The control of progesterone receptor expression in MCF-7 breast cancer cells: effects of estradiol and sex hormone-binding globulin (SHBG).

Estradiol controls the gene transcription and expression of many proteins in breast cancer cells, like the progesterone receptor, PR, that is up-regulated by the hormone. Moreover, estradiol is one of the crucial factors inducing the proliferation of breast cancer cells. Sex Hormone-Binding Globulin (SHBG), the plasma carrier for both estradiol and androgens, inhibits the estradiol-induced growth of MCF-7 cells (estrogen-dependent breast cancer cells), through its membrane receptor (SHBG-R), cAMP and PKA. The anti-estrogenic effect of SHBG, which has been described only as far as cell proliferation is concerned, could also play a meaningful role in the estradiol control of other factors in breast cancer cells. In the present study, the effect of SHBG on the estradiol control of PR expression (both mRNA and protein) and function (receptor binding capacity) in MCF-7 cells was examined. SHBG inhibited the estradiol-induced up-regulation of PR mRNA as well as protein level and function. Moreover, the effect of SHBG on estradiol control of PR expression and function was showed to be specific and mediated by PKA. The intermediacy of PKA in the PR expression control, together with the observation that it is effective in the condition in which the SHBG receptor is activated, supports the hypothesis that the anti-estrogenic effect of SHBG could be receptor-mediated. The ability of SHBG to inhibit estradiol action in a specific way in estrogen-dependent breast cancer cells has, therefore, to be taken into account for the development of future therapeutic strategies.

Estradiol induction of cAMP in breast cancer cells is mediated by foetal calf serum (FCS) and sex hormone-binding globulin (SHBG).

Plasma sex hormone-binding globulin (SHBG or SBP), the specific carrier for estradiol and androgens, after binding to its membrane receptor (SHBG-R), causes a significant increase of cAMP in the presence of estradiol, in both breast (MCF-7) and prostate (LNCaP) cancer cells maintained in serum-free medium. On the other hand, it has been proposed that estrogens, in addition to the well-known nuclear receptor pathway, exert their biological effect inducing cAMP, as a consequence of a direct membrane action, in breast cancer and uterine cells. The aim of the present study was to clarify this controversial issue by verifying if the cAMP increase in MCF-7 cells was a direct effect of estradiol, or if it was mediated by FCS proteins, such as bovine sex hormone-binding globulin; and to reevaluate the effect of human SHBG on cAMP induction in the presence of FCS. MCF-7 cells were maintained in DCC-FCS (treated with DCC to remove steroids), in SHBG-FREE/DCC-FCS (treated with DCC and with a specific affinity chromatography to remove bovine sex hormone-binding globulin), or in serum-free medium (SFM). It was observed that estradiol determined a significant time-dependent increase of cAMP only in MCF-7 cells maintained in 10% DCC-FCS. When cells were maintained in 10% SHBG-FREE/DCC-FCS, estradiol had no detectable effect. However, its ability to increase cAMP was observed again after the addition of human SHBG, in doses ranging from 5 to 50 nM. Moreover, in the presence of 10% SHBG-FREE/DCC-FCS, SHBG, even in the absence of estradiol, caused a significant increase of cAMP. In conclusion, the data reported in the present study suggest that the ability of estradiol to induce cAMP in MCF-7 cells is not due to a direct membrane effect of the hormone, but rather it is mediated by FCS. SHBG is one of the serum factors mediating estradiol action. Lastly, it was proven that SHBG triggers the cAMP pathway in MCF-7 cells in a physiologic culture condition and at physiologic concentrations.

Sex hormone-binding globulin, its membrane receptor, and breast cancer: a new approach to the modulation of estradiol action in neoplastic cells.

