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Sex-hormone binding globulin receptor-mediated growth inhibition in breast cancer cells: a proteomics approach


Both estrogen (E2) and growth factors stimulate proliferation of E2-dependent breast tumor cells. Functional cross-talk exists between E2-directed and growth factor-directed pathways. Convergence between these pathways may lead to a synergistic feed-forward circuit, resulting in a stronger or more sustained proliferative response in breast cancer cells [1]. Another signaling pathway involved in the modulation of breast cancer cell proliferation is mediated by the receptor for sex hormone-binding globulin (SHBG-R). SHBG has been found to function as part of a novel steroid signaling system that is independent of the 'classical pathway' for intracellular steroid receptors. This 'alternative pathway' (Fig. 1) involves the activation of membrane SHBG-Rs by SHBG and E2. It has been demonstrated that SHBG, through cAMP and protein kinase A (PKA), can inhibit the proliferative effect of E2 on breast cancer cells [24]. Its role in E2-dependent cancer cell proliferation may in the longer term be exploited for therapeutic strategies. The pathways involved have not yet been elucidated. Indeed, the receptor has not yet been identified, although it is known to exist from binding kinetics studies.

Figure 1
figure 1

Model for SHBG-mediated signaling. The plasma membrane binding site for SHBG is preferentially expressed in ER+ cells with a reduced proliferative index [2-4]. SHBG-R only binds steroid-free or unliganded SHBG. Following binding of unliganded SHBG to SHBG-R on the cell membrane, E2 binds to the SHBG–SHBG-R complex, thereby activating it. Activation induces the synthesis of cAMP, which, in turn, triggers downstream signaling via PKA [2-4]. The biological outcome of this signaling pathway in cells of the human breast carcinoma cell line MCF-7 is decreased E2-mediated cell growth.


We are applying proteomics techniques to identify the plasma membrane SHBG-R, and to elucidate the key signaling proteins involved in pathway(s) mediated by SHBG/SHBG-R binding. The following cell lines are the source material for membrane and cytosolic preparations: MCF-7, estrogen-dependent (estrogen receptor positive [ER+]) breast cancer cultured cells; MDA-MB 231, estrogen-insensitive (ER-) breast cancer cultured cells; MCF-10A, non-neoplastic mammary cells [control]. The steps for identification of SHBG-R are as follows. MCF-7 cells are known to have SHBG-R (a transmembrane protein) on their surfaces. Cell membranes are prepared from MCF-7 cells and then, following extraction, the membrane proteins are separated via two-dimensional electrophoresis (2D-E). Identification of SHBG-R on immunoblots involves a ligand binding assay using SHBG as the primary binder followed by anti-SHBG (to detect bound SHBG) then a tertiary antibody conjugated to an enzyme system. Identification and partial characterization of SHBG-R involves peptide mass fingerprinting and sequencing of amino acids, and international database searching. The design of experiments for identification of signaling components of SHBG-mediated pathway(s) is as shown in Fig. 2.

Figure 2
figure 2

Identification of key signaling components of SHBG-mediated pathway(s). A flow-chart of the experimental design and method (involving comparative proteomic analyses) for identification of key regulated signaling components in SHBG-mediated pathways in breast cancer cells. Each cell type will be exposed to SHBG (vs not exposed to SHBG), and to human estradiol (E2) (vs not exposed to E2). Differential analysis of proteomes for the different cell lines/treatments will then be carried out. Differences in expression/post-translational modifications of specific proteins will determine what proteins are of interest. End effects will be examined as well as the results of time-dependent experiments.


Progress will be reported on the identification and characterization of the SHBG receptor.


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The authors wish to acknowledge a Macquarie University research grant.

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Bowers-Morrow, V., Ali, S. Sex-hormone binding globulin receptor-mediated growth inhibition in breast cancer cells: a proteomics approach. Breast Cancer Res 7 (Suppl 2), P2.10 (2005).

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  • Breast Cancer Cell
  • Cancer Cell Proliferation
  • Breast Tumor Cell
  • Peptide Mass Fingerprinting
  • Breast Cancer Cell Proliferation