HER-2 overexpression differentially alters transforming growth factor-β responses in luminal versus mesenchymal human breast cancer cells

INTRODUCTION: Amplification of the HER-2 receptor tyrosine kinase has been implicated in the pathogenesis and aggressive behavior of approximately 25% of invasive human breast cancers. Clinical and experimental evidence suggest that aberrant HER-2 signaling contributes to tumor initiation and disease progression. Transforming growth factor beta (TGF-β) is the dominant factor opposing growth stimulatory factors and early oncogene activation in many tissues, including the mammary gland. Thus, to better understand the mechanisms by which HER-2 overexpression promotes the early stages of breast cancer, we directly assayed the cellular and molecular effects of TGF-β1 on breast cancer cells in the presence or absence of overexpressed HER-2. METHODS: Cell proliferation assays were used to determine the effect of TGF-β on the growth of breast cancer cells with normal or high level expression of HER-2. Affymetrix microarrays combined with Northern and western blot analysis were used to monitor the transcriptional responses to exogenous TGF-β1 in luminal and mesenchymal-like breast cancer cells. The activity of the core TGF-β signaling pathway was assessed using TGF-β1 binding assays, phospho-specific Smad antibodies, immunofluorescent staining of Smad and Smad DNA binding assays. RESULTS: We demonstrate that cells engineered to over-express HER-2 are resistant to the anti-proliferative effect of TGF-β1. HER-2 overexpression profoundly diminishes the transcriptional responses induced by TGF-β in the luminal MCF-7 breast cancer cell line and prevents target gene induction by a novel mechanism that does not involve the abrogation of Smad nuclear accumulation, DNA binding or changes in c-myc repression. Conversely, HER-2 overexpression in the context of the mesenchymal MDA-MB-231 breast cell line potentiated the TGF-β induced pro-invasive and pro-metastatic gene signature. CONCLUSION: HER-2 overexpression promotes the growth and malignancy of mammary epithelial cells, in part, by conferring resistance to the growth inhibitory effects of TGF-β. In contrast, HER-2 and TGF-β signaling pathways can cooperate to promote especially aggressive disease behavior in the context of a highly invasive breast tumor model

Transformation of mouse mammary epithelial cells with the Ha- ras but not with the neu oncogene results in a gene dosage-dependent increase in transforming growth factor-α production

An enhanced expression of transforming growth factor-α (TGFα) was demonstrated in two clones of NOG-8 mouse mammary epithelial cells, NOG-8 SR1 and NOG-8 SR2, that have been transformed by a v-Ha- ras oncogene. The amount of TGFα production in NOG-8 SR1 and NOG-8 SR2 cells was dependent on the level of p21 ras expression in these clones, which directly correlated with their cloning efficiency in soft agar. There was also a decrease in the number of epidermal growth factor (EGF) receptors on the NOG-8 SR1 and NOG-8 SR2 cells that is proportional to the amount of TGFα secreted. These effects were specific for ras because neu-transformed NOG-8 cells grew in soft agar at a comparable level to NOG-8 SR2 cells yet did not show any increase in TGFα production or change in EGF receptor expression

Modulation by Estrogen and Growth Factors of Transforming Growth Factor-Alpha and Epidermal Growth Factor Receptor Expression in Normal and Malignant Human Mammary Epithelial Cells

A number of well-characterized growth factors, such as epidermal growth factor (EGF) and the insulin-like growth factors (IGFs), can stimulate the proliferation and modify the differentiation of normal and malignant rodent and human mammary epithelial cells (Salomon et al. 1986 a, c). For example, EGF or transforming growth factor-alpha (TGFa) can stimulate the lobulo-alveolar development of mouse mammary glands in organ culture or in vivo (Tonelli and Soroff 1980; Okamoto and Oka 1984; Vonderhaar 1987). In addition, EGF may perform a physiological role in the development of the mouse mammary gland during pregnancy and lactation and in the spontaneous formation of mammary tumors in mice (Okamoto and Oka 1984; Oka et al. 1987; Tsutsumi et al. 1987). Several of these growth factors, such as IGF-I, platelet-derived growth factor (PDGF), TGFa, and TGFß, have been found in the conditioned medium (CM) from several human breast cancer cell lines (Salomon et al. 1984; Dickson et al. 1986; Huff et al. 1986; Bronzert et al. 1987; Knabbe et al. 1987; Peres et al. 1987). The production of TGFa, IGF-I, and TGF/3 can be modulated by estrogens or anti-estrogens, suggesting that the growth-promoting effects of estrogen may be mediated in part by TGFa and/or IGF-I and that the growth-inhibitory effects of anti-estrogen may be mediated by TGF/3 (Dickson et al. 1985; Perroteau et al. 1986; Knabbe et al. 1987; Liu et al. 1987; Peres et al. 1987; Huff et al. 1988). However, it is still unclear whether these or other growth factors are present in mammary tumor cells in vivo and, if so, whether they are involved in the etiology of tumor development and progression. Likewise, there is little information on the distribution and level of expression of similar growth factors in normal mammary epithelial cells at specific times during the development of the mammary gland