Interaction of growth factors during progression towards steroid independence in T-47-D human breast cancer cells

When deprived of steroid in the long term, T-47-D human breast cancer cells lose estrogen sensitivity of cell growth. This loss of response results from an increased basal growth rate in the absence of steroid, not from a loss of estrogen-stimulated growth, and it occurs without any loss of estrogen receptor number or function. Growth factor gene expression and sensitivity have been investigated in this model system in an attempt to unravel the molecular mechanisms underlying the progression to steroid autonomy. The transition was accompanied by a decreased dependence on added serum and by a loss of the stimulatory effects of insulin and basic fibroblast growth factor, but also by an acquired sensitivity to stimulation by transforming growth factor-beta (TGF-beta). An increase in TGF-beta 1 mRNA was detected following loss of steroid sensitivity. There was no increase in epidermal growth factor (EGF) receptor number. These findings are discussed in relation to current knowledge concerning the mechanisms by which estrogens stimulate breast cancer cell proliferation.     

Transition of human breast cancer cells from an oestrogen responsive to unresponsive state

An in vitro model system is described for studying the problem of loss of steroid sensitivity in breast cancer cells. Growth of cloned oestrogen-sensitive human breast cancer cells in the long-term absence of steroid gives rise to a population of oestrogen-insensitive cells. In ZR-75-1 cells, the effect is clonal but occurs at high frequency suggesting a mechanism affecting a wide proportion of the cell population synchronously. This does not involve any reduction in oestrogen receptor number. Furthermore, there is no coordinated loss of oestrogen-sensitive molecular markers, showing that oestrogen receptors remain not only present but functional. These growth changes are not accompanied by any loss of growth inhibition by antioestrogen. Although steroid deprivation does not result in loss of oestrogen-sensitive markers, this does not hold true for other steroids. There was a reduction in progestin, androgen and glucocorticoid regulation on transfected LTRs. Loss of steroid-sensitive growth was accompanied by changes in response to exogenous growth factors and altered endogenous growth factor mRNA production. Steroid-deprived T-47-D cells acquire sensitivity to stimulation by TGFβ and have raised TGFβ₁ and TGFβ₂ mRNA levels. ZR-75-1 cells are growth inhibited by TGFβ and have reduced TGFβ₁mRNA levels. In MCF-7 cells, increased IGFII mRNA, following transfection, can result in an increased basal cell growth rate in the absence of steroid. These findings are discussed in relation to possible autocrine mechanisms in the loss of steroid sensitivity of breast cancer cells.     

Cyclic AMP-dependent protein kinase modulation of the glucocorticoid-induced cytolytic response in murine T-lymphoma cells

The antiproliferative effect of glucocorticoid hormones on lymphoid tissue serves as the basis for their use in chemotherapy of lymphomas and leukemias. The effectiveness of the steroid-mediated response is potentially contingent upon a variety of factors, including the cellular level of glucocorticoid receptors. This report demonstrates that differences in the expression of the glucocorticoid receptor gene can modulate steroid sensitivity of individuals within a population of lymphoma cells. We have also found that loss of cAMP-dependent protein kinase activity caused a measurable decrease of steroid sensitivity in the murine T-lymphoma WEHI-7 without producing a significant change in steroid binding capacity. However, the extent of this change in sensitivity was dependent upon the level of glucocorticoid receptor expression. Lymphoma cells containing few spare steroid receptors became significantly resistant to glucocorticoids through loss of cAMP-dependent kinase function. On the other hand, elevated levels of cAMP were found to cause an increase in glucocorticoid receptor mRNA concentrations. Thus, cAMP-dependent protein kinase activity has the potential to modulate a lymphoma cell's steroid sensitivity by affecting the level of glucocorticoid receptor expression as well as the receptor's efficiency in producing a cytolytic response.     

