Two-dimensional gel electrophoresis analyses identify nucleophosmin as an estrogen regulated protein associated with acquired estrogen-independence in human breast cancer cells

We have used two-dimensional gel electrophoresis to identify proteins associated with estrogen-induced proliferation in MCF-7 breast cancer cells and their progression to estrogen-independent proliferation. We compared the total cellular proteins from MCF-7 cells and an estrogen independent derivative of the MCF-7 cells MCF-7/LCC1 (Brunner et al. Cancer Research 1993, 53, 283-290), each grown with and without estradiol. These comparisons reveal seven estrogen-regulated proteins. Three of these proteins (HI-1: 36 kDa/pI 4.5, HI-10: 40 kDa/pI 5.5 and HI-19: 62 kDa/pI 5.0) exhibit a 'progression-like' pattern, being induced by estradiol in MCF-7 cells and constitutively present/upregulated in the MCF-7/LCC1 growing without estradiol. HI-11 (65 kDa/pI 5.5) is strongly induced by estradiol in MCF-7 cells but constitutively downregulated and unresponsive to estradiol in the MCF-7/LCC1 cells. Two proteins exhibit a suppressor pattern and are downregulated by estradiol in the estrogen-dependent MCF-7 cells (HI-3: 44 kDa/pI 4.4 and HI-4: 56 kDa/pI 5.2) and present in MCF-7/LCC1 cells growing without estradiol at levels comparable to that seen in estrogen-treated MCF-7 cells. One protein (HI-9: 68 kDa/pI 5.5) exhibits a marked estrogen regulated pi shift, rather than changes in abundance. We purified and sequenced the HI-10 protein, which we identified as the nucleolar protein, nucleophosmin (NPM). One- and two-dimensional Western blot analyses of MCF-7/LCC1 cell lysates confirmed that HI-10 is immunoreactive with an antinucleophosmin antibody. Western blotting also confirmed the estrogenic regulation of NPM seen in the initial two-dimensional gel electrophoresis studies. Thus, NPM is induced by estradiol in the MCF-7 cells and upregulated in the MCF-7/LCC1 cells growing without estrogen, clearly associating its expression with an acquired estrogen-independent phenotype. NPM has several potentially important roles in regulating cell function and signaling. It is a substrate for phosphorylation by p34(cdc2) kinase, protein kinase C and nuclear kinase II, and a repressor of the transcriptional regulating activities of both the IRF-1 tumor suppressor protein and the YY1 transcription factor. Studies are currently underway to determine which of these NPM functions may be involved in the hormonal progression of breast cancer. (C) 1999 Elsevier Science Ltd. All rights reserved.