Progesterone receptor replenishment in T47D human breast cancer cells. Roles of protein synthesis and hormone metabolism

T47D are unusual human breast cancer cells that do not require estrogen to synthesize high levels of progesterone receptors. These cells can, therefore, be used to study the mechanisms by which progesterone, freed of estrogen interference, controls the synthesis of its receptors. In a recent paper we described progesterone receptor translocation and a subsequent very rapid nuclear processing step that results in an apparent loss of 60 to 80% of cellular progesterone receptors, 30 to 60 min after progesterone treatment. This paper deals with the replenishment of cellular receptors following processing. If progesterone is removed from cells after 60 min of treatment, cytoplasmic progesterone receptors replenish in 16 to 20 h. However, replenishment occurs even during chronic progesterone treatment; this is an artifact created by the extremely rapid (t1/2 approximately 2 h) metabolism of progesterone in media exposed to cells. If progesterone metabolism is blocked, then replenishment is not seen, probably because the hormone continuously retranslocates the newly replenished sites. There is an early protein synthesis-dependent step; cycloheximide in the first 4 h inhibits replenishment 24 h later, but if cycloheximide is slightly delayed (beyond 4 h), replenishment proceeds normally. In contrast to progesterone, the synthetic progestin R5020 completely suppresses progesterone receptor replenishment even 96 h after its removal from the medium. This compound can bind covalently to receptors and may be very difficult to remove from cells. Clearly, progestin treatment, and by analogy, circulating progesterone, will have profound effects on cytoplasmic and nuclear progesterone receptor levels when these are measured in biopsied human tumors as an adjunct to endocrine therapy.     

Progesterone antagonists and progesterone receptor modulators in the treatment of breast cancer

Progesterone antagonists (PAs) (antiprogestins) or progesterone receptor modulators (PRMs) form an interesting category of new hormonal agents in the treatment of breast cancer. In vitro, antiproliferative effects of different PAs are mainly observed in estrogen-stimulated growth of PR-positive tumor cell lines. Both progestin agonist/antagonist actions on mammary tumor cells are dependent on the type of cell line, culture medium and concentrations of the PAs used, and type of biologic response measured. In various experimental animal tumor models, different PAs showed a greater antitumor activity than tamoxifen or high-dose progestins. Most interestingly, combination treatment of different PAs (mifepristone, ORG 31710, onapristone) or PRMs with different antiestrogens (tamoxifen, droloxifen, ICI 164384) or with an aromatase inhibitor (atemestane) showed greater antitumor efficacy than treatment with each single type of drug alone. These additive antiproliferative effects were demonstrated in various experimental in vitro and in vivo models. In some studies, these effects were accompanied by additive effects on several cell biologic parameters. In pretreated postmenopausal patients with metastatic breast cancer, objective responses have been observed in 10-12%, and stable disease in 42-46% of the patients; in previously untreated patients objective response rates of 11 and 56% have been reported. The clinical development of onapristone was stopped because of liver toxicity. At the present time, apart from development of new pure potent PAs, clinical investigation of combined therapy of PAs with antiestrogens are urgently needed.     

Progesterone receptor regulation by retinoic acid in the human breast cancer cell line T-47D

Data are presented which document the first known effect of retinoic acid on progesterone receptor (PR) gene expression. Treatment of T-47D human breast cancer cells with retinoic acid for 48 h resulted in a marked concentration-dependent decrease in the level of PR mRNA and immunoreactive protein which was similar to the known effect of progestins on these parameters. Retinoic acid, however, did not bind to PR, nor did it cause the previously demonstrated increase in PR molecular weight observed after progestin exposure. When T-47D cells were treated with retinoic acid for 6 h rather than 48 h, no reduction in the level of PR protein was noted at any retinoic acid concentration whereas the effects of retinoic acid on PR mRNA at 6 and 48 h were the same. Examination of the time course of the effects of retinoic acid revealed a rapid decrease in PR mRNA levels detectable 1 h after and maximal 6 h after treatment of T-47D cells with retinoic acid. These effects of retinoic acid contrasted with previously demonstrated progestin effects on PR mRNA which were not apparent until 3 h after and were not maximal until 12 h after treatment. As expected, the PR protein concentration was unaffected for at least 6 h but was maximally decreased 24-48 h after retinoic acid treatment. In summary, retinoic acid treatment of T-47D cells caused a decrease in the cellular PR concentration by decreasing levels of receptor mRNA and protein, suggesting that retinoic acid is capable of modulating sensitivity to progestins in human breast cancer cells.     

