PBR-mediated Cell Proliferation and Nuclear Transport of Phenotype with PBR Expression, Nuclear Localization, and Breast Cancer : Correlation of Breast Cancer Cell Aggressive Peripheral-Type Benzodiazepine Receptor (PBR) in Human

Aberrant cell proliferation and increased invasive and metastatic behavior are hallmarks of the advancement of breast cancer. Numerous studies implicate a role for cholesterol in the mechanisms underlying cell proliferation and cancer progression. The peripheral-type benzodiazepine receptor (PBR) is an Mr 18,000 protein primarily localized to the mitochondria. PBR mediates cholesterol transport across the mitochondrial membranes in steroidogenic cells. A role for PBR in the regulation of tumor cell proliferation has also been shown. In this study, we examined the expression, characteristics, localization, and function of PBR in a battery of human breast cancer cell lines differing in their invasive and chemotactic potential as well as in several human tissue biopsies. Expression of PBR ligand binding and mRNA was dramatically increased in the highly aggressive cell lines, such as MDA-231, relative to nonaggressive cell lines, such as MCF-7. PBR was also found to be expressed at high levels in aggressive metastatic human breast tumor biopsies compared with normal breast tissues. Subcellular localization with both antibodies and a fluorescent PBR drug ligand revealed that PBR from the MDA-231 cell line as well as from aggressive metastatic human breast tumor biopsies localized primarily in and around the nucleus. This localization is in direct contrast to the largely cytoplasmic localization seen in MCF-7 cells, normal breast tissue, and to the typical mitochondrial localization seen in mouse tumor Leydig cells. Pharmacological characterization of the receptor and partial nucleotide sequencing of PBR cDNA revealed that the MDA-231 PBR is similar, although not identical, to previously described PBR. Addition of high affinity PBR drug ligands to MDA-231 cells increased the incorporation of bromodeoxyuridine into the cells in a dose-dependent manner, suggesting a role for PBR in the regulation of MDA-231 cell proliferation. Cholesterol uptake into isolated MDA-231 nuclei was found to be 30% greater than into MCF-7 nuclei. High-affinity PBR drug ligands regulated the levels of cholesterol present in MDA-231 nuclei but not in MCF-7. In addition, the PBR-dependent MDA-231 cell proliferation was found to highly correlate (r 5 20.99) with the PBRmediated changes in nuclear membrane cholesterol levels. In conclusion, these data suggest that PBR expression, nuclear localization, and PBRmediated cholesterol transport into the nucleus are involved in human breast cancer cell proliferation and aggressive phenotype expression, thus participating in the advancement of the disease. INTRODUCTION The PBR was originally discovered as an alternative binding site for the benzodiazepine diazepam (Valium; Ref. 1). PBR is a multimeric receptor composed of the Mr 18,000 receptor protein and the Mr 34,000 voltage-dependent anion channel protein. The Mr 18,000 subunit is thought to be responsible for the binding of isoquinolines, whereas both subunits are required for the binding of benzodiazepines (2). Expression of PBR has been found in every tissue examined; however, it is most abundant in steroid-producing tissues such as the adrenals, testes, ovaries, placenta, and brain, where it is primarily localized in the outer mitochondrial membrane (3). In steroid-producing tissues, PBR is known to regulate the transport of cholesterol from the outer to the inner mitochondrial membrane, the rate-determining step in steroid biosynthesis (4). Although the primary function of PBR is the regulation of steroidogenesis, its existence in nonsteroidogenic tissues as well as in other cellular compartments including the plasma membrane (3) suggests that there may be other roles for PBR. In the early 1980s it was shown that diazepam (Valium), a ligand for both the central (GABAA) and PBR receptors, induced murine Friend erythroleukaemia (MEL) cell differentiation and inhibited 3T3 cell proliferation at micromolar concentrations (5). A series of 15 benzodiazepines, including diazepam, also inhibited thymoma cell proliferation at micromolar concentrations (6) and further suggested that this effect was mediated by PBR rather than the GABAA receptor. Stimulation of cell proliferation was shown to occur when glioma cells were incubated with nanomolar concentrations of Ro5-4864 and PK 11195, both high-affinity PBR ligands (7). In this same study, it was shown that Ro5-4864 and PK 11195 inhibited glioma cell proliferation at micromolar concentrations. Similar results were obtained in testicular Leydig tumor cells (8). The effect of PK 11195, an exclusive ligand of PBR, provided unequivocal evidence that the effects seen were mediated exclusively by PBR. Inhibition of cell proliferation by micromolar concentrations of Ro5-4864 and PK 11195 have also been reported in astrocytes and V79 Chinese Hamster lung cells (9, 10). In addition, micromolar concentrations of PBR ligands have also been shown to inhibit growth factor-induced cell proliferation in both astrocytes and lymphoma cells (11, 12). Considering the abundance of PBR in tumors (6, 7, 9–12) and the proposed correlation between PBR expression and human brain tumor aggression (13, 14), we examined PBR expression and, if present, its function in breast cancer. Breast cancer is the most common neoplasm and the leading cause of cancer-related deaths for women in most developing countries (15). The American Cancer Society estimates that in 1998 nearly 180,000 new cases of invasive breast cancer will be diagnosed among women, with over 43,000 deaths, in the United States alone. Two fundamental Received 8/5/98; accepted 12/15/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by Grant ES-07747 from the National Institute of Environmental Health Sciences and SPORE Grant P50-CA-58185 from the National Cancer Institute, NIH. This work was partially presented at the 88th Annual Meeting of the American Association for Cancer Research (San Diego, CA) and the Xth International Meeting on Hormonal Steroids (Quebec City, Canada). 2 Supported by a Research Career Development Award HD-01031 from the National Institute of Child Health and Human Development, NIH. 3 To whom requests for reprints should be addressed, at Department of Cell Biology, Georgetown University Medical Center, 3900 Reservoir Road, Washington, DC 20007. Phone: (202) 687-8991; Fax: (202) 687-1823; E-mail: [email protected]. 4 The abbreviations used are: PBR, peripheral-type benzodiazepine receptor; hPBR, human PBR; FBS, fetal bovine serum; BrdUrd, 5-bromo-29-deoxyuridine; HRP, horseradish peroxidase; DBI, diazepam binding inhibitor; SREBP, sterol regulatory element binding protein; SSC, sodium chloride/sodium citrate.