^Kip1 protein levels were examined by immunoblotting (A). After 36 h, cells were trypsinized and treated with propidium iodide, and cell cycle distribution was analyzed by flow cytometry (B). Cells treated with LipofectAMINE alone followed by no addition of MAb 4D5 were included as controls. The LipofectAMINE procedure itself had no effect on cell cycle distribution compared to untreated controls, as assessed after 36 h of incubation (not shown).

FIG. 13

Effect of antisense-mediated inhibition of MAb 4D5-induced p27^Kip1 accumulation on Cdk2 activity and p27^Kip1-Cdk2 complex formation in BT474 cells. BT474 cells were seeded, treated with either LipofectAMINE alone (Lipo), antisense p27^Kip1 oligonucleotide (AS), or mismatch (MM) control oligonucleotide, and treated with MAb 4D5 (+ 4D5) as outlined in Fig. 12. After 24 and 36 h of incubation, cells were extracted and immunoprecipitated with Cdk2-specific antibodies followed by in vitro histone H1 kinase assay (A). After 24 h of incubation, the same extracts were analyzed for p27^Kip1 protein levels by immunoblotting (WB) directly or after immunoprecipitation (IP) of Cdk2 complexes. Cells treated with LipofectAMINE alone followed by no addition of MAb 4D5 were used as controls, and Cdk2 activity is expressed as a percentage of this control.