Progression to steroid autonomy in S115 mouse mammary tumor cells: role of DNA methylation

Although monoclonal in origin, mammary tumors acquire a marked heterogeneity of cell phenotypes, including a mixture of steroid hormone-sensitive cells and insensitive cells. We describe here long- term studies on the effects of androgen withdrawal on cloned androgen- responsive S115 mouse mammary tumor cells as a model system to investigate mechanisms by which tumor cells lose their steroid sensitivity. In the prolonged absence of androgen, the cells lost hormone-sensitive parameters reproducibly, including loss of proliferative response, saturation density response, cell morphology response, and mouse mammary tumor virus long terminal repeat (MMTV-LTR)- related RNA. These experiments have demonstrated that when deprived of hormone in the long term, a clone of responsive cells gives rise reproducibly to a population of unresponsive cells in an ordered series of phenotypic changes. At the time when the cells lost all androgen response in terms of cell biology and MMTV-LTR-RNA, increased methylation of MMTV-LTR sequences in the DNA was detected. Thereafter recovery of androgen sensitivity has not been achieved in any of these parameters. The possible role of de novo DNA methylation in the progression to androgen autonomy of S115 cells is discussed.     

Steroid regulation of transfected genes in mouse mammary tumour cells

The regulation of mouse mammary tumour virus (MMTV) RNA by glucocorticoid hormones is well-established and has provided much information on how steroid hormones work. However, we have shown that androgens can also control MMTV RNA accumulation in S115 mouse mammary tumour cells. This novel androgen action could be explained on the basis that the MMTV long terminal repeat (LTR) can respond to several classes of steroid if appropriate receptors are present in the cells. We have used transfection experiments to demonstrate that androgens can act directly on the LTR in S115 cells. Hormonal regulation of transfected chimaeric genes into these cells was effected by androgen and glucocorticoid but not by oestrogen or progesterone, corresponding to the receptor status of the cells. Furthermore, hormonal control was also conferred by the LTR on expression of an independent cotransfected adjacent gene under its own separate promoter, suggesting that effects of an LTR can stretch to neighbouring genes in a type of hormone-enhancer insertion mechanism.     

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.     

Approaches to studying the role of growth factors in the progression of breast tumours from the steroid sensitive to insensitive state

Progression from steroid sensitive to autonomous proliferation can be modelled in several cultured mammary tumour cell lines by long-term withdrawal of steroids. A feature of all the four systems studied thus far is that the basal growth in the absence of steroid increases with duration of steroid withdrawal until it reaches that obtained in the presence of steroid. It cannot be assumed that the increased proliferation in the absence of steroid is modulated by the same pathways as those stimulated by steroids in sensitive cells. Therefore, we feel that mechanisms of progression can best be studied via cell behaviour in the absence of steroid. With both the mouse S115 and human T-47-D systems, changes in sensitivity to several growth factors accompany progression; responses to TGF beta 1 are of particular interest in the T-47-D cells where this growth factor becomes stimulatory in the steroid insensitive state. This is accompanied by upregulation of TGF beta 1 mRNA. This upregulation of TGF beta agrees with the finding that ER - PR - primary human breast tumours contain more TGF beta 1 than do ER + PR + tumours; TGF alpha has the opposite pattern. Furthermore, only 40 and 30 kDa TGF beta species have been detected within cultured cells and primary tumours; TGF alpha exists in a 30 kDa form. The functions of these large forms of TGF alpha and TGF beta are unclear. Our conclusions from these experiments is that the increased proliferation in the absence of steroid accompanying progression may not be mediated by the same pathways as those perturbed by steroids in sensitive cells. Furthermore, TGF beta 1 may have different effects in steroid responsive and unresponsive cells.     

