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. 2000 Aug 15;97(17):9609-14.
doi: 10.1073/pnas.160564197.

Blockade of the epidermal growth factor receptor tyrosine kinase suppresses tumorigenesis in MMTV/Neu + MMTV/TGF-alpha bigenic mice

A E Lenferink  1 , J F Simpson, L K Shawver, R J Coffey, J T Forbes, C L Arteaga
Affiliations

Blockade of the epidermal growth factor receptor tyrosine kinase suppresses tumorigenesis in MMTV/Neu + MMTV/TGF-alpha bigenic mice

A E Lenferink et al. Proc Natl Acad Sci U S A. .

Abstract

Overexpression of ErbB-2/Neu has been causally associated with mammary epithelial transformation. Here we report that blockade of the epidermal growth factor receptor (EGFR) kinase with AG-1478 markedly delays breast tumor formation in mouse mammary tumor virus (MMTV)/Neu + MMTV/transforming growth factor alpha bigenic mice. This delay was associated with inhibition of EGFR and Neu signaling, reduction of cyclin-dependent kinase 2 (Cdk2) and mitogen-activated protein kinase (MAPK) activities and cyclin D1, and an increase in the levels of the Cdk inhibitor p27(Kip1). In addition, BrdUrd incorporation into tumor cell nuclei was prevented with no signs of tumor cell apoptosis. These observations prompted us to investigate the stability of p27. Recombinant p27 was degraded rapidly in vitro by untreated but not by AG-1478-treated tumor lysates. Proteasome depletion of the tumor lysates, addition of the specific MEK1/2 inhibitor U-0126, or a T187A mutation in recombinant p27 all prevented p27 degradation. Cdk2 and MAPK precipitates from untreated tumor lysates phosphorylated recombinant wild-type p27 but not the T187A mutant in vitro. Cdk2 and MAPK precipitates from AG-1478-treated tumors were unable to phosphorylate p27 in vitro. These data suggest that increased signaling by ErbB receptors up-regulates MAPK activity, which, in turn, phosphorylates and destabilizes p27, thus contributing to dysregulated cell cycle progression.