The role of human Sex Hormone-Binding Globulin (SHBG), the plasma carrier of sex steroids, and its membrane receptor, SHBG-R, in estrogen-dependent breast cancer has been investigated in our laboratory in the past few years. SHBG-R is expressed in MCF-10 A cells (not neoplastic mammary cells), MCF-7 cells (breast cancer, ER positive) and in tissue samples from patients affected with ER positive breast cancer, but not in estrogen-insensitive MDA-MB 231 cells. The SHBG/SHBG-R interaction, followed by the binding of estradiol to the complex protein/receptor, causes a significant increase of the intracellular levels of cAMP, but does not modify the amount of estradiol entering MCF-7 cells. The estradiol-induced proliferation of MCF-7 cells is inhibited by SHBG, through SHBG-R, cAMP and PKA. Similarly, the proliferation rate of tissue samples positive for SHBG-R was significantly lower than the proliferation rate of negative samples. SHBG and SHBG-R could thus trigger a 'biologic' anti-estrogenic pathway. In order to get a more detailed knowledge of this system, we first examined the frequence of the reported mutated form of SHBG in 255 breast cancer patients. The mutated SHBG is characterized by a point mutation (Asp 327 --> Asn) causing an additional N-glycosylation site, which does not affect the binding of steroids to SHBG. The frequence of the mutation was significantly higher (24.5%) in estrogen-dependent breast cancers than in healthy control subjects (11.6%). This observation confirms the close relationship between SHBG and estrogen-dependent breast cancer and suggests that the mutation could modify SHBG activity at cell site. Lastly, the possibility of using SHBG to modulate the estradiol action in breast cancer was further studied by transfecting MCF-7 cells with an expression vector carrying the SHBG cDNA (study in collaboration with G.L. Hammond). Transfected cells are able to produce significant amount of SHBG in their medium, but their SHBG-R is reduced to undetectable levels. The SHBG produced by transfected MCF-7 cells is, however, able to inhibit estradiol-induced proliferation of MCF-7 cells expressing a functional receptor. Thus, the local production of SHBG obtained with transfection could be a useful tool to control cell growth in estrogen-dependent breast cancer.

The additionally glycosylated variant of human sex hormone-binding globulin (SHBG) is linked to estrogen-dependence of breast cancer.

Sex Hormone-Binding Globulin (SHBG), the plasma carrier for androgens and estradiol, inhibits the estradiol-induced proliferation of breast cancer cells through its membrane receptor, cAMP, and PKA. In addition, the SHBG membrane receptor is preferentially expressed in estrogen-dependent (ER+/PR+) breast cancers which are also characterized by a lower proliferative rate than tumors negative for the SHBG receptor. A variant SHBG with a point mutation in exon 8, causing an aminoacid substitution (Asp 327-->Asn) and thus, the introduction of an additional N-glycosylation site, has been reported. In this work, the distribution of the SHBG variant was studied in 255 breast cancer patients, 32 benign mammary disease patients, and 120 healthy women. The presence of the SHBG mutation was evaluated with PCR amplification of SHBG exon 8 and Hinf I restriction fragment length polymorphism (RFLP) procedure. This technique allowed us to identify 54 SHBG variants (53 W/v and 1 v/v) in breast cancer patients (21.2%), 5 variants (4 W/v and 1 v/v) in benign mammary disease patients (15.6%), and 14 variants (W/v) in the control group (11.6%). The results of PCR and RFLP were confirmed both by nucleotide sequence of SHBG exon 8 and western blot of the plasma SHBG. No differences in the mean plasma level of the protein were observed in the three populations. The frequency of the SHBG variant was significantly higher in ER+/PR+ tumors and in tumors diagnosed in patients over 50 years of age than in the control group. This observation suggests the existence of a close link between the estrogen-dependence of breast cancer and the additionally glycosylated SHBG, further supporting a critical role of the protein in the neoplasm.

Overexpression of androgen-binding protein/sex hormone-binding globulin in male transgenic mice: tissue distribution and phenotypic disorders.