Endocrine treatment of prostate cancer

Although androgen deprivation as a treatment for patients with prostate cancer was described more than 60 years ago its optimal use remains controversial. The widespread use of prostate-specific (PSA) assay has lead to earlier diagnosis and earlier detection of recurrent disease. This means that the systemic side effects of androgen deprivation and quality of life have become more important. Debates continue regarding the proper use and timing of endocrine therapy with orchiectomy, oestrogen agonists, gonadotropin hormone-releasing hormone (GnRH) agonists, GnRH antagonists, and androgen antagonists. A critical review of the literature was performed. Data support that androgen deprivation is an effective treatment for patients with advanced prostate cancer. However, although it improves survival, it is not curative, and creates a spectrum of unwanted effects that influence quality of life. Castration remains the frontline treatment for metastatic prostate cancer, where orchiectomy, oestrogen agonists and GnRH agonists produce equivalent clinical responses. Maximum androgen blockade (MAB) is not significantly more effective than single agent GnRH agonist or orchiectomy. Nonsteroidal antiandrogen monotherapy is as effective as castration in treatment of locally advanced prostate cancer offering quality of life benefits. Adjuvant endocrine treatment is able to delay disease progression at any stage. There is, however, controversy of the possible survival benefit of such treatment, including patients having PSA relapse after definitive local treatment for prostate cancer. Neoadjuvant endocrine treatment has its place mainly in the external beam radiotherapy setting. Intermittent androgen blockade is still considered experimental. The decision regarding the type of androgen deprivation should be made individually after informing the patient of all available treatment options, including watchful waiting, and on the basis of potential benefits and adverse effects. Several large studies are under way to investigate the role of adjuvant endocrine treatment in the field of early prostate cancer, intermittent androgen deprivation and endocrine therapy alone compared with endocrine therapy with radiotherapy. The real challenge, however, is to develop better means to avert hormone-refractory prostate cancer and better treatments for patients with hormone-refractory disease when it occurs.     

Combination therapy with flutamide and medical (LHRH agonist) or surgical castration in advanced prostate cancer: 7-year clinical experience

Three hundred and sixty-three patients with clinical stage D2 prostate cancer who had not received previous endocrine therapy or chemotherapy were treated with the combination therapy using the pure antiandrogen Flutamide and the LHRH agonist [ -Trp⁶,des-Gly-NH₂¹⁰]LHRH ethylamide (or orchiectomy) for an average of 771 days (24–2607 days). Only 31 of the 308 evaluable patients (10.1%) did not show an objective positive response at the start of the combination therapy compared with an average of 18% in five recent studies using monotherapy. The median survival achieved using monotherapy is approximately 24 months while, in the present study, it is increased to 41.2 months, thus giving an additional 17 months of survival with the combination therapy. It should be mentioned that at the time of relapse, combination therapy is continued and, addition, further blockade of adrenal androgen secretion is achieved with aminoglutethimide and hydrocortisone. While our studies showing the advantages of combination therapy with pure antiandrogen in advanced prostate cancer have been confirmed by independent large-scale randomized studies, our preliminary data clearly suggest the interest of downstaging early stage prostate cancer by temporary combination therapy prior to radical prostatectomy.     

Expression of the E-cadherin repressors Snail, Slug and Zeb1 in urothelial carcinoma of the urinary bladder: relation to stromal fibroblast activation and invasive behaviour of carcinoma cells

Epithelial–mesenchymal transition (EMT) is regulated by interaction of carcinoma and stromal cells and crucial for progression of urinary bladder carcinoma (UBC). Therefore, the influence of activated fibroblasts on the expression of E-cadherin repressors as well as EMT and invasion in UBC was investigated. A correlative analysis of the immunohistochemical expression of fibroblast (ASMA, S100A4, FAP, SDF1, PDGFRβ) and EMT (Snail, Slug, Zeb1, E-cadherin) markers was performed on 49 UBC cases of different stages. The impact of distinguishable growth factor stimulated fibroblasts on invasion, EMT, and E-cadherin repressor expression was investigated in an invasion model. In situ, invasiveness was significantly correlated to the loss of membranous E-cadherin (E-cad_m) and increased Snail, Slug, Zeb1 in tumour cells, as well as to increased ASMA, S100A4, and PDGFRβ in stromal cells. A significant correlation to nodal metastasis could be evidenced for the loss of E-Cad_m, and for an increase in S100A4 and PDGFRβ. Comparison of stromal and EMT markers revealed significant correlations of ASMA to Snail and Slug; of S100A4 to the loss of E-cad_m and Zeb1; and of PDGFRβ to the loss of E-Cad_m, Slug and Zeb1. In vitro, TGFβ1 induced myofibroblasts were the strongest attractants, while aFGF or TGFβ1/aFGF stimulated fibroblasts were the most potent EMT inductors. As shown here for the first time, distinct sub-populations of fibroblasts are to various extents associated with EMT and tumour progression in UBC. These relevant findings might be the basis for the identification of new diagnostic markers and therapeutic targets selectively affecting tumour supporting CAF effects.     