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Figures

Figure 1
Figure 1
Blockade of the EGFR kinase inhibits the proliferation of ErbB-2/Neu-overexpressing cells. (a) MCF-10A/TE cells with or without AG-1478. After 24 h, cell lysates were prepared in EBC buffer and subjected to immunoblot procedures for EGFR, ErbB-2/Neu, and P-Tyr. Levels of a 180-kDa P-Tyr band, probably representing EGFR and ErbB-2/Neu, were reduced by AG-1478 without any change in receptor protein content. (b) MCF-10A/TE cells were seeded in 6-well plates at a density of 0.5 × 106 cells per well and treated either continuously for 5 days or daily for 1 h for 5 consecutive days with 10 μM AG-1478. Each bar represents the mean number of cells ± SE of three wells after the 5-day experiment. The hatched bar represents the number of cells per well 24 h postplating. (c) Exponentially growing BT-474 and SKBR-3 breast tumor cells were incubated overnight with or without 10 μM AG-1478, solubilized in EBC buffer, and then analyzed by immunoblot procedures for ErbB-2/Neu, P-Tyr, Rb, cyclin D1, and p27. (d) BT-474 and SKBR-3 cells (3 × 104) were plated in 35-mm dishes in IMEM/10% FCS/0.8% agarose/10 mM Hepes in the absence or presence of 1–10 μM AG-1478. After 7 days, colonies measuring 50 μm were counted. Each bar represents the mean colony number ± SE of three dishes. (e) BT-474 and SKBR-3 cells were incubated overnight with 10 μM AG-1478 and then subjected to flow cytometric analysis. Numbers represent the percentage of cells in G1, G2M, and S phases.
Figure 2
Figure 2
Blockade of the EGFR kinase prolongs the latency of MMTV/Neu + TGF-α bigenic mammary tumors. (a) Eight-week-old bigenic mice were allocated randomly to daily i.p. injections with 50 mg/kg AG-1478 (○) or DMSO (▴). After 28 weeks, 9 of 10 (90%) mice in the control group developed at least one mammary tumor, whereas only 2 of 10 (20%) of the AG-1478-treated mice developed palpable tumors (P = 0.0007 on day 250; P = 0.006 on day 300 by log-rank test). (b and c) Whole-mount preparations from control and 6-month-old bigenic mice treated with DMSO (b) or AG1478 (c). (d and e) Hematoxylin/eosin-stained ×400 histological sections of mammary glands from 6-month-old bigenic mice treated with DMSO (d) or AG-1478 (e).
Figure 3
Figure 3
AG-1478 inhibits DNA synthesis in mammary epithelium from MMTV/Neu + TGF-α bigenic mice. (a and b) Mice were injected daily for 5 days with DMSO (a) or AG-1478 (b). On the fifth day, BrdUrd was injected i.p. 2 h before isolation of one of the inguinal mammary glands. Tissue sections were prepared and subjected to immunohistochemistry with a BrdUrd antibody as described in Materials and Methods. (Magnification: ×400.)
Figure 4
Figure 4
AG-1478 treatment blocks EGFR and ErbB-2/Neu signaling and modulates molecules involved in G1/S traverse. (a) EGFR and ErbB-2/Neu proteins were precipitated from tumor tissue taken from two mice (I, II) immediately before (−) and after (+) five i.p. doses of AG-1478 (50 mg/kg per day). The immune complexes were subjected to immunoblot procedures by using EGFR, ErbB-2/Neu, P-Tyr, Shc, Grb-2, and PLC-γ1 antibodies. (b) Protein (100 μg/lane) from the mammary tumor lysates from the same mice before (−) and after (+) treatment was subjected to immunoblot analyses with antibodies against active MAPK, Rb, cyclin D1, cyclin A, p27, and p21.
Figure 5
Figure 5
Phosphorylation/degradation of recombinant p27 by MMTV/Neu + TGF-α tumor lysates. (a) Cdk2 was precipitated from tumor lysates from mice that had been treated or not with AG-1478 and added to a kinase reaction containing either HH1, wild-type p27, or T187A p27 as substrates for [γ-32P]ATP incorporation. Phosphorylated products were resolved by SDS/PAGE and visualized by autoradiography. (b and c) Twenty micrograms of total protein from the tumor lysates was incubated with 250 ng of either wild-type p27 (b) or T187A p27 (c) at 30°C for the indicated times. Where indicated, tumor lysates were depleted of their proteasome complex by differential centrifugation before incubation with p27. To monitor for spontaneous degradation, wild-type p27-His was incubated under the same conditions but in the absence of a tumor lysate (b Bottom). Degradation was assessed by SDS/PAGE and p27 immunoblot as indicated in Materials and Methods. The endogenous p27 is undetectable by immunoblot analysis of this amount of total protein (20 μg) from the tumor lysates. Because of the histidine tag, both wild-type and mutant p27 molecules migrate as 33-kDa proteins.
Figure 6
Figure 6
MAPK-mediated phosphorylation/degradation of p27. (a) Cdk2, MEK1, and MAPK were precipitated from a tumor lysate from a treatment-naïve mouse (control and U-0126 lanes) or from a mouse that had been treated with AG-1478 for 5 days. Immune complexes then were added to kinase reactions containing HH1, glutathione S-transferase-MAPK, or MBP, respectively. Where indicated, 10 μM U-0126 was added to the kinase reaction. Phosphorylated species were resolved by SDS/PAGE and visualized by autoradiography. (b) Twenty micrograms of total protein from a proteasome-containing tumor lysate was incubated with 250 ng of p27-His at 30°C for the indicated times in the presence of 10 μM U-0126 or DMSO (control). Degradation of p27 was monitored by immunoblot analysis as described in Fig. 5. (c) MAPK was precipitated from tumor lysates from mice that had been treated or not with AG-1478. Immune complexes then were tested for kinase activity against MBP, p27-His, or T187A p27 substrates. After SDS/PAGE, phosphorylated substrates were visualized by autoradiography.
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