The rat androgen-binding protein/sex hormone-binding globulin (ABP/SHBG) gene in transgenic mice was previously shown to be specifically expressed in the testes. This study verifies a Sertoli cell location of ABP and translation of testicular ABP mRNA in the transgenic mice by dihydrotestosterone (DHT)-binding assays and immunohistochemistry. DHT-binding activities in the testis and epididymis of the hemizygous transgenic mice were elevated 20-fold as compared to activity in the wild-type tissues. DHT-binding activities were also elevated in blood plasma at least 25- to 50-fold in the transgenic mice; binding was undetectable in the plasma from control mice. Immunohistochemical analysis revealed that the transgenic testicular ABP was primarily in the cytoplasm of Sertoli cells and lumen of the seminiferous tubules. In some tubules, intense staining also was associated with spermatids. After transport to the epididymis, there were large amounts of immunoreactive ABP internalized in the epithelium of the initial segment and proximal caput. The increased levels of plasma and testicular ABP had no effect on levels of testosterone; there was a 30-fold range of plasma and testicular testosterone levels in the wild-type and transgenic mice. Increased ABP levels in the transgenic mice were associated with structural and functional abnormalities in the testis. Abnormal spermatogenesis resulted in extensive structural changes in the transgenic testis; the degree of the defect varied from near normality to the loss of most germ cells. In the affected mice, seminiferous tubules had smaller diameters and decreased numbers of germ cells, particularly in the spermatid stages of differentiation. Pyknotic nuclei and multinucleated cells were associated with the spermatids in the defective tubules, but not in the wild-type tubules. Consequently, mice with the spermatogenic disorder had reduced epididymal sperm numbers. The variable spermatogenic disorder was associated with variable male fertility. The homozygous transgenic male and female mice also had a serious motor dysfunction affecting their hind limbs. This study demonstrates how the transgenic mouse model can be used to study ABP's function, and the data support several hypotheses on its function in the testis and epididymis.

Distribution of immunoreactive androgen-binding protein/sex hormone-binding globulin in tissues of the fetal rat.

Androgen-binding protein/sex hormone-binding globulin (ABP/SHBG) is an extracellular carrier protein that binds androgens and estrogens with high affinity. In the adult, ABP/SHBG is thought to function in the male reproductive system and the general circulation in both sexes to modulate the actions of sex steroids. The ABP/SHBG gene is also expressed in the embryonic rat liver, where SHBG is secreted into the fetal blood of male and female rats. The embryo also expresses an alternative SHBG with a unique N-terminal sequence. In this study, the distribution of immunoreactive SHBG in the 17-day-old male fetal rat was determined with six antisera. In general, all of the antisera reacted with the same structures. Specific tissue immunoreactivity was mostly cytoplasmic and/or extracellular. By far the most prominent immunoreactive structures were the mesoderm-derived tissues: connective tissue, striated and cardiac muscle, cartilage, and the liver hematopoietic system. In addition, all regions of the fetal brain contained immunoreactive neurons. In the developing male reproductive system, there was minor reactivity in the testicular cords, whereas the connective tissue in the differentiating Wolffian duct stained with all of the antisera. The Wolffian duct epithelium and epithelia in other developing organs contained small amounts of immunoreactive SHBG, except for the lung, which stained in the epithelial extracellular matrix. An antibody raised against a unique N-terminal peptide specific for the alternative SHBG protein revealed that it was also present in many tissues. These data suggest that SHBG is important for the differentiation of mesodermal tissues. SHBG may modulate the action of androgens in embryonic stroma, thereby regulating development of the epithelium in hormone-dependent tissues.

The alternate N-terminal sequence of rat androgen-binding protein/sex hormone-binding globulin contains a nuclear targeting signal.

Androgen-binding protein/sex hormone-binding globulin (ABP/SHBG) is an extracellular carrier protein of androgens and estrogens. The protein regulates the bioavailability of sex steroids, and expanding evidence suggests that it also acts as a hormone. ABP/SHBG is secreted by Sertoli cells and hepatocytes using a signal peptide. An alternate messenger RNA encodes a nonsecreted form of ABP/SHBG (Alt-ABP/SHBG) that has a unique N-terminal amino acid sequence. In this study, we report that the alternate N-terminal sequence targets Alt-ABP/SHBG to the nucleus instead of the endoplasmic reticulum. The recombinant Alt-ABP/SHBG expressed in COS-7 cells was located in the nucleus, whereas recombinant cellular ABP/SHBG was primarily cytoplasmic. Neither dihydrotestosterone nor estradiol had any detectable effect on the ABP/SHBG or Alt-ABP/SHBG cellular location. Although the function of the nuclear Alt-ABP/SHBG is unknown, it may act as a modulator of androgen receptor- and/or estrogen receptor-mediated gene regulation.

