Id-1 promotes cell proliferation through the activation of EGFR, NF-κB p50 homodimer and Bcl-3 in MCF-7 breast cancer cell line

Abstract

Progression of breast cancer from hormone-dependent to hormone-independent causes a major problem in breast cancer therapy. The possible involvement of inhibitor of differentiation type 1 (Id-1) in this progression was suggested by the findings in our group that expression of Id-1 was higher in patients with a lower percentage of estrogen receptors (ER). Therefore, in this study, we over-expressed Id-1 in the hormone-dependent breast cancer cell line, MCF-7, to examine whether Id-1 confers growth advantage to these cells in the absence of estrogen. Our results showed that cell growth was increased in the Id-1 transfectants compared with the mock-transfected clone control over a 120 hour period. Cell growth of the transfectant was similar to the mock-transfected control after transient transfection of the Id-1 antisense oligonucleotide. Moreover, elevated levels of epidermal growth factor receptor (EGFR) which is associated with hormone-independent breast cancer, were found in Id-1 transfectants using Western blot and reverse-transcription (RT) - PCR analyses. The level of EGFR expression was decreased after transient transfection of Id-1 antisense oligonucleotide compared with the same Id-1 transfectant without Id-1 antisense oligonucleotide. The involvement of nuclear factor-kappa B (NF-κB) pathway which is one of the downstream pathways of EGFR, suggested to be activated in ER negative breast cells, was investigated. After applying an inhibitor of NF-κB, parthenolide, cell growth and cells in the S phases of the cell cycle were significantly decreased by more than 50%. Results also showed that the percentage inhibitions were positively associated with the expression levels of Id-1, implying that Id-1 may be able to activate the NF-κB pathway. Interestingly, elevated expression of nuclear fragment p50 and Bcl-3, but not p65, were observed with increased level of Id-1 using Western blotting. By contrast, the expression of IκB-α, the corresponding inhibitor of the complex of NF-κB p50/p65 heterodimers, was not correlated with the expression of Id-1. Moreover, results of electrophoretic mobility shift assay (EMSA) showed that levels of NF-κB p50 were significantly increased in Id-1 transfectants and positively associated with levels of Id-1. Our results suggest that Id-1 may be able to modulate cell growth in the absence of estrogen, possibly through the activation of EGFR signaling pathways and through the activation of NF-κB p50/p50 homodimer and Bcl-3 but not NF-κB p50/p65 heterodimer. Although further study is needed, Id-1 may serve as a biomarker in the progression of breast cancer from hormone-dependent to hormone-independent. Inactivation of Id-1 may be a potential therapeutic target for breast cancer patients who have developed resistance to hormonal therapy.