Expression of sex steroid receptors and IGF-1 mRNA in breast tissue--effects of hormonal treatment

The mechanisms behind increased breast tissue proliferation and a possibly increased breast cancer risk in women using hormonal contraception (HC) and hormonal replacement therapy (HRT) are incompletely understood. We analyzed breast tissue from 20 premenopausal and seven postmenopausal women undergoing reduction mammoplasties for estrogen receptor (ER) and progesterone receptor (PR) content as well as mRNA levels for ER, PR and insulin-like growth factor-1 (IGF-1). The receptor values were correlated to IGF-1 mRNA concentrations and levels of steroid and peptide hormones and SHBG. In women using HC, we found significantly lower ER values (p=0.02) but non-significantly lower ER mRNA levels compared to those in naturally cycling women. PR and PR mRNA were no different. Women on HC displayed a higher breast tissue proliferation (p=0.05) expressed as Ki-67, MIB-1 positivity, which was correlated with IGF-1 mRNA (r_s=0.82, p=0.04). Since the concentration of sex steroid receptors in breast tissue is comparatively low and steroid receptors are down-regulated during hormonal treatment, mechanisms other than direct sex steroid receptor action are likely to be present. Our results suggest a role for IGF-1 in the proliferative response of breast tissue during exogenous hormonal treatment.     

Cell cycle control in breast cancer cells

In breast cancer, cyclins D1 and E and the cyclin-dependent kinase inhibitors p21 (Waf1/Cip1)and p27 (Kip1) are important in cell-cycle control and as potential oncogenes or tumor suppressor genes. They are regulated in breast cancer cells following mitogenic stimuli including activation of receptor tyrosine kinases and steroid hormone receptors, and their deregulation frequently impacts on breast cancer outcome, including response to therapy. The cyclin-dependent kinase inhibitor p16 (INK4A) also has a critical role in transformation of mammary epithelial cells. In addition to their roles in cell cycle control, some of these molecules, particularly cyclin D1, have actions that are not mediated through regulation of cyclin-dependent kinase activity but may be important for loss of proliferative control during mammary oncogenesis.     

Sodium molybdate increases the amount of progesterone and estrogen receptor detected in certain human breast cancer cytosols

When sodium molybdate is added at a final concentration of 20 mM, additional 85 and 4S progesterone (³ H-R5020) receptor can be detected in the cytosols from a number of human breast cancers. Additional estrogen receptor also could be measured in some cytosols, and a quantitative temperaturedependent conversion of 8S to 4S binding molecules achieved. Sodium molybdate also prevented the loss of binding activity that occurred when cytosols were incubated at 30° in the absence of added estradiol. In addition to increasing the amount of progesterone receptor, and to a lesser extent estrogen receptor that may be detected, elucidation of the mechanism by which this salt stabilized receptors should contribute to further understanding of how cytosol steroid receptor content and function is regulated.     

Progression to steroid insensitivity can occur irrespective of the presence of functional steroid receptors

A major problem in treatment of cancers arising in steroid-sensitive cells is their inevitable progression to a steroid-insensitive state; current therapies are based on the assumption that hormone insensitivity is associated with loss of receptor. We demonstrate for the first time that breast tumor cells can progress to steroid insensitivity in spite of functional steroid receptors. Transfection of the steroid-inducible LTR-C3 gene into unresponsive S115 mouse mammary tumor cells results in full inducibility of that gene with both androgen and glucocorticoid. Thus, although all known endogenous inducible parameters are lost, the steroid sensitivity of a transfected exogenous gene demonstrates that the machinery for steroid responsiveness is still fully functional. Furthermore, these transfected genes retain steroid sensitivity only while steroid is present; on prolonged withdrawal of steroid, they lose responsiveness, implying an epigenetic mechanism is involved.     

Calcitonin receptors in a cloned human breast cancer cell line (MCF 7)

A human breast cancer cell line, MCF 7, is shown to possess a specific calcitonin receptor and calcitonin responsive adenylate cyclase, and calcitonin treatment results in activation of cyclic AMP-dependent protein kinase. Studies with several analogues of calcitonin show that the receptor and adenylate cyclase response preserve the ability to discriminate among the structure-function relationships of the calcitonin molecule. The same cell line has been shown recently to possess a receptor for the steroid hormone, 1,25(OH)₂-vitamin D. Coexistence in MCF 7 cells of receptors for two calcium-regulating hormones may be related to the osteoclast-like properties of these cells.     