Structural basis for androgen receptor agonists and antagonists: interaction of SPEED 98-listed chemicals and related compounds with the androgen receptor based on an in vitro reporter gene assay and 3D-QSAR

The androgen receptor (AR) activity of listed chemicals, so called SPEED 98, by the Ministry of the Environment, Japan, and structurally related chemicals was characterized using MDA-kb2 human breast cancer cells stably expressing an androgen-responsive luciferase reporter gene, MMTV-luc. Since our results suggested that chemicals with diverse chemical structures were capable of disrupting the endocrine systems mediated by AR, a comparative molecular field analysis (CoMFA) model was developed to analyze the structural requirements necessary to disrupt AR function. A significant CoMFA model with r(2)=0.825 and q(2)=0.332 was developed for AR antagonist activity of 35 pure antagonists excluding procymidone. On the other hand, a good CoMFA model with r(2)=0.983 and q(2)=0.555 was obtained for antagonist activity of 13 chemicals with both agonist and antagonist activities. The steric and electrostatic properties were sufficient to describe the structural requirements for AR antagonist activity. In addition, the structural difference of AR agonists and antagonists was explained based on CoMFA results and the AR-LBD crystal structure. As several ERalpha agonists such as diethylstilbestrol (DES) acted as AR antagonists, the surface area of the AR ligand-binding domain (LBD) was compared with that of the ERalpha-LBD based on their reported crystal structures to analyze how those ligands interact with LBDs. The surface area of AR-LBD was shown to be smaller than that of ERalpha-LBD and therefore compounds with both estrogenic and antiandrogenic activities can fit well into the ERalpha-LBD but may protrude from the AR-LBD. It is likely that this subtle difference of the surface areas of the LBDs determines whether an ERalpha agonist acts as an AR antagonist or an agonist.     

Amine dependence of proliferative activity in two transplantable lines of mouse colonic carcinoma

Serotonin, histamine and their antagonists have previously been shown to influence both the cell proliferation rate and the volumetric growth rate of colonic tumours. Of these earlier studies, those on cell proliferation could not distinguish between direct effects on tumour cells and indirect effects on the host, whereas those on the volumetric growth rate of tumours, whilst suggesting an outcome related to the individual properties of the tumour rather than the host, could not distinguish between influences on cell gain, cell loss or stromal changes. In an attempt to distinguish between these possibilities the current experiments on the mitotic rate in two lines of transplantable mouse colonic carcinoma were undertaken. One line of tumour proved to be sensitive to inhibition by a histamine H2 receptor antagonist and a dopamine D2 antagonist but resistant to serotonin antagonists; the inhibition by histamine antagonists was surmountable by co-administration of histamine. The other line proved to be highly sensitive to the inhibitory effects of serotonin antagonist and less so to antagonists of the other two amines and in this case the effect of serotonin antagonists was surmountable by serotonin. These results suggest that the variations between different colonic tumours in the response to amine antagonists is due to differences in the extent of inhibition of cell proliferation rather than differences in cell loss or stromal effects. Thus it appears likely that amine antagonists are able to directly interfere with the proliferation of some colonic tumour cells.     

Differential androgen sensitivity is associated with clonal heterogeneity in steroid metabolism, ornithine decarboxylase regulation and IL-1alpha action in mouse mammary tumor cells

