Endogenous human prolactin and not exogenous human prolactin induces estrogen receptor alpha and prolactin receptor expression and increases estrogen responsiveness in breast cancer cells

Prolactin (PRL) and estrogen act synergistically to increase mammary gland growth, development, and differentiation. Based on their roles in the normal gland, these hormones have been studied to determine their interactions in the development and progression of breast cancer. However, most studies have evaluated only endocrine PRL and did not take into account the recent discovery that PRL is synthesized by human mammary cells, permitting autocrine/paracrine activity. To examine the effects of this endogenous PRL, we engineered MCF7 cells to inducibly overexpress human prolactin (hPRL). Using this Tet-On MCF7hPRL cell line, we studied effects on cell growth, PRLR, ER alpha, and PgR levels, and estrogen target genes. Induced endogenous hPRL, but not exogenous hPRL, increased ER alpha levels as well as estrogen responsiveness in these cells, suggesting that effects on breast cancer development and progression by estrogen may be amplified by cross-regulation of ER alpha levels by endogenous hPRL. The long PRLR isoform was also upregulated by endogenous, but not exogenous PRL. This model will allow investigation of endogenous hPRL in mammary epithelial cells and will enable further dissection of PRL effects on other hormone signaling pathways to determine the role of PRL in breast cancer.     

Indole-3-carbinol prevents PTEN loss in cervical cancer in vivo

Indole-3-carbinol (I3C) is a phytochemical (derived from broccoli, cabbage, and other cruciferous vegetables) with proven anticancer efficacy including the reduction of cervical intraepithelial neoplasia (CIN) and its progression to cervical cancer. In a breast cancer cell line, I3C inhibited cell adhesion, spreading, and invasion associated with an upregulation of the tumor suppressor gene PTEN, suggesting that PTEN is important in inhibition of late stages in the development of cancer. The goal of this study was to determine the expression of PTEN during the development of cervical cancer and whether I3C affected expression of PTEN in vivo. We show diminished PTEN expression during the progression from low-grade to high-grade cervical dysplasia in humans and in a mouse model for cervical cancer, the K14HPV16 transgenic mice promoted with estrogen. The implication is that loss of PTEN function is required for this transition. Additionally, dietary I3C increased PTEN expression in the cervical epithelium of the transgenic mouse, an observation that suggests PTEN upregulation by I3C is one mechanism by which I3C inhibits development of cervical cancer.     

Electromagnetic fields alter the expression of estrogen receptor cofactors in breast cancer cells

Breast cancer is the most common malignancy of women in Western societies. The increasing exposure to electromagnetic fields has been suspected to contribute to the rising incidence of breast cancer in industrialized countries. The majority of breast tumors is treated with the partial antiestrogen tamoxifen. Most tumors become resistant to tamoxifen in the course of treatment resulting in treatment failure. Electromagnetic fields reduce the efficacy of tamoxifen similar to tamoxifen resistance. In this study we investigated the mechanism by which electromagnetic fields influence the sensitivity to tamoxifen. In cells exposed to 1.2 microT of a 50 Hz electromagnetic field gene expression of cofactors of the estrogen receptors was compared to sham exposed cells. Using a gene array technology several cofactors were found to be differentially expressed. The expression of the coactivators, SRC-1 and AIB1, and of two corepressors, N-Cor and SMRT, was quantified by RT-PCR. Both coactivators were expressed more strongly in the exposed cells while the expression of two corepressors decreased. The RNA analysis was confirmed by Western blots. The contradirectional changes in gene expression of coactivators and corepressors by electromagnetic fields results in a lower sensitivity to tamoxifen. Electromagnetic fields may contribute to the induction of tamoxifen resistance in vivo.     

Effects of analogs of indole-3-carbinol cyclic trimerization product in human breast cancer cells

5,6,11,12,17,18-Hexahydrocyclonona[1,2-b:4,5-b*:7,8-b**]triindole (CTr) is a major digestive product of indole-3-carbinol (I3C) from Brassica vegetables and exhibits strong estrogenic activities. CTr increases proliferation of estrogen-dependent breast tumor cells, binds with strong affinity for the estrogen receptor-alpha (ERalpha), and activates expression of estrogen (E(2))-dependent genes. To begin to examine the structural features that determine the biological activity of CTr, we prepared and studied the effects of two analogs, 9,18-dihydro-12H-[1,2,5]trithionino[3,4-b:6,7-b*:9,8-b**]triindole (S(3)CTr) and 5,6,11,12,17,18-hexahydro-5,11,17-trimethylcyclonona[1,2-b:4,5-b*:7,8-b**]triindole (Me(3)CTr). N-Methylation of CTr completely ablated the estrogenic activities of CTr. In the dose range in which CTr was clearly estrogenic, Me(3)CTr exhibited no detectable effect on cell growth, ERalpha binding to E(2), or ERalpha-responsive gene expression. S(3)CTr showed mixed ERalpha agonist activities. It bound to the ERalpha and activated receptor binding with DNA, weakly activated expression of transfected E(2)-responsive reporter gene constructs, and strongly inhibited the E(2)-induced activation of these reporter constructs. S(3)CTr activated aryl hydrocarbon receptor (AhR)-mediated pathways, consistent with the moderately strong binding affinity of S(3)CTr for the AhR. Comparisons of the conformational characteristics among CTr and its two analogs indicated that the estrogenic effects of CTr are highly sensitive to apparently minor structural modifications, and further supported the hypothesis for a central role of hydrogen bonding around the nitrogen atom in CTr binding to the ligand binding site of ERalpha.     

