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.     

Evidence that transforming growth factor-beta is a hormonally regulated negative growth factor in human breast cancer cells

The hormone-dependent human breast cancer cell line MCF-7 secretes transforming growth factor-beta (TGF-beta), which can be detected in the culture medium in a biologically active form. These polypeptides compete with human platelet-derived TGF-beta for binding to its receptor, are biologically active in TGF-beta-specific growth assays, and are recognized and inactivated by TGF-beta-specific antibodies. Secretion of active TGF-beta is induced 8 to 27-fold under treatment of MCF-7 cells with growth inhibitory concentrations of antiestrogens. Antiestrogen-induced TGF-beta from MCF-7 cells inhibits the growth of an estrogen receptor-negative human breast cancer cell line in coculture experiments; growth inhibition is reversed with anti-TGF-beta antibodies. We conclude that in MCF-7 cells, TGF-beta is a hormonally regulated growth inhibitor with possible autocrine and paracrine functions in breast cancer 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.     

BG-1 ovarian cell line: An alternative model for examining estrogen-dependent growth in vitro

Summary Examination of estrogen-responsive processes in cell culture is used to investigate hormonal influence on cancer cell growth and gene expression. Most experimental studies have used breast cancer cell lines, in particular MCF7 cells, to investigate estrogen responsiveness. In this study we examined an ovarian cancer cell line, BG-1, which is highly estrogen-responsive in vitro. This observation, plus the fact that the cells are of ovarian rather than mammary gland origin, makes it an attractive alternative model. 17β-Estradiol, epidermal growth factor, and insulin-like growth factor induced proliferation of BG-1 and MCF7 cells. Viability was dependent on these growth factors in BG-1 cells, but not in MCF7 cells. Therefore, we examined the differences between these two cell lines with respect to estrogen and growth factor receptors. BG-1 cells have twice as many estrogen receptors as MCF7 cells, and BG-1 cells have higher insulin-like growth factor-1 and epidermal growth factor receptor levels than MCF7 cells. This may also explain why BG-1 cells proliferate 56% more robustly in serum and show more serum dependence in culture. In both BG-1 and MCF7 cells, epidermal growth factor receptor number is low (<20 000/cell), while insulin-like growth factor-1 receptor level was highest in estrogen receptor positive cell lines. For example, insulin-like growth factor-1 receptor was higher in BG-1 and MCF7 cells than in estrogen receptor negative cells (HeLa>MDA-MB-435>HBL100). In conclusion, BG-1 cells are an excellent model for understanding hormone responsiveness in ovarian tissue and an alternative for examining estrogen receptor-mediated and insulin-like growth factor-1/epidermal growth factor/estrogen cross-talk processes because of their sensitivity to these factors.     

Regulation of growth of LNCaP human prostate tumor cells by growth factors and steroid hormones

The mitogenic activity of several growth factors on androgen responsive LNCaP human prostate tumor cells was studied. A two-fold stimulation of cell proliferation was observed after a culture period of 6 days in 1 ng EGF/ml, 10 ng TGF-alpha/ml or 20 ng basic FGF/ml. TGF-beta (0.02 ng/ml), which did not affect cell proliferation when added alone to the culture medium, inhibited the EGF- and TGF-alpha-induced growth. The synthetic androgen R1881 (0.1 nM) stimulated cell proliferation three-fold and increased the number of EGF receptors from 11500 to 28500 sites/cell. One of the mechanisms involved in androgen action on these cells is therefore an increased EGF receptor expression and increased sensitivity to EGF. TGF-beta did not directly affect androgen-responsive growth but inhibited the synergistic effect of EGF. A considerable expression of TGF alpha (precursors) could be demonstrated on the cells by immunohistochemical staining. However the staining intensity was not affected by androgens. These results make it less likely that androgen-responsive growth is mediated by regulation of secretion of an EGF- or TGF alpha-like activity, which in turn acts in an autocrine manner to stimulate growth. Estrogens, progestagens and antiandrogens do not inhibit androgen responsive growth of LNCaP cells but have striking growth stimulatory effects, increase EGF receptor level and increase acid phosphatase secretion. LNCaP cells contain a modified androgen receptor system with respect to both steroid specificity and antiandrogen sensitivity. It has recently been shown that the stimulatory effects are due to a mutated amino acid in the steroid binding domain of the androgen receptor.     

Ribozyme targeting demonstrates that the nuclear receptor coactivator AIB1 is a rate-limiting factor for estrogen-dependent growth of human MCF-7 breast cancer cells

Human breast tumorigenesis is promoted by the estrogen receptor pathway, and nuclear receptor coactivators are thought to participate in this process. Here we studied whether one of these coactivators, AIB1 (amplified in breast cancer 1), was rate-limiting for hormone-dependent growth of human MCF-7 breast cancer cells. We developed MCF-7 breast cancer cell lines in which the expression of AIB1 can be modulated by regulatable ribozymes directed against AIB1 mRNA. We found that depletion of endogenous AIB1 levels reduced steroid hormone signaling via the estrogen receptor alpha or progesterone receptor beta on transiently transfected reporter templates. Down-regulation of AIB1 levels in MCF-7 cells did not affect estrogen-stimulated cell cycle progression but reduced estrogen-mediated inhibition of apoptosis and cell growth. Finally, upon reduction of endogenous AIB1 expression, estrogen-dependent colony formation in soft agar and tumor growth of MCF-7 cells in nude mice was decreased. From these findings we conclude that, despite the presence of different estrogen receptor coactivators in breast cancer cells, AIB1 exerts a rate-limiting role for hormone-dependent human breast tumor growth.     