SYK expression in human breast cancer

BACKGROUND: Syk (Splenic Tyrosine Kinase) is an intracellular receptor protein kinase involved in cell proliferation, differentiation and phagocytosis. It has been studied in T and B lymphocytes, NK cells and platelets. The strong expression of Syk in mammary gland prompted research into its potential role in mammary carcinogenesis. There have been very few studies about its role in breast cancer with conflicting results. This study aims to investigate the hypothesis that Syk expression is down-regulated in breast cancer compared with ANCT and the association between its expression and clinicopathological parameters. MATERIALS AND METHODS: mRNA was extracted from 48 breast cancer specimens. Relative Syk to ribosomal RNA expression was determined by RT-PCR and Taqman methodology. Mann-Whitney U test was used to examine the association between Syk expression in cancer and ANCT. Spearman's rank correlation test was used to examine the association between Syk expression in tumours and patients' age, tumour size, tumour grade, estrogen and progesterone receptor status, lymph node metastasis, vascular invasion and clinical outcome. RESULTS: The median for the relative value of Syk expression was 0.17 and 0.18 (range: 0.12 - 0.56 and 0.0 - 1.77) for tumours and ANCT respectively. There was no significant association between Syk expression in cancers and ANCT (p= 0.598) nor between Syk expression in tumours and patients' age, tumour size, tumour grade, estrogen and progesterone receptor status, lymph node metastasis, vascular invasion or prognosis. CONCLUSION: This study shows that Syk mRNA expression does not seem to vary between breast tumours and ANCT. Furthermore, we observed no significant association between Syk expression and clinicopathological parameters.     

Lactoferrin expression in human breast cancer

We analyzed lactoferrin expression in 78 samples from patients with sporadic breast cancer and found 31/78 negative for mRNA expression. Similar results were obtained by immuno-histochemical localization of the lactoferrin protein. We did not find relationship between lactoferrin expression and clinical parameters. We investigated for the absent lactoferrin expression in some cases of breast cancer. In 68 of the samples analyzed, we found an inverse correlation between estrogen receptor expression and lactoferrin expression (P < 0,0001), thus indicating that regulation by the estrogen receptor is not the main element responsible for the expression of lactoferrin in breast cancer. Analysis of methylation of the lactoferrin genomic DNA extracted from the same patients revealed that the degree of methylation does not explain the observed absence of lactoferrin. The 937 bp lactoferrin promoter was investigated for possible mutations. By single-strand conformation polymorphism analysis one polymorphic site was found and characterized.     

Estrogen-regulated protein in human breast cancer

An estrogen-regulated cytoplasmic protein has been purified from MCF-7 human breast cancer cells with anion exchange and monoclonal antibody affinity chromatographies. The purified protein has a monomeric mol. wt of 28,000 and isoelectric species with pI's between 5.9 and 6.0. Amino acid analysis indicates the protein is acidic, is probably hydrophilic, and contains unusually low amounts of methionine and half cystine. The monoclonal antibodies are of the IgG₁ subclass, exhibit a high affinity for the 28K protein, and recognize an antigenic site that is stable to sodium dodecyl sulfate (SDS) gel electrophoresis and electrophoretic transfer techniques. These monoclonal antibodies have been used to detect 28K in certain human breast tumors.     

Functional expression of the human breast cancer resistance protein in Pichia pastoris

We report functional expression of BCRP in Pichia pastoris in which BCRP was produced as a 62 kDa underglycosylated protein. BCRP expression level in P. pastoris was comparable to that in HEK cells. The basal BCRP ATPase activity in the yeast membranes was approximately 40-80 nmol Pi/min/mg protein, which can be modulated by known BCRP substrates and inhibitors. Photolabeling of BCRP with 8-azido[alpha-32P]ATP was dependent preferentially on the presence of Co2+ than Mg2+ and could be inhibited by a molar excess of ATP. Vanadate-induced trapping of 8-azido[alpha-32P]ADP by BCRP was much more significant in the presence of Co2+ than that with Mg2+. The Km and Vmax values of BCRP for [3H]E1S transport were 3.6+/-0.3 microM and 55.2+/-1.6 pmol/min/mg protein, respectively. This efficient and cost-effective expression system should facilitate large scale production and purification of BCRP for further structural and functional analyses.     

