Defining the ATM-mediated barrier to tumorigenesis in somatic mammary cells following ErbB2 activation

Abstract

p53, apoptosis, and senescence are frequently activated in preneoplastic lesions and are barriers to progression to malignancy. These barriers have been suggested to result from an ATM-mediated DNA damage response (DDR), which may follow oncogene-induced hyperproliferation and ensuing DNA replication stress. To elucidate the currently untested role of DDR in breast cancer initiation, we examined the effect of oncogene expression in several murine models of breast cancer. We did not observe a detectable DDR in early hyperplastic lesions arising in transgenic mice expressing several different oncogenes. However, DDR signaling was strongly induced in preneoplastic lesions arising from individual mammary cells transduced in vivo by retroviruses expressing either PyMT or ErbB2. Thus, activation of an oncogene after normal tissue development causes a DDR. Furthermore, in this somatic ErbB2 tumor model, ATM, and thus DDR, is required for p53 stabilization, apoptosis, and senescence. In palpable tumors in this model, p53 stabilization and apoptosis are lost, but unexpectedly senescence remains in many tumor cells. Thus, this murine model fully recapitulates early DDR signaling; the eventual suppression of its endpoints in tumorigenesis provides compelling evidence that ErbB2-induced aberrant mammary cell proliferation leads to an ATM-mediated DDR that activates apoptosis and senescence, and at least the former must be overcome to progress to malignancy. This in vivo study also uncovers an unexpected effect of ErbB2 activation previously known for its prosurvival roles, and suggests that protection of the ATM-mediated DDR-p53 signaling pathway may be important in breast cancer prevention.

Fig. 1.

Poor DDR induction in transgenic mouse models of mammary cancer. Early lesions in mammary glands from 7 to 9-week-old MMTV-ErbB2 (n = 4), MMTV-c-Myc (n = 4), MMTV-H-RasG12D (n = 3), and MMTV-PyMT (n = 4) transgenic mice were stained for the proteins indicated, by immunofluorescence or immunohistochemistry. Brightfield images are 40× with 20 μm scale bar. Deconvolution immunofluorescent images are 100× (53BP1) or 60× (p-S1981-ATM) with 5-μm scale bar.

Fig. 2.

Somatic activation of ErbB2 induces a DDR that becomes inactivated during tumor progression. (A) Normal mammary ducts and hyperplasias in RCAS-ErbB2–infected mammary glands from 14-week-old MMTV-tva mice (n = 4) and mammary tumors from infected mice (n = 4) were stained for the proteins indicated and by TUNEL. Arrows indicate Ki67 cells (brown), whereas arrowheads indicate SA-β-Gal-positive cells (blue). Dashed lines, when present, outline normal ducts. Brightfield images are 40× with 20-μm scale bar. 53BP1 and p-S1981-ATM deconvolution immunofluorescent images are 100× with 5-μm scale bar. ARF and TUNEL immunofluorescent images are 40× with 20 μm scale bar. (B and C) Percentages of p53-positive nuclei (B) and TUNEL-positive cells (C) in early lesions (n = 4) and tumors (n = 4) arising in 12-week-old MMTV-tva mice injected with RCAS-ErbB2. Columns represent mean ± SEM. *P = 0.01–0.05; **P = 0.001–0.01.

Fig. 3.

ATM ablation results in reduced DDR signaling, apoptosis, and senescence in response to ErbB2 activation. (A) Hyperplastic mammary glands from RCAS-ErbB2–infected 10- to 12-week-old MMTV-tva mice with the ATM status indicated at the top were stained for the proteins indicated and by TUNEL. Brightfield images are 40× with 20-μm scale bar. 53BP1 deconvolution immunofluorescent images are 100× with 5 μm scale bar. ARF and TUNEL immunofluorescent images are 40× with 20 μm scale bar. (B and C) Percentages of p53-positive nuclei (B) and TUNEL-positive cells (C) in early lesions arising in 10–12 week-old MMTV-tva/ATM^+/+ (n = 3), MMTV-tva/ATM^+/− (n = 3), or MMTV-tva/ATM^−/− mice (n = 3) injected with RCAS-ErbB2. Columns represent mean ± SEM. Statistical analysis was performed using univariate ANOVA. *P = 0.01–0.05; **P = 0.001–0.01.