Id-1 down-regulates Bax expression via NF-κB activation in breast cancer

Abstract

Inhibitors of differentiation and DNA binding-1 (Id-1) have been suggested to play a positive role in cell proliferation and tumorigenesis in many types of human cancers. Increasing evidence supports the function of the helix-loop-helix protein Id-1 as an oncogene and ectopic Id-1 expression not only regulates cell proliferation, but also has protective effect on anticancer drug-induced apoptosis in prostate cancer and nasopharyngeal cancer cell lines. However, little is known about the molecular function of Id-1 in breast cancer cells. Therefore, we tested the role of Id-1 in breast cancer cell line MCF-7 by ectopically overexpressing or inactivating Id-1 by stable transfection or retroviral infection with an antisense construct, respectively. In MCF-7 cells stably overexpressing Id-1, the anchorage-dependent cell growth tested by MTT assay in low serum condition was enhanced. We made a three-fold approach to elucidate the molecular mechanism involved in such enhanced growth. First, we tested whether cell proliferation was enhanced by activating the extracellular signal-regulated kinase (ERK) pathway. At the protein level, the expression of epidermal growth factor receptor (EGFR), ErbB2 (her-2/neu), early growth response factor-1 (Egr-1) were all enhanced by Id-1 as confirmed by Western analysis. In addition, more activated forms of ERKs were detected by using the phospho-specific ERK antibodies. Second, we examined if cell survival, mediated by nuclear factor-?B (NF-?B) signaling pathway, was enhanced by Id-1. We observed an increase in the p65 subunit of NF- B and the down-regulation of I B and p53 proteins by Id-1. However, c-Myc remained unchanged in Id-1 overexpressing cells. Third, we made a novel approach to examine whether the apoptotic pathways were affected by Id-1 in breast cancer cells. Upon quantifying the expression levels of known apoptotic regulators, we found that Bax protein expression was significantly reduced in Id-1 overexpressing MCF-7 stable clones. The reduction of Bax protein expression was due to Id-1 overexpression, because such reduction was reversed upon reduced Id-1 expression by retroviral antisense expression. Over-expression of Id-1 also led to reduction of Bax mRNA level as well as Bax promoter activity. Such changes in Id-1-expressing cells suggest that the Id-1-induced cellular protection against apoptosis in breast cancer is mediated through Bax suppression. Taken together, we report that in breast cancer cell MCF-7, Id-1 regulates cell proliferation through activation of ERK signaling pathway, promotes cell survival through activation of NF- B signaling pathway, and suppresses cell apoptosis through down-regulation of Bax. In this regard, inactivation of Id-1 may provide a potential therapeutic strategy leading to inhibition of breast cancer cell progression.