Long Chain Fatty Acyl-CoA Synthetase 4 Is a Biomarker for and Mediator of Hormone Resistance in Human Breast Cancer

The purpose of this study was to determine the role of long-chain fatty acyl-CoA synthetase 4 (ACSL4) in breast cancer. Public databases were utilized to analyze the relationship between ACSL4 mRNA expression and the presence of steroid hormone and human epidermal growth factor receptor 2 (HER2) in both breast cancer cell lines and tissue samples. In addition, cell lines were utilized to assess the consequences of either increased or decreased levels of ACSL4 expression. Proliferation, migration, anchorage-independent growth and apoptosis were used as biological end points. Effects on mRNA expression and signal transduction pathways were also monitored. A meta-analysis of public gene expression databases indicated that ACSL4 expression is positively correlated with a unique subtype of triple negative breast cancer (TNBC), characterized by the absence of androgen receptor (AR) and therefore referred to as quadruple negative breast cancer (QNBC). Results of experiments in breast cancer cell lines suggest that simultaneous expression of ACSL4 and a receptor is associated with hormone resistance. Forced expression of ACSL4 in ACSL4-negative, estrogen receptor α (ER)-positive MCF-7 cells resulted in increased growth, invasion and anchorage independent growth, as well as a loss of dependence on estrogen that was accompanied by a reduction in the levels of steroid hormone receptors. Sensitivity to tamoxifen, triacsin C and etoposide was also attenuated. Similarly, when HER2-positive, ACSL4-negative, SKBr3 breast cancer cells were induced to express ACSL4, the proliferation rate increased and the apoptotic effect of lapatinib was reduced. The growth stimulatory effect of ACSL4 expression was also observed in vivo in nude mice when MCF-7 control and ACSL4-expressing cells were utilized to induce tumors. Our data strongly suggest that ACSL4 can serve as both a biomarker for, and mediator of, an aggressive breast cancer phenotype.     

Multihormone regulation of MMTV-LTR in transfected T-47-D human breast cancer cells

Multihormonal regulation on the long terminal repeat (LTR) region of mouse mammary tumour virus (MMTV) has been studied using T-47-D human breast cancer cells stably transfected with the steroid sensitive LTR-C3 chimaeric gene. The specificity of steroid action on transfected LTR sequences has been compared with regulation of endogenous cellular markers. We conclude that the hormone response element of the LTR can be induced by physiological concentrations of androgen, progestin and glucocorticoid. 17 beta-Oestradiol did not regulate the LTR at physiological levels but an effect was found at 10(-6) M. This effect was not inhibited by antioestrogen nor was it reproduced by the synthetic oestrogen diethylstilboestrol suggesting such effects do not occur via the oestrogen receptor. The antioestrogens tamoxifen and transhydroxytamoxifen do not induce the LTR. No significant steroid competition was found in LTR regulation: whilst oestradiol did not act at physiological concentration it did not interfere with induction by androgen, progestin or glucocorticoid. Such gene regulation did not simply follow receptor status of the cells nor was it reflected in patterns of growth regulation by steroids. The implications of these findings on the mechanism of steroid hormone action are discussed.     

Natural history of estrogen receptor-negative,progesterone receptor-positive breast cancer

The biological significance of estrogen receptor-negative but progesterone receptor-positive breast carcinomas is not clear. In the present study the aggressiveness of breast carcinomas in relation to ER and PgR status has been investigated. The probability of disease-free survival in 297 node-negative breast carcinoma patients was monitored during a follow-up ranging from six to 96 months (median 45 months). Steroid hormone receptor content was assayed with the biochemical method recommended by the EORTC. The probability of disease-free survival was significantly worse for patients with ER-negative, PgR-positive carcinomas compared to the other three steroid hormone receptor phenotypes. Our results suggest that ER-negative, PgR-positive breast carcinomas are biologically different in terms of aggressiveness from the other steroid hormone receptor phenotypes.     