Relation between cytochrome P450IA1 expression and estrogen receptor content of human breast cancer cells

Multidrug resistance (MDR) in an MCF-7 human breast cancer cell line (MCF7/Adr) is associated with decreased drug accumulation and overexpression of P-glycoprotein as well as alterations in the levels of specific drug-metabolizing enzymes, including decreased activity of the phase I drug-metabolizing enzyme aryl hydrocarbon hydroxylase (AHH) and increased expression of the anionic form of the phase II drug-metabolizing enzyme glutathione S-transferase. Since the development of MDR in this MCF-7 cell line is also associated with a loss of estrogen receptors (ER), we have examined the expression of cytochrome P450IA 1, the gene encoding AHH activity, in other breast cancer cell lines not selected for drug resistance but expressing various levels of ER. These studies show that a relationship exists between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible AHH activity and the ER content in a series of breast cancer cell lines. In these cell lines expression of AHH activity is regulated, at least in part, at the level of P450IA 1 RNA. While TCDD-specific binding proteins (Ah receptors) were found in each of the breast cancer cell lines, there was no apparent relation between the level of nuclear TCDD-binding proteins and the level of TCDD-inducible P450IA 1 expression. Previous studies from our laboratory have described an inverse relationship between levels of the anionic form of glutathione S-transferase and ER in breast cancer.(ABSTRACT TRUNCATED AT 250 WORDS)     

An estradiol-porphyrin conjugate selectively localizes into estrogen receptor-positive breast cancer cells

A conjugate of a C(11)-beta-derivative of estradiol and an asymmetric tetraphenylporphyrin was synthesized to study its potential selective uptake by breast cancer cells naturally over-expressing the nuclear receptor for estrogen (ER). Competitive radioligand binding assays of this conjugate with recombinant ER showed that the conjugate bound to ER in a dose-dependent manner with an EC50 of 274 nM, compared with 1 nM for estradiol, the natural ligand. Cellular uptake studies with ER-positive MCF-7 and ER-negative HS578t human breast cancer cells revealed that, the conjugate was taken up by MCF-7 cells in a dose-dependent manner, which was obliterated by co-incubation with a large excess of estradiol. On the other hand there was very little uptake of the un-conjugated porphyrin by MCF-7 and Hs578t cells. HS578t cells also showed insignificant uptake of the conjugate under the conditions of our experiment. These results strongly suggested that specific interaction between the endogenous ER in MCF-7 cells and the estrogen part of the conjugate enabled these cells to selectively internalize the conjugate over the un-conjugated porphyrin. Therefore, ER-binding conjugates of estradiol and porphyrins could potentially be used for ER-targeted photodynamic therapy of hormone-sensitive cancers of breast, ovary, gonads etc.     

Modeling the estrogen receptor to growth factor receptor signaling switch in human breast cancer cells

Breast cancer cells develop resistance to endocrine therapies by shifting between estrogen receptor (ER)-regulated and growth factor receptor (GFR)-regulated survival signaling pathways. To study this switch, we propose a mathematical model of crosstalk between these pathways. The model explains why MCF7 sub-clones transfected with HER2 or EGFR show three GFR-distribution patterns, and why the bimodal distribution pattern can be reversibly modulated by estrogen. The model illustrates how transient overexpression of ER activates GFR signaling and promotes estrogen-independent growth. Understanding this survival-signaling switch can help in the design of future therapies to overcome resistance in breast cancer.     

Sex-specific differences in the expression levels of estrogen receptor subtypes in colorectal cancer

Background: Altered expression of estrogen receptors alpha and beta (ERα and ERβ) has been hypothesized to play a role in carcinogenesis. However, little is known about the sex-specific differences of ER expression in colorectal cancer (CRC).Objective: This study examined ERα and ERβ protein levels in male and female patients with CRC.Methods: Using Western blot analysis, the intensity of ERα and ERβ protein levels was determined in tumor tissue and in corresponding normal colon mucosa from patients with CRC.Results: All 64 white patients (33 men, mean [SEM] age 64.1 [13.1] years, age range 26-90 years; 31 women, mean age 68.5 [14.5] years, age range 39-91 years [4 were premenopausal at time of surgery]) expressed ERα and ERβ protein in normal colon mucosa, and there were no significant differences between men and women. In tumor tissue, a significantly increased ERα protein level was observed in men (P = 0.02 vs normal tissue), whereas in women, the ERα level did not differ significantly between tumor and normal tissue. The level of ERβ protein in CRC was significantly reduced in both men and women, but more so in men (P = 0.04 vs women). Furthermore, in men, the ERβ level was significantly lower in poorly differentiated tumors than in moderately differentiated tumors (P < 0.03), whereas in women, poor differentiation of the tumor was not associated with a significant decrease of ERβ level.Conclusions: Altered levels of ER subtypes resulting in an increased ERα:ERβ ratio were found in patients with CRC. The observation of significantly greater alterations in men than in women supports the hypothesis of sex-specific differences in the pathogenesis of CRC.