Estrogen and progesterone receptor proteins in breast cancer.

This article reviews the contribution of steroid hormone receptor studies to the resolution of a basic clinical problem: how to determine which cancers are hormone dependent without an actual treatment trial. Previously published studies on hormone receptor assays and analyses are reviewed, the current status of knowledge in the area is summarized in terms of its relevance to breast cancer cells, and future scenarios are proffered. Assay methods and available clinical results for cytoplasmic estrogen receptor identification in human breast cancer comprise the first section. Information on assaying nuclear estrogen receptor, and its relative clinical importance, is presented in Part 2. Assays to determine the presence of progesterone receptor in human breast cancer; the estogen regulation of such progesterone receptors; and clinical findings comprise Part 3. Studies of the mechanisms of estrogen action in the MCF-7 human breast cancer cell line are the subject of Part 4. Recent experiments in animals on the efficacy of antiestrogen treatments, such treatments in human in vitro cell cultures, and the few clinical trials available are presented in Part 5. It is emphasized that the simple presence or absence of estrogen receptors in tumors does not absolutely indicate whether growth of a particular tumor is sensitive to estrogens. Experimental appraoches, designed to further delineate this problem, are outlined, based on observations such as the finding that the probability of tumor regression correlates better with quantitative rather than with qualitative assessment of estrogen receptors; that the specific end product of hormone action is unknown and without this information an ideal biochemical marker to a tumor's sensitivity of hormones is unavailable; and that the complex sequence of biochemical events in the actions of estrogen needs more complete elucidation.     

Minireview: nuclear receptors and breast cancer

Until recently, the study of nuclear receptor (NR) function in breast cancer biology has been largely limited to estrogen and progesterone receptors. The development of reliable gene expression arrays, real-time quantitative RT-PCR, and immunohistochemical techniques for studying NR superfamily members in primary human breast cancers has now revealed the presence and potential importance of several additional NRs in the biology of breast cancer. These include receptors for steroid hormones (including androgens and corticosteroids), fat-soluble vitamins A and D, fatty acids, and xenobiotic lipids derived from diet. It is now clear that after NR activation, both genomic and nongenomic NR pathways can coordinately activate growth factor signaling pathways. Advances in our understanding of both NR functional networks and epithelial cell growth factor signaling pathways have revealed a frequent interplay between NR and epithelial cell growth factor family signaling that is clinically relevant to breast cancer. Understanding how growth factor receptors and their downstream kinases are activated by NRs (and vice-versa) is a central goal for maximizing treatment opportunities in breast cancer. In addition to the estrogen receptor, it is predicted that modulating the activity of other NRs will soon provide novel prevention and treatment approaches for breast cancer patients.     

Autocrine transforming growth factor-beta signaling mediates Smad-independent motility in human cancer cells

Transforming growth factor-beta (TGF-beta) is a pleiotropic growth factor that plays a critical role in modulating cell growth, differentiation, and plasticity. There is increasing evidence that after cells lose their sensitivity to TGF-beta-mediated growth inhibition, autocrine TGF-beta signaling may potentially promote tumor cell motility and invasiveness. To understand the molecular mechanisms by which autocrine TGF-beta may selectively contribute to tumor cell motility, we have generated MDA-MB-231 breast cancer cells stably expressing a kinase-inactive type II TGF-beta receptor (T beta RII-K277R). Our data indicate that T beta RII-K277R is expressed, can associate with the type I TGF-beta receptor, and block both Smad-dependent and -independent signaling pathways activated by TGF-beta. In addition, wound closure and transwell migration assays indicated that the basal migratory potential of T beta RII-K277R expressing cells was impaired. The impaired motility of T beta RII-K277R cells could be restored by reconstituting TGF-beta signaling with a constitutively active TGF-beta type I receptor (ALK5(TD)) but not by reconstituting Smad signaling with Smad2/4 or Smad3/4 expression. In addition, the levels of ALK5(TD) expression sufficient to restore motility in the cells expressing T beta RII-K277R were associated with an increase in phosphorylation of Akt and extracellular signal-regulated kinase 1/2 but not Smad2. These data indicate that different signaling pathways require different thresholds of TGF-beta activation and suggest that TGF-beta promotes motility through mechanisms independent of Smad signaling, possibly involving activation of the phosphatidylinositol 3-kinase/Akt and/or mitogen-activated protein kinase pathways.