Expression of Long-chain Fatty Acyl-CoA Synthetase 4 in Breast and Prostate Cancers Is Associated with Sex Steroid Hormone Receptor Negativity

Previous studies have shown that key enzymes involved in lipid metabolic pathways are differentially expressed in normal compared with tumor tissues. However, the precise role played by dysregulated expression of lipid metabolic enzymes and altered lipid homeostasis in carcinogenesis remains to be established. Fatty acid synthase is overexpressed in a variety of cancers, including breast and prostate. The purpose of the present study was to examine the expression patterns of additional lipid metabolic enzymes in human breast and prostate cancers. This was accomplished by analysis of published expression databases, with confirmation by immunoblot assays. Our results indicate that the fatty acid-activating enzyme, long-chain fatty acyl-CoA synthetase 4 (ACSL4), is differentially expressed in human breast cancer as a function of estrogen receptor alpha (ER) status. In 10 separate studies, ACSL4 messenger RNA (mRNA) was overexpressed in ER-negative breast tumors. Of 50 breast cancer cell lines examined, 17 (89%) of 19 ER-positive lines were negative for ACSL4 mRNA expression and 20 (65%) of 31 ER-negative lines expressed ACSL4 mRNA. The inverse relationship between ER expression and ACSL4 expression was also observed for androgen receptor status in both breast and prostate cancers. Furthermore, loss of steroid hormone sensitivity, such as that observed in Raf1-transfected MCF-7 cells and LNCaP-AI cells, was associated with induction of ACSL4 expression. Ablation of ACSL4 expression inMDA-MB-231 breast cancer cells had no effect on cell proliferation; however, sensitivity to the cytotoxic effects of triacsin C was increased three-fold in the cells lacking ACSL4.     

From the structure of steroid receptors to their assessment by immunocytochemistry in target cells

Immunohistochemical studies with antibodies to steroid hormone receptors provide new insight in the mechanism of action of steroid hormones. Immunologically reactive estrogen and progesterone receptors are found exclusively in cell nuclei of target cells even in the absence of the hormonal ligand. A hormonal treatment inducing receptor transformation and "translocation" to the nucleus does not modify the intracellular distribution of the receptor. This result is in contradiction with most biochemical studies which show a displacement of receptor from the cytosolic fraction to the nuclear fraction after hormone-receptor complex formation. We propose that different affinity levels of the non-transformed and hormone-complexed receptor molecules for nuclear structure produce unequal losses of nuclear receptor during homogenization. A lesser loss appears as an increase in nuclear binding sites or immunologically reactive receptor. The glucocorticosteroid receptor differs from the others in that it shows an increase of nuclear immunoreactive receptor after hormone administration. This result was accepted as evidence for a nuclear translocation in the sense initially proposed for all steroid hormones. Alternatively, one may propose another explanation based on the same experimental artefact as invoked for the estrogen and progesterone cytosol receptors. A higher affinity of the hormone-complexed receptor entails a lesser loss from the nucleus during tissue processing, and consequently an apparent increase in nuclear staining. Such a possibility is currently tested in parallel with the progesterone receptor.     

Regucalcin is under‐expressed in human breast and prostate cancers: Effect of sex steroid hormones

Regucalcin plays an important role in maintenance of intracellular Ca²⁺ homeostasis, suppresses cell proliferation, inhibits expression of oncogenes, and increases the expression of tumour suppressor genes. This suggests that regucalcin functions may be altered in cancer tissues. In this study the regucalcin expression in breast and prostate cancer cases was analysed by RT‐PCR and immunohistochemistry showing that the mRNA and/or protein are under‐expressed in these tumors. The effect of sex steroid hormones on regucalcin expression in breast and prostate cancer cells was determined by real‐time PCR. MCF‐7 and LNCaP cells were stimulated with 0, 1, and 10 nM of 17β‐estradiol (E₂) or 5α‐dihydrotestosterone (DHT), respectively, for 0, 6, 12, 24, and 48 h. MCF‐7 cells were also stimulated with E₂ conjugated to BSA (E₂‐BSA). To explore the mechanisms underlying the sex steroid regulation of regucalcin expression, control treatments with ICI 182,780, flutamide and cyclohexamide were carried out. E₂ effects regulating regucalcin expression were not abrogated in the presence of ICI 182,780, and were similar to those observed with E₂‐BSA, which suggests the involvement of a membrane‐bound estrogen receptor. In LNCaP cells, DHT down‐regulated regucalcin expression, an effect inhibited by the presence of both flutamide and cyclohexamide, suggesting the involvement of androgen receptor and de novo protein synthesis. The loss of regucalcin expression in breast and prostate cancer cases and the regulation of its expression by sex steroid hormones suggest that it may be associated with development and progression of these human tumors. J. Cell. Biochem. 107: 667–676, 2009. © 2009 Wiley‐Liss, Inc.