Sex steroid binding protein exerts a negative control on estradiol action in MCF-7 cells (human breast cancer) through cyclic adenosine 3',5'-monophosphate and protein kinase A.

Estradiol is considered to be a critical factor in the growth induction of some breast cancer cells, like MCF-7 cell line. Among other compounds involved in the control of neoplastic mammary cell growth, cAMP has been suggested, on the other hand, to exert an antiproliferative effect. Sex steroid binding protein (SBP) sex hormone binding globulin (SHBG), the plasma carrier for both androgens and estradiol, recognizes a specific receptor located on membranes of estrogen- and androgen-sensitive tissue and cultured cells (e.g. MCF-7 cell). The interaction of estradiol with the receptor-bound SBP has been reported to induce a significant accumulation of cAMP in MCF-7 cells; in addition, a negative modulation of estradiol induced proliferation of these cells has been described after treatment with SBP. We report here a more detailed observation about the effect of SBP on MCF-7 cell estradiol-induced growth as well as the possible linkage between SBP and its membrane receptor and protein kinase A activity. MCF-7 cell growth was induced by estradiol, but the effect of estradiol was completely abolished by cell treatment with both SBP and estradiol. The inhibitory effect of SBP was highly specific. Because it was suggested that SBP might act through cAMP, we investigated the effect of SBP and estradiol in cells treated with protein kinase A inhibitor peptide (6-22) amide, a specific inhibitor of the cAMP target protein kinase A. The blockade of PKA had no effect on estradiol action on cell growth but masked completely the effect of SBP because MCF-7 increased growth sustained by estradiol was fully detectable also in the presence of SBP. We also observed that MCF-7 cells treated with increasing doses of 8Br-cAMP, cAMP analog and PKA activator, showed a progressive reduction of their growth. 8Br-cAMP was also able to inhibit estradiol promotion of MCF-7 cell growth. The inhibitory effect of 8Br-cAMP on estradiol-induced proliferation was already detectable at analog concentration of 100 nM, which has been reported to be the level reached by cAMP in MCF-7 cells treated with SBP and estradiol. In conclusion, the present study strongly confirms our previous observation that SBP inhibits the estradiol induction of MCF-7 cell growth, appropriately suggesting that this SBP action, a consequence of the interaction with the receptor, is likely to be mediated by cAMP and PKA. In addition, the study implies a significant role of cAMP in the control of breast cancer cell growth.

Sex steroid-binding protein and its membrane receptor in estrogen-dependent breast cancer: biological and pathophysiological impact.

Data obtained in our laboratory about the membrane receptor for sex steroid-binding protein (SBP-R) in human breast cancer are reported. SBP-R was detected in MCF-7 cells (estrogen receptor positive, ER+), while MDA-MB 231 cells (ER-) did not bind SBP. MCF-7 cells treated with SBP and E2 showed a marked increase of intracellular cAMP, and a significant reduction of both E2 induced cell proliferation and E2-mediated increase of progesterone receptor (PGR). The inhibition of E2 effects in MCF-7 cells was shown to be highly specific for SBP and mediated by protein kinase A, the target of cAMP. Membrane SBP-R was also evaluated in primary breast cancers. SBP-R was detectable only on ER+/PR+ samples and SBP-R+ samples presented a lower proliferation rate than negative samples. Our data, thus suggest that SBP-R and ER could be functionally related and also that SBP could modulate estrogen action at target cell site.

The hepatic receptor for sex steroid-binding protein: study on a non-malignant cell line (Chang liver).

The binding of human sex steroid-binding protein (SBP), labelled with 125I, was studied on whole cultured liver cells (Chang liver cells). SBP was shown to bind to a receptor site on normal hepatocytes. The binding was time- and temperature-dependent, highly specific and of high affinity. The liver receptor recognizing SBP was demonstrated to be different from the asialoglycoprotein receptor; in addition, laminin, which is structurally related to SBP, could not bind to the receptor. SBP was shown to recognize two binding sites with different affinities; the low affinity site was shown to possess a remarkably high capacity. This characteristic, which until now has been described only for hepatocytes, could be related to the unique role of the liver with regard to both SBP and sex steroids.