Upon androgen deprivation, Shionogi (SC-115) mouse mammary tumors undergo phenotypic changes enabling their escape from growth dependence on androgens. Even within androgen-responsive cell populations, marked clonal heterogeneity is observed in the trophic effects of androgens. The present study compares several parameters of androgen action between three SC-115 cell clonal subpopulations exhibiting high (clone 107), low (clone S1A2) and no trophic response (clone 415) to androgens. These parameters pertain to (1) kinetics of androgen binding, (2) metabolism of 5alpha-dihydrotestosterone (DHT), 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) and 5alpha-androstane-3beta,17beta-diol (3beta-diol), (3) ornithine decarboxylase (ODC) activity and (4) interleukin-1alpha (IL-1alpha) action on cell proliferation. Only marginal differences in the affinity and abundance of androgen-specific binding sites were detected between the three clones. While clone S1A2 degraded DHT to 3alpha-diol at a much faster rate than the highly androgen-sensitive 107 cells and androgen-insensitive 415 cells, differences in the rates of intracrine conversion of 3alpha-diol and 3beta-diol to DHT did not correlate with the ability of these steroids to stimulate cell proliferation. Induction of ODC activity at the onset of exponential growth was strongly DHT-dependent in 107 cells, whereas this dependence was markedly attenuated in androgen-hyposensitive cells. Unexpectedly, DHT strongly repressed the marked ODC induction resulting from fresh medium addition in 415 cells which show no growth response to androgens. Low IL-1alpha concentrations were mitogenic in all three SC-115 clones. Whereas the mitogenic action of IL-1alpha was completely androgen-dependent in 107 cells, this dependence was relieved in S1A2 cells, which responded to DHT and IL-1alpha in an additive fashion. Thus, clonal heterogeneity in the pattern of steroid metabolism within Shionogi tumors cannot solely account for loss of androgen dependence, which may rather correlate with the constitutive activation of transduction pathways controlling the expression of growth-associated genes (e.g. ODC) by serum growth factors, including IL-1alpha.     

Steroid hormone responsive cells in serum-free culture--analyses of hormone-mediated gene expression and cell growth

Steroid hormone-responsive cell lines were clones from mouse mammary cancer (Shionogi Carcinoma 115) and Leydig cell tumor. SC-3 and SC-4 cells from Shionogi Carcinoma were androgen-responsive and -unresponsive in a serum-free medium, respectively. SC-3 cells secreted FGF-like growth factor as well as 24 K glycoprotein in response to androgen stimuli. B-1 and B-1F cells from mouse Leydig cell tumor were growth-stimulated in a serum-free medium by estrogen, androgen or retinoic acid. Transfection of ERE-TK-CAT gene into B-1F cells revealed that both estrogen and retinoic acid activated the CAT activity. In addition, the presence of corresponding receptors for steroid hormones or retinoic acid was demonstrated by hormone binding assays and/or Northern blot analysis. Thus, these serum-free culture systems seem to be very useful for analysing hormone action mechanisms in vitro.     

The metabolism of testosterone and dihydrotestosterone in an androgen-dependent tumour. A possible correlation between dihydrotestosterone and tumour growth in vivo

The effects of dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) and testosterone on the growth of the androgen-dependent Shionogi SC-115 tumour in mice have been compared and the metabolites in the tumour arising from each steroid have been identified. After the transfer of SC-115 tumour cells to castrated male mice, treatment of the recipients with dihydrotestosterone produced a striking proliferative response that enabled earlier tumour detection and led to a higher tumour incidence than obtained with testosterone. At short intervals after the intravenous injection of 200muCi of [1,2-(3)H]testosterone the amounts of radioactivity in tumour, muscle and seminal vesicles were almost equal. The metabolism of [1,2-(3)H]testosterone in tumour and muscle was slight in comparison with the extensive metabolism in seminal vesciles. Whereas up to 7% of the total neutral steroid recovered from whole tumour tissue and isolated nuclei was in the form of [1,2-(3)H]dihydrotestosterone, the amount of this compound in the corresponding preparations from seminal vesciles was several times greater. When the metabolism of [1,2-(3)H]dihydrotestosterone in tumour tissue was studied, it was found that more than 60% of the total neutral steroid in both cytoplasm and nuclei consisted of [1,2-(3)H]dihydrotestosterone. Thus much higher intracellular concentrations of dihydrotestosterone occurred with the administration of this steroid than with testosterone. Tumour cell proliferation was suppressed by oestradiol and the amount of androgen in nuclei was significantly decreased by high doses of this hormone.     

Thymic hormone containing cells--IX. Steroids in vitro modulate thymulin secretion by human and murine thymic epithelial cells

We investigated the in vitro effects (kinetics and dose-response) of adrenal and sexual steroid hormones on the secretion of thymulin, a thymic hormone, by human thymic epithelial cells in primary cultures as well as in a rat epithelial cell line. We demonstrated that all steroids tested, in a range of physiological doses, stimulated thymulin production to various extents. Progesterone and estradiol, however, were revealed to be the most efficient. Specific steroid antagonists abrogated the steroid-induced stimulation of thymulin production. These findings confirm our previous in vivo results and demonstrate that steroid hormones can act directly on thymic epithelial cells to modulate their endocrine production.