Enhanced phosphorylation of progesterone receptor by protein kinase C in human breast cancer cells

Affinity-isolated progesterone receptor (PR) from human breast cancer cells incubated with [32P]orthophosphate was shown to exist as a phosphoprotein. Exposure of the cells to 10 nM phorbol-12-myristate-13-acetate (PMA) for 10 min increased by 30-40% the amount of label incorporated into the 116-kDa receptor protein. A two-fold increase in the total number of steroid binding sites was also observed in cells receiving PMA treatment. This apparent unmasking of PR binding sites by phosphorylation probably involved conformational changes to existing receptor complexes and affected the eventual state of receptor dissociation or transformation. An increase primarily in the 8 S sedimenting molecular species was observed but PMA treatment also led to the appearance of a smaller, 2-3 S form of receptor (10% of total) that was not present in control samples. When cytosols were partially transformed in vitro by ATP and salt, all molecular species of receptor (8, 4, and 2-3 S) from the PMA-treated samples consistently migrated faster in sucrose gradients. The larger amount of 2-3 S receptor in PMA-treated samples disappeared when ATP, but not salt, was the transforming agent. These results suggest a major role for phosphorylating reactions in the receptor-mediated action of steroids by regulating hormone-binding and influencing receptor transformation. Tumor promoters such as the phorbol esters may act by artificially increasing the level of processing of steroid receptor.     

Altered expression of insulin receptor isoforms in breast cancer

Purpose

Insulin-like growth factor (IGF) signaling through human insulin receptor isoform A (IR-A) contributes to tumorigenesis and intrinsic resistance to anti-IGF1R therapy. In the present study, we (a) developed quantitative TaqMan real time-PCR-based assays (qRT-PCR) to measure human insulin receptor isoforms with high specificity, (b) evaluated isoform expression levels in molecularly-defined breast cancer subtypes, and (c) identified the IR-A:IR-B mRNA ratio as a potential biomarker guiding patient stratification for anti-IGF therapies.

Experimental Design

mRNA expression levels of IR-A and IR-B were measured in 42 primary breast cancers and 19 matched adjacent normal tissues with TaqMan qRT-PCR assays. The results were further confirmed in 165 breast cancers. The tumor samples were profiled using whole genome microarrays and subsequently subtyped using the PAM50 breast cancer gene signature. The relationship between the IR-A:IR-B ratio and cancer subtype, as well as markers of proliferation were characterized.

Results

The mRNA expression levels of IR-A in the breast tumors were similar to those observed in the adjacent normal tissues, while the mRNA levels of IR-B were significantly decreased in tumors. The IR-A:IR-B ratio was significantly higher in luminal B breast cancer than in luminal A. Strong concordance between the IR-A:IR-B ratio and the composite Oncotype DX proliferation score was observed for stratifying the latter two breast cancer subtypes.

Conclusions

The reduction in IR-B expression is the key to the altered IR-A:IR-B ratio observed in breast cancer. The IR-A:IR-B ratio may have biomarker utility in guiding a patient stratification strategy for an anti-IGF therapeutic.     

Progesterone receptor gene and protein expression in the anterior preoptic area and hypothalamus of defeminized rats

Progesterone receptor (PR) plays an important role during sexual differentiation of the rat brain. The objective of the present study was to determine PR protein and gene expression pattern in preoptic-anterior hypothalamic area (POA-AHA) and hypothalamus (HYP), after estradiol or testosterone treatment during the postnatal critical period of sexual differentiation of the rat brain (defeminized animals). Three-day-old female rats were subcutaneously (s.c.) injected with a single dose of 17beta-estradiol (200 microg), or testosterone enanthate (200 microg), or vehicle (corn oil). POA-AHA and HYP were dissected 3 h, 24 h, and 14 days, as well as on the day of vaginal opening (VO) after treatments. Other animals, previously treated as above, were acutely injected with 17beta-estradiol (5 microg) on the day of VO; POA-AHA and HYP were obtained 3 h later. Total RNA was extracted and processed for semiquantitative RT-PCR and tissue slices were prepared for protein detection by immunohistochemistry. We observed that PR mRNA expression was increased in POA-AHA and HYP of the animals treated with estradiol or testosterone 3 hours after treatments, compared with the vehicle-treated control group. We also found a significant increase in PR mRNA and protein expression in POA-AHA and HYP on the day of VO in both estradiol and testosterone defeminized rats. Interestingly, the acute administration of estradiol on the day of VO (VO + E(2)) did not increase PR mRNA or protein expression in POA-AHA and HYP of either estradiol or testosterone defeminized animals, as opposed to the marked induction observed in the intact animals of the control group. The overall results suggest that estradiol and testosterone treatment during the postnatal critical period of sexual differentiation of the brain modifies the regulation of the PR mRNA and protein expression during early onset of maturity.