Estrogen receptor of primary breast cancers: evidence for intracellular proteolysis

Abstract

Iodinated oestradiol-labeled oestrogen receptor (ER) isoforms devoid of amino-terminal ABC domains represent about two-thirds of the whole receptor population detected in cytosol samples from human breast cancers. This high frequency could not be ascribed to the expression of truncated mRNAs, or to the proteolysis of the native ER peptide at the time of homogenization or assay, suggesting an intracellular proteolysis. Free amino-terminal and ligand-binding domains maintained together within oligomeric structure(s); increase of ionic strength separated them. The amino-terminal region was consistently detected in the cell nucleus by specific immunohistochemistry leading to the concept of a potential intranuclear association between ER cleavage products and/or other regulatory proteins.

Figure 1

Comparison of [³H]oestradiol (E₂)-binding capacities of a series of human breast cancer cytosols simultaneously measured by DCC and hydroxylapatite (HAP) assays (ethanolic extraction). The ordinate corresponds to values established by DCC, and the abscissa to those by hydroxylapatite (data established by Scatchard plot analysis). A significant correlation was obtained between the two assays in 97 out of 102 (95%) samples; five outlayers are represented by closed symbols in the insert.

Figure 2

Comparison of [¹²⁵I]TAZ-binding capacities (5 nmol/l) of a series of human breast cancer cytosols simultaneously measured by DCC and hydroxylapatite (HAP) assays (phosphate extraction).

Figure 3

(A) Molecular weight and monoclonals anti-ER recognition of [¹²⁵I]TAZ-labelled ER isoforms extracted from hydroxylapatite (HAP) with KCl. (left) Part of human breast cancer cytosol pool, after labelling with 1 nmol/l [¹²⁵I]TAZ for 1 h at 0°C in the presence or absence of a 200-fold excess of radioinert oestradiol, was immunoprecipitated with H222, H226 or ER1D5 anti-ER monoclonal antibodies, and then analyzed by SDS-PAGE and autoradiography. (right) Another part of this cytosol pool was adsorbed onto HAP, labelled with 1 nmol/l [¹²⁵I]TAZ, extracted with 0.5 mol/l KCl, and immunoprecipitated before being subjected to electrophoresis. (B) Presumed structure of ER isoforms extracted from HAP with KCl. Potential sites of covalent attachment of TAZ [40,41] are indicated by open circles; antigenic sites for anti-ER monoclonal antibodies are shown above ER structure. The predicted ER isoforms extracted from HAP as well as their sizes determined by SDS-PAGE are shown below.

Figure 4

Correlation between ER mRNA and ER (by DCC assay). Breast tumours mRNA abundance (intensity of the 6.6-kb band) was expressed relatively to mRNA levels in MCF-7 cells (MCF-7 = 1).

Figure 5

Effect of heat treatment on the relative expression of ER isoforms. Human breast cancer cytosols were heated at 37°C for 2 min in the presence or absence of protease inhibitors. They were then labelled with [¹²⁵I]TAZ in the presence or absence of a 200-fold excess of radioinert oestradiol, immunoprecipitated with H-222 anti-ER monoclonal antibodies, and then subjected to SDS-PAGE. Lane 1, unheated control; lane 2, plus an excess of oestradiol; lane 3, 2 min heating in the absence of protease inhibitors; lane 4, 2 min heating in the presence of protease inhibitors.

Figure 6

In situ labelling of ER with [¹²⁵I]TAZ. Breast tissues slices (samples 1-4, ER positive; sample 5, ER-negative) were incubated with 1 nmol/l [¹²⁵I]TAZ for 1 h at 0°C and the unbound ligand was removed. Then, all tissues were mixed with Krebs-Ringer phosphate buffer containing 1% SDS, 1.6 mmol/l EDTA and 2% β-mercaptoethanol, and briefly homogenized. After lysis at 100°C, proteins were extracted with phenol, precipitated by acetone and were finally analyzed by SDS-PAGE. The figure shows the electrophoretic patterns of